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Sample records for acanthamoeba polyphaga mimivirus

  1. Acanthamoeba polyphaga mimivirus and other giant viruses: an open field to outstanding discoveries

    PubMed Central

    2014-01-01

    In 2003, Acanthamoeba polyphaga mimivirus (APMV) was first described and began to impact researchers around the world, due to its structural and genetic complexity. This virus founded the family Mimiviridae. In recent years, several new giant viruses have been isolated from different environments and specimens. Giant virus research is in its initial phase and information that may arise in the coming years may change current conceptions of life, diversity and evolution. Thus, this review aims to condense the studies conducted so far about the features and peculiarities of APMV, from its discovery to its clinical relevance. PMID:24976356

  2. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    PubMed Central

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P213, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities. PMID:16511098

  3. Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

    PubMed Central

    Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

    2014-01-01

    ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside

  4. Acanthamoeba polyphaga mimivirus Stability in Environmental and Clinical Substrates: Implications for Virus Detection and Isolation

    PubMed Central

    de Almeida, Gabriel M.; Campos, Rafael K.; Boratto, Paulo V. M.; Franco-Luiz, Ana P. M.; La Scola, Bernard; Ferreira, Paulo C. P.; Kroon, Erna G.; Abrahão, Jônatas S.

    2014-01-01

    Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies. PMID:24498379

  5. The N-terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRS(apm)) is a monomer in solution.

    PubMed

    Choudhury, Aparajita; Banerjee, Rajat

    2013-03-18

    Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRSapm) was the first reported aminoacyl-tRNA synthetase of viral origin. The previous crystal structure of TyrRSapm showed a non-canonical orientation of the dimer conformation and the CP1 domain, responsible for dimer formation, displays a major modification of a motif structurally conserved in other TyrRS structures. An earlier study reported that Bacillus stearothermophilus N-terminal TyrRS exists as a dimer under native conditions. N-terminal TyrRSapm (ΔTyrRSapm, 1-234 aa) was constructed to remove the C-terminal anticodon-binding domain. Here we show by Ferguson plot analysis and analytical ultracentrifugation that ΔTyrRSapm exists as a monomer and contains a disulfide-bridge. The ΔTyrRSapm loses the ability to bind tRNA(Tyr), however it remains active in pyrophosphate exchange with similar ligand dissociation constants as the full-length enzyme. PMID:23384724

  6. Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

    PubMed

    Caumo, Karin Silva; Monteiro, Karina Mariante; Ott, Thiely Rodrigues; Maschio, Vinicius José; Wagner, Glauber; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2014-12-01

    Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae. PMID:25149354

  7. Acanthamoeba polyphaga-Enhanced Growth of Mycobacterium smegmatis

    PubMed Central

    Lamrabet, Otmane; Medie, Felix Mba; Drancourt, Michel

    2012-01-01

    Background Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival. Methodology/Principal Findings Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc2 155, M. smegmatis ATCC 19420T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth. Conclusions/Significance Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria. PMID:22253795

  8. Related giant viruses in distant locations and different habitats: Acanthamoeba polyphaga moumouvirus represents a third lineage of the Mimiviridae that is close to the megavirus lineage.

    PubMed

    Yoosuf, Niyaz; Yutin, Natalya; Colson, Philippe; Shabalina, Svetlana A; Pagnier, Isabelle; Robert, Catherine; Azza, Said; Klose, Thomas; Wong, Jimson; Rossmann, Michael G; La Scola, Bernard; Raoult, Didier; Koonin, Eugene V

    2012-01-01

    The 1,021,348 base pair genome sequence of the Acanthamoeba polyphaga moumouvirus, a new member of the Mimiviridae family infecting Acanthamoeba polyphaga, is reported. The moumouvirus represents a third lineage beside mimivirus and megavirus. Thereby, it is a new member of the recently proposed Megavirales order. This giant virus was isolated from a cooling tower water in southeastern France but is most closely related to Megavirus chiliensis, which was isolated from ocean water off the coast of Chile. The moumouvirus is predicted to encode 930 proteins, of which 879 have detectable homologs. Among these predicted proteins, for 702 the closest homolog was detected in Megavirus chiliensis, with the median amino acid sequence identity of 62%. The evolutionary affinity of moumouvirus and megavirus was further supported by phylogenetic tree analysis of conserved genes. The moumouvirus and megavirus genomes share near perfect orthologous gene collinearity in the central part of the genome, with the variations concentrated in the terminal regions. In addition, genomic comparisons of the Mimiviridae reveal substantial gene loss in the moumouvirus lineage. The majority of the remaining moumouvirus proteins are most similar to homologs from other Mimiviridae members, and for 27 genes the closest homolog was found in bacteria. Phylogenetic analysis of these genes supported gene acquisition from diverse bacteria after the separation of the moumouvirus and megavirus lineages. Comparative genome analysis of the three lineages of the Mimiviridae revealed significant mobility of Group I self-splicing introns, with the highest intron content observed in the moumouvirus genome. PMID:23221609

  9. Related Giant Viruses in Distant Locations and Different Habitats: Acanthamoeba polyphaga moumouvirus Represents a Third Lineage of the Mimiviridae That Is Close to the Megavirus Lineage

    PubMed Central

    Yoosuf, Niyaz; Yutin, Natalya; Colson, Philippe; Shabalina, Svetlana A.; Pagnier, Isabelle; Robert, Catherine; Azza, Said; Klose, Thomas; Wong, Jimson; Rossmann, Michael G.; La Scola, Bernard; Raoult, Didier; Koonin, Eugene V.

    2012-01-01

    The 1,021,348 base pair genome sequence of the Acanthamoeba polyphaga moumouvirus, a new member of the Mimiviridae family infecting Acanthamoeba polyphaga, is reported. The moumouvirus represents a third lineage beside mimivirus and megavirus. Thereby, it is a new member of the recently proposed Megavirales order. This giant virus was isolated from a cooling tower water in southeastern France but is most closely related to Megavirus chiliensis, which was isolated from ocean water off the coast of Chile. The moumouvirus is predicted to encode 930 proteins, of which 879 have detectable homologs. Among these predicted proteins, for 702 the closest homolog was detected in Megavirus chiliensis, with the median amino acid sequence identity of 62%. The evolutionary affinity of moumouvirus and megavirus was further supported by phylogenetic tree analysis of conserved genes. The moumouvirus and megavirus genomes share near perfect orthologous gene collinearity in the central part of the genome, with the variations concentrated in the terminal regions. In addition, genomic comparisons of the Mimiviridae reveal substantial gene loss in the moumouvirus lineage. The majority of the remaining moumouvirus proteins are most similar to homologs from other Mimiviridae members, and for 27 genes the closest homolog was found in bacteria. Phylogenetic analysis of these genes supported gene acquisition from diverse bacteria after the separation of the moumouvirus and megavirus lineages. Comparative genome analysis of the three lineages of the Mimiviridae revealed significant mobility of Group I self-splicing introns, with the highest intron content observed in the moumouvirus genome. PMID:23221609

  10. Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles

    PubMed Central

    Olofsson, Jenny; Axelsson-Olsson, Diana; Brudin, Lars; Olsen, Björn; Ellström, Patrik

    2013-01-01

    The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81–176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced. PMID:24223169

  11. Photodynamic inactivation of Acanthamoeba polyphaga with curcuminoids: an in vitro study

    NASA Astrophysics Data System (ADS)

    Corrêa, Thaila Q.; Geralde, Mariana C.; Carvalho, Mariana T.; Bagnato, Vanderlei S.; Kurachi, Cristina; de Souza, Clovis W. O.

    2016-03-01

    Acanthamoeba polyphaga are free-living amoebae that can be considered potentially pathogenic organisms by cause serious human infections, including keratitis and granulomatous amoebic encephalitis that usually results in death. Photodynamic inactivation (PDI) has been used for the biological control of microorganisms and can be promise in the control of Acanthamoeba infections. This study evaluated the in vitro effectiveness of PDI in A. polyphaga using curcuminoids salt as photosensitizer (PS) besides observing morphological changes caused by this PS in this organism, in confocal microscopy. A. polyphaga trophozoites were grown at 37°C in PYG medium for 48 to 72 hours. After, the trophozoites were incubated with PS solution during one hour and the samples were irradiated using light-emitting diodes at 460 nm at light doses 30 and 50 J/cm2. The results revealed reduction of 27.7%, 61.4% and 82.5% at 30 J/cm2 and 75.2%, 85.0% and 95.9% at 50 J/cm2, respectively, at curcuminoid salt concentrations of 500, 1000 and 1500 μg/mL. Through fluorescence images, it was possible to visualize the curcuminoid salt's uptake by the trophozoites. The PS showed toxicity to amoebae, in the dark, but the irradiation in PDI contributed to amoebae death effect. These data suggest that PDI may be an application of therapeutic intervention against Acanthamoeba infections, since it was effective in the inactivation of these amoebae.

  12. Investigating the role of Acanthamoeba polyphaga in protecting Human Adenovirus from water disinfection treatment.

    PubMed

    Verani, Marco; Di Giuseppe, Graziano; Tammaro, Carmine; Carducci, Annalaura

    2016-06-01

    Human adenoviruses are responsible for a wide range of clinical infections and are present in aquatic environments, including river, seawater, drinking-water and sewage. Free-living amoebae (Acanthamoeba) in the same environments may internalize them and other microorganisms can act as a reservoir for the internalized viruses. In this study, we studied the interaction between Acanthamoeba polyphaga and Human Adenovirus type 5 (HAdV 5) to determine whether the amoeba played a role in protecting the internalized viruses from chemical disinfection. The efficacy of sodium hypochlorite disinfection against A. polyphaga and HAdV 5 either singly or in combination was assessed at three different concentrations. Individually, the amoeba were more resistant to chemical disinfection than HAdV 5 and remained alive after exposure to 5mg/l of sodium hypochlorite. In contrast, HAdV 5 lost infectivity following exposure to 2.5mg/l of sodium hypochlorite. When the amoeba and HAdV 5 were co-cultured, infectious virus was found in the cytoplasm of the amoeba at 5mg/l disinfectant concentration. These findings suggest that the A. polyphaga is providing protection for the HAdV 5. PMID:26999560

  13. Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

    PubMed Central

    Lamb, David C.; Warrilow, Andrew G. S.; Rolley, Nicola J.; Parker, Josie E.; Nes, W. David; Smith, Stephen N.; Kelly, Diane E.

    2015-01-01

    In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections. PMID:26014948

  14. Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51).

    PubMed

    Lamb, David C; Warrilow, Andrew G S; Rolley, Nicola J; Parker, Josie E; Nes, W David; Smith, Stephen N; Kelly, Diane E; Kelly, Steven L

    2015-08-01

    In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections. PMID:26014948

  15. Raman spectroscopic study on the excystation process in a single unicellular organism amoeba (Acanthamoeba polyphaga)

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Chung; Perevedentseva, Elena; Cheng, Chia-Liang

    2015-05-01

    An in vivo Raman spectroscopic study of amoeba (Acanthamoeba polyphaga) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms in vivo and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

  16. Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological status and susceptibility to chemical inactivation

    SciTech Connect

    Barker, J.; Farrell, I. ); Brown, M.R.W.; Collier, P.J.; Gilbert, P. )

    1992-08-01

    Survival studies were conducted on Legionella pneumophila cells that had been grown intracellulary in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 [times] MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure of PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.

  17. Exposure to Mimivirus Collagen Promotes Arthritis

    PubMed Central

    Shah, Nikunj; Hülsmeier, Andreas J.; Hochhold, Nina; Neidhart, Michel; Gay, Steffen

    2014-01-01

    Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens. PMID:24173233

  18. The abundant free-living amoeba, Acanthamoeba polyphaga, increases the survival of Campylobacter jejuni in milk and orange juice

    PubMed Central

    Olofsson, Jenny; Berglund, Petra Griekspoor; Olsen, Björn; Ellström, Patrik; Axelsson-Olsson, Diana

    2015-01-01

    Background Campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world. Most C. jejuni infections are acquired from contaminated poultry, milk, and water. Due to health care costs and human suffering, it is important to identify all possible sources of infection. Unpasteurized milk has been associated with several outbreaks of C. jejuni infection. Campylobacter has been identified on fresh fruit, and other gastrointestinal pathogens such as Salmonella, E. coli O157:H7 and Cryptosporidium have been involved in fruit juice outbreaks. C. jejuni is sensitive to the acidic environment of fruit juice, but co-cultures with the amoeba, Acanthamoeba polyphaga, have previously been shown to protect C. jejuni at low pH. Methods To study the influence of A. polyphaga on the survival of C. jejuni in milk and juice, the bacteria were incubated in the two products at room temperature and at 4°C with the following treatments: A) C. jejuni preincubated with A. polyphaga before the addition of product, B) C. jejuni mixed with A. polyphaga after the addition of product, and C) C. jejuni in product without A. polyphaga. Bacterial survival was assessed by colony counts on blood agar plates. Results Co-culture with A. polyphaga prolonged the C. jejuni survival both in milk and juice. The effect of co-culture was most pronounced in juice stored at room temperature. On the other hand, A. polyphaga did not have any effect on C. jejuni survival during pasteurization of milk or orange juice, indicating that this is a good method for eliminating C. jejuni in these products. Conclusion Amoebae-associated C. jejuni in milk and juice might cause C. jejuni infections. PMID:26387556

  19. Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

    PubMed

    Santos, Israel Gomes de Amorim; Scher, Ricardo; Rott, Marilise Brittes; Menezes, Leociley Rocha; Costa, Emmanoel Vilaça; Cavalcanti, Sócrates Cabral de Holanda; Blank, Arie Fitzgerald; Aguiar, Jaciana dos Santos; da Silva, Teresinha Gonçalves; Dolabella, Silvio Santana

    2016-02-01

    Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 μg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 μg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency. PMID:26446087

  20. Detection limits of Legionella pneumophila in environmental samples after co-culture with Acanthamoeba polyphaga

    PubMed Central

    2013-01-01

    Background The efficiency of recovery and the detection limit of Legionella after co-culture with Acanthamoeba polyphaga are not known and so far no investigations have been carried out to determine the efficiency of the recovery of Legionella spp. by co-culture and compare it with that of conventional culturing methods. This study aimed to assess the detection limits of co-culture compared to culture for Legionella pneumophila in compost and air samples. Compost and air samples were spiked with known concentrations of L. pneumophila. Direct culturing and co-culture with amoebae were used in parallel to isolate L. pneumophila and recovery standard curves for both methods were produced for each sample. Results The co-culture proved to be more sensitive than the reference method, detecting 102-103 L. pneumophila cells in 1 g of spiked compost or 1 m3 of spiked air, as compared to 105-106 cells in 1 g of spiked compost and 1 m3 of spiked air. Conclusions Co-culture with amoebae is a useful, sensitive and reliable technique to enrich L. pneumophila in environmental samples that contain only low amounts of bacterial cells. PMID:23442526

  1. The fate of Helicobacter pylori phagocytized by Acanthamoeba polyphaga demonstrated by fluorescent in situ hybridization and quantitative polymerization chain reaction tests

    EPA Science Inventory

    Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pyl...

  2. Marine mimivirus relatives are probably large algal viruses

    PubMed Central

    Monier, Adam; Larsen, Jens Borggaard; Sandaa, Ruth-Anne; Bratbak, Gunnar; Claverie, Jean-Michel; Ogata, Hiroyuki

    2008-01-01

    Background Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data. Results We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in mimivirus. Conclusion Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae. PMID:18215256

  3. Mimivirus Collagen Is Modified by Bifunctional Lysyl Hydroxylase and Glycosyltransferase Enzyme*

    PubMed Central

    Luther, Kelvin B.; Hülsmeier, Andreas J.; Schegg, Belinda; Deuber, Stefan A.; Raoult, Didier; Hennet, Thierry

    2011-01-01

    Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology. PMID:22045808

  4. Evaluation of the activity of new cationic carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga.

    PubMed

    Heredero-Bermejo, Irene; Copa-Patiño, Jose Luis; Soliveri, Juan; Fuentes-Paniagua, Elena; de la Mata, Francisco Javier; Gomez, Rafael; Perez-Serrano, Jorge

    2015-02-01

    Dendrimers are repetitively branched molecules with a broad spectrum of applications, mainly for their antimicrobial properties and as nanocarriers for other molecules. Recently, our research group have synthesized and studied their activity against Acanthamoeba sp., causative agent of a severe ocular disease in humans: Acanthamoeba keratitis. New cationic carbosilane dendrimers were tested against the protozoa forms at different concentrations and for different incubation times. Trophozoite viability was determined by manual counting and cyst viability by observing excystment in microplates with fresh culture medium. Cytotoxicity was checked on HeLa cells using the microculture tetrazolium assay. Alterations were observed by optical microscopy and by flow cytometry staining with propidium iodide. Six out of the 18 dendrimers tested were non-cytotoxic and effective against the trophozoite form, having one of them (dendrimer 14 with an IC50 of 2.4 + 0.1 mg/L) a similar activity to chlorhexidine digluconate (IC50 1.7 + 0.1 mg/L). This dendrimer has a polyphenoxo core and a sulphur atom close to the six -NH3+ terminal groups. On the other hand, only two dendrimers showed some effect against cysts (dendrimers 14 and 17). However, their minimum cysticidal concentrations were cytotoxic and less effective than the control drug. The alterations on the amoeba morphology produced by the treatment with dendrimers were size reduction, increased complexity, loss of acanthopodia and cell membrane disruption. In conclusion, these results suggest that some dendrimers may be studied in animal models to test their effect and that new dendrimers with similar features should be synthesized. PMID:25358240

  5. Ameobal Pathogen Mimivirus Infects Macrophages through Phagocytosis

    PubMed Central

    Ghigo, Eric; Kartenbeck, Jürgen; Lien, Pham; Pelkmans, Lucas; Capo, Christian; Mege, Jean-Louis; Raoult, Didier

    2008-01-01

    Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages. PMID:18551172

  6. Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon

    PubMed Central

    2014-01-01

    Background The identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas. Methods/results Based on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs. Conclusion This discovery expands our knowledge of Mimiviridae evolution and ecology. PMID:24886672

  7. The virophage as a unique parasite of the giant mimivirus.

    PubMed

    La Scola, Bernard; Desnues, Christelle; Pagnier, Isabelle; Robert, Catherine; Barrassi, Lina; Fournous, Ghislain; Merchat, Michèle; Suzan-Monti, Marie; Forterre, Patrick; Koonin, Eugene; Raoult, Didier

    2008-09-01

    Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses. PMID:18690211

  8. Mimiviruses: Replication, Purification, and Quantification.

    PubMed

    Abrahão, Jônatas Santos; Oliveira, Graziele Pereira; Ferreira da Silva, Lorena Christine; Dos Santos Silva, Ludmila Karen; Kroon, Erna Geessien; La Scola, Bernard

    2016-01-01

    The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc. PMID:27153385

  9. Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire

    PubMed Central

    Rodrigues, Rodrigo Araújo Lima; dos Santos Silva, Ludmila Karen; Dornas, Fábio Pio; de Oliveira, Danilo Bretas; Magalhães, Thais Furtado Ferreira; Santos, Daniel Assis; Costa, Adriana Oliveira; de Macêdo Farias, Luiz; Magalhães, Paula Prazeres; Bonjardim, Cláudio Antônio; Kroon, Erna Geessien; La Scola, Bernard; Cortines, Juliana Reis

    2015-01-01

    ABSTRACT Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion

  10. Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses

    PubMed Central

    Assis, Felipe L.; Bajrai, Leena; Abrahao, Jonatas S.; Kroon, Erna G.; Dornas, Fabio P.; Andrade, Kétyllen R.; Boratto, Paulo V. M.; Pilotto, Mariana R.; Robert, Catherine; Benamar, Samia; La Scola, Bernard; Colson, Philippe

    2015-01-01

    Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR) and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%–99% similar, whereas Kroon virus had a low similarity (90%–91%) with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment. PMID:26131958

  11. Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses.

    PubMed

    Assis, Felipe L; Bajrai, Leena; Abrahao, Jonatas S; Kroon, Erna G; Dornas, Fabio P; Andrade, Kétyllen R; Boratto, Paulo V M; Pilotto, Mariana R; Robert, Catherine; Benamar, Samia; Scola, Bernard La; Colson, Philippe

    2015-07-01

    Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR) and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%-99% similar, whereas Kroon virus had a low similarity (90%-91%) with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment. PMID:26131958

  12. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

    PubMed

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-05-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

  13. Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

    PubMed

    O'Day, Danton H; Suhre, Karsten; Myre, Michael A; Chatterjee-Chakraborty, Munmun; Chavez, Sara E

    2006-08-01

    A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis. PMID:16777069

  14. Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

    PubMed Central

    Zade, Amrutraj; Sengupta, Malavi; Kondabagil, Kiran

    2015-01-01

    Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV) is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV). The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene). Our thorough in silico analysis of the subfamily specific (SF) region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B, and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III PI3K homolog, coded by APMV L615), Atg-8 and dynamin (host proteins) are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly. PMID:26441866

  15. Acanthamoeba keratitis.

    PubMed

    Pogson, C

    1993-01-01

    1. Acanthamoeba keratitis is an uncommon but increasingly prevalent infection with the potential to cause severe ocular damage. Acanthamoeba is a nonflagellated free-living amoeba that is ubiquitous in the environment. The most common type is A castellani, but A polyphagia, A rhysodes, A culbertson, and A hatchetti have been isolated from infected eyes. 2. Clinical features include foreign body sensation, blurred vision, tearing, and photophobia. There is minimal pain in the early stages of infection, but severe pain is a manifestation of the advanced stages of the disease. 3. The incidence of Acanthamoeba keratitis appears to be decreasing because of increased awareness, but education emphasizing proper lens sterilization is essential and should be carried out when contact lenses are first dispensed. PMID:8158668

  16. Isolation and complete genome sequencing of Mimivirus bombay, a Giant Virus in sewage of Mumbai, India.

    PubMed

    Chatterjee, Anirvan; Ali, Farhan; Bange, Disha; Kondabagil, Kiran

    2016-09-01

    We report the isolation and complete genome sequencing of a new Mimiviridae family member, infecting Acanthamoeba castellanii, from sewage in Mumbai, India. The isolated virus has a particle size of about 435 nm and a 1,182,200-bp genome. A phylogeny based on the DNA polymerase sequence placed the isolate as a new member of the Mimiviridae family lineage A and was named as Mimivirus bombay. Extensive presence of Mimiviridae family members in different environmental niches, with remarkably similar genome size and genetic makeup, point towards an evolutionary advantage that needs to be further investigated. The complete genome sequence of Mimivirus bombay was deposited at GenBank/EMBL/DDBJ under the accession number KU761889. PMID:27330993

  17. Killing acanthamoebae with polyaminopropyl biguanide: quantitation and kinetics.

    PubMed Central

    Burger, R M; Franco, R J; Drlica, K

    1994-01-01

    The two Acanthamoeba species most often implicated in corneal keratitis, A. castellanii and A. polyphaga, were exposed as cysts to polyaminopropyl biguanide (PAPB), a commonly used antimicrobial agent. Killing of amoeba cysts was rapid and extensive, with fewer than 2% of either species surviving 30 s of exposure to > or = 45 ppm of PAPB. Killing kinetics were biphasic, and further exposures of 15 min to 1 h killed greater than 90% of those surviving initial killing. This potency of PAPB, together with its low toxicity to humans when ingested or applied topically, underscores the potential of PAPB as an antiamoebic agent. PMID:8031066

  18. Acanthamoeba royreba sp. n. from a human tumor cell culture.

    PubMed

    Willaert, E; Stevens, A R; Tyndall, R L

    1978-02-01

    A new species of Acanthamoeba was isolated from a culture of an established line of human choriocarcinoma cells. The identification of this strain, originally called the Oak Ridge strain, and the establishment of a new species for it were based on morphologic, serologic, and immunochemical studies. In general, the structure of the trophozoite did not differ significantly from that of other species of Acanthamoeba, except that a body which more closely resembled a centriole than material described previously as centriolar satellites was observed in trophozoites examined with the electron microscope. The dimensions of the trophozoite were the smallest among the species of Acanthamoeba. The cyst was typical of the genus, but differed from those of other species by its smaller size and the presence of numerous ostioles. Studies of the Oak Ridge strain by immunofluorescence using antisera developed against the isolate and Acanthamoeba culbertsoni, A. castellanii, A. polyphaga, A. rhysodes, A. astronyxis, and A. palestinensis revealed the antigenic uniqueness of the Oak Ridge strain. It was demonstrated by immunoelectrophoretic analyses of the soluble proteins of the Oak Ridge strain that shared approximately 1/2 of its antigenic structure with A. castellanii and A. culbertsoni. The antigenic differences of the isolate from other species of Acanthamoeba were deduced from comparison of the antigenic constitution of these species and the Oak Ridge strain with A. culbertsoni and A. castellanii. Although the strain was initially recognized by its cytopathogenicity for cultures, it did not produce acute infections in mice after intranasal inoculation of 1 X 10(4) ameba/mouse. The foregoing results constituted the basis for the establishment of the Oak Ridge strain as a new species, A. royreba sp. n., in the genus Acanthamoeba. PMID:566323

  19. Inactivation of Acanthamoeba spp. and Other Ocular Pathogens by Application of Cold Atmospheric Gas Plasma.

    PubMed

    Heaselgrave, Wayne; Shama, Gilbert; Andrew, Peter W; Kong, Michael G

    2016-05-15

    Currently there are estimated to be approximately 3.7 million contact lens wearers in the United Kingdom and 39.2 million in North America. Contact lens wear is a major risk factor for developing an infection of the cornea known as keratitis due to poor lens hygiene practices. While there is an international standard for testing disinfection methods against bacteria and fungi (ISO 14729), no such guidelines exist for the protozoan Acanthamoeba, which causes a potentially blinding keratitis most commonly seen in contact lens wearers, and as a result, many commercially available disinfecting solutions show incomplete disinfection after 6 and 24 h of exposure. Challenge test assays based on international standard ISO 14729 were used to determine the antimicrobial activity of cold atmospheric gas plasma (CAP) against Pseudomonas aeruginosa, Candida albicans, and trophozoites and cysts of Acanthamoeba polyphaga and Acanthamoeba castellanii P. aeruginosa and C. albicans were completely inactivated in 0.5 min and 2 min, respectively, and trophozoites of A. polyphaga and A. castellanii were completely inactivated in 1 min and 2 min, respectively. Furthermore, for the highly resistant cyst stage of both species, complete inactivation was achieved after 4 min of exposure to CAP. This study demonstrates that the CAP technology is highly effective against bacterial, fungal, and protozoan pathogens. The further development of this technology has enormous potential, as this approach is able to deliver the complete inactivation of ocular pathogens in minutes, in contrast to commercial multipurpose disinfecting solutions that require a minimum of 6 h. PMID:26994079

  20. In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms.

    PubMed

    Heredero-Bermejo, I; Sánchez-Nieves, J; Soliveri, J; Gómez, R; de la Mata, F J; Copa-Patiño, J L; Pérez-Serrano, J

    2016-07-25

    Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage. PMID:27173821

  1. Immunity to Acanthamoeba.

    PubMed

    Ferrante, A

    1991-01-01

    Human serum contains antibodies, mainly of the IgM and IgG isotypes, to pathogenic species of Acanthamoeba. This, as well as the capacity of these amebas to activate complement via the alternative pathway, may be a first-line defense against acanthamoeba infections in humans. Both antibody and complement appear to be important in promoting recognition of these amebas by phagocytic cells such as neutrophils. However, killing of amebas by neutrophils is dependent on lymphokine/monokine priming of the neutrophil. This priming augments the respiratory-burst activity and release of lysosomal enzymes of neutrophils in their response to the ameba. The products of the oxygen-dependent respiratory burst appear to be of prime importance in the killing of this free-living ameba. Antibodies also may prevent tissue invasion by Acanthamoeba by inhibiting its adherence, phagocytic activity, and migration and by neutralizing cytopathogenic amebic agents. Studies on experimental Acanthamoeba infections in mice showed marked species and strain specificity with regard to induction of protection with amebic antigens. Immune compromise or, alternatively, invasion at unique body sites in healthy individuals may form the basis for human infection with Acanthamoeba. PMID:2047675

  2. Mimivirus circulation among wild and domestic mammals, Amazon Region, Brazil.

    PubMed

    Dornas, Fábio P; Rodrigues, Felipe P; Boratto, Paulo V M; Silva, Lorena C F; Ferreira, Paulo C P; Bonjardim, Cláudio A; Trindade, Giliane S; Kroon, Erna G; La Scola, Bernard; Abrahão, Jônatas S

    2014-03-01

    To investigate circulation of mimiviruses in the Amazon Region of Brazil, we surveyed 513 serum samples from domestic and wild mammals. Neutralizing antibodies were detected in 15 sample pools, and mimivirus DNA was detected in 9 pools of serum from capuchin monkeys and in 16 pools of serum from cattle. PMID:24564967

  3. Ecology of Acanthamoeba.

    PubMed

    De Jonckheere, J F

    1991-01-01

    Acanthamoeba is a free-living ameba that is present in all types of environments throughout the world. The recent increase in cases of keratitis, especially in relation to an increase in the use of contact lenses, is probably due to the omnipresence of the organism as a result of the pronounced resistance of its cysts to disinfection and desiccation. The temperature of the eye is lower than that of the rest of the human body. Therefore, the presence of Acanthamoeba strains that grow at lower temperatures may also contribute to infection, thereby increasing the number of possibly infectious amebas. Recent evidence, however, indicates that perhaps only a limited number of species cause ocular disease. Delineation of the exact species of Acanthamoeba that cause keratitis is a prerequisite for the study of the ecology of the keratitis-producing amebas. PMID:2047667

  4. Amoebae as battlefields for bacteria, giant viruses, and virophages.

    PubMed

    Slimani, Meriem; Pagnier, Isabelle; Raoult, Didier; La Scola, Bernard

    2013-04-01

    When amoebae are simultaneously infected with Acanthamoeba polyphaga Mimivirus (APM) and the strictly intracellular BABL1 bacterium, the latter is always lost after serial subculturing. We showed that the virophage Sputnik 1, by reducing APM fitness, preserved BABL1 growth in acute and chronic models. This capability of a virophage to modulate the virulence of mimiviruses highlights the competition that occurs between them during natural host infection. PMID:23388714

  5. Biology and pathogenesis of Acanthamoeba

    PubMed Central

    2012-01-01

    Acanthamoeba is a free-living protist pathogen, capable of causing a blinding keratitis and fatal granulomatous encephalitis. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread and host susceptibility, and are highlighted together with potential therapeutic and preventative measures. The use of Acanthamoeba in the study of cellular differentiation mechanisms, motility and phagocytosis, bacterial pathogenesis and evolutionary processes makes it an attractive model organism. There is a significant emphasis on Acanthamoeba as a Trojan horse of other microbes including viral, bacterial, protists and yeast pathogens. PMID:22229971

  6. A Mimivirus Enzyme that Participates in Viral Entry.

    PubMed

    Klose, Thomas; Herbst, Dominik A; Zhu, Hanyu; Max, Joann P; Kenttämaa, Hilkka I; Rossmann, Michael G

    2015-06-01

    Mimivirus was initially identified as a bacterium because its dense, 125-nm-long fibers stained Gram-positively. These fibers probably play a role during the infection of some host cells. The normal hosts of Mimivirus are unknown, but in the laboratory Mimivirus is usually propagated in amoeba. The structure of R135, a major component of the fibrous outer layer of Mimivirus, has been determined to 2-Å resolution. The protein's structure is similar to that of members of the glucose-methanol-choline oxidoreductase family, which have an N-terminal FAD binding domain and a C-terminal substrate recognition domain. The closest homolog to R135 is an aryl-alcohol oxidase that participates in lignin biodegradation of plant cell walls. Thus R135 might participate in the degradation of their normal hosts, including some lignin-containing algae. PMID:25982526

  7. A Mimivirus enzyme that participates in viral entry

    PubMed Central

    Klose, Thomas; Herbst, Dominik A.; Zhu, Hanyu; Max, Joann P.; Kenttämaa, Hilkka I.; Rossmann, Michael G.

    2015-01-01

    Summary Mimivirus was initially identified as a bacterium because its dense, 125nm-long fibers stained Gram-positive. These fibers probably play a role during the infection of some host cells. The normal hosts of Mimivirus are unknown, but in the laboratory Mimivirus is usually propagated in amoeba. The structure of R135, a major component of the fibrous outer layer of Mimivirus, has been determined to 2Å resolution. The protein's structure is similar to members of the glucose-methanol-cholin oxidoreductase family, which have an N-terminal FAD binding domain and a C-terminal substrate recognition domain. The closest homologue to R135 is an aryl alcohol oxidase that participates in lignin biodegradation of plant cell walls. Thus R135 might participate in the degradation of their normal hosts, including some lignin-containing algae. PMID:25982526

  8. Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

    PubMed Central

    Hsueh, Tun-Yun

    2015-01-01

    Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)—ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce—could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce. PMID:25841006

  9. Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

    PubMed

    Heredero-Bermejo, I; Criado-Fornelio, A; Soliveri, J; Díaz-Martín, J A; Matilla-Fuentes, J; Sánchez-Arias, J A; Copa-Patiño, J L; Pérez-Serrano, J

    2015-08-01

    A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism. PMID:25956947

  10. Acanthamoeba feature a unique backpacking strategy to trap and feed on Listeria monocytogenes and other motile bacteria.

    PubMed

    Doyscher, Dominik; Fieseler, Lars; Dons, Lone; Loessner, Martin J; Schuppler, Markus

    2013-02-01

    Despite its prominent role as an intracellular human pathogen, Listeria monocytogenes normally features a saprophytic lifestyle, and shares many environmental habitats with predatory protozoa. Earlier studies claimed that Acanthamoeba may act as environmental reservoirs for L. monocytogenes, whereas others failed to confirm this hypothesis. Our findings support the latter and provide clear evidence that L. monocytogenes is unable to persist in Acanthamoeba castellanii and A. polyphaga. Instead, external Listeria cells are rapidly immobilized on the surface of Acanthamoeba trophozoites, forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulent L. monocytogenes mutants, non-pathogenic Listeria, and other motile bacteria, indicating that formation of backpacks is not specific for L. monocytogenes, and independent of bacterial pathogenicity or virulence. Hence, backpacking appears to represent a unique and highly effective strategy of Acanthamoeba to trap and feed on motile bacteria. PMID:22925311

  11. Giant DNA Virus Mimivirus Encodes Pathway for Biosynthesis of Unusual Sugar 4-Amino-4,6-dideoxy-d-glucose (Viosamine)*

    PubMed Central

    Piacente, Francesco; Marin, Margherita; Molinaro, Antonio; De Castro, Cristina; Seltzer, Virginie; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Claverie, Jean-Michel; Abergel, Chantal; Tonetti, Michela

    2012-01-01

    Mimivirus is one the largest DNA virus identified so far, infecting several Acanthamoeba species. Analysis of its genome revealed the presence of a nine-gene cluster containing genes potentially involved in glycan formation. All of these genes are co-expressed at late stages of infection, suggesting their role in the formation of the long fibers covering the viral surface. Among them, we identified the L136 gene as a pyridoxal phosphate-dependent sugar aminotransferase. This enzyme was shown to catalyze the formation of UDP-4-amino-4,6-dideoxy-d-glucose (UDP-viosamine) from UDP-4-keto-6-deoxy-d-glucose, a key compound involved also in the biosynthesis of l-rhamnose. This finding further supports the hypothesis that Mimivirus encodes a glycosylation system that is completely independent of the amoebal host. Viosamine, together with rhamnose, (N-acetyl)glucosamine, and glucose, was found as a major component of the viral glycans. Most of the sugars were associated with the fibers, confirming a capsular-like nature of the viral surface. Phylogenetic analysis clearly indicated that L136 was not a recent acquisition from bacteria through horizontal gene transfer, but it was acquired very early during evolution. Implications for the origin of the glycosylation machinery in giant DNA virus are also discussed. PMID:22157758

  12. Identification of giant Mimivirus protein functions using RNA interference

    PubMed Central

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases. PMID:25972846

  13. Photochemotherapeutic strategies against Acanthamoeba keratitis

    PubMed Central

    2012-01-01

    Here, we determined the potential of photochemotherapy, namely the application of photodynamic compounds followed by exposure to a suitable source of UV-visible radiation against corneal pathogen, Acanthamoeba. Organometallic macromolecule, tin porphyrin [Sn(IV)porphyrin] was synthesized and purity confirmed using nuclear magnetic resonance spectroscopy. The Sn(IV)porphyrin was tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype using growth and viability assays. The effects of Sn(IV)porphyrin on A. castellanii binding to and cytopathogenicity of human corneal epithelial cells in vitro were tested. The metalloporphyrin showed potent amoebistatic effects. The tin porphyrin inhibited amoebae binding to and cytopathogenicity of corneal epithelial cells. By using derivatives of photodynamic compounds [Sn(IV)porphyrin-antibody conjugates] for selective targeting of the parasite together with appropriate selection of light source will determine the potential of photochemotherapy against Acanthamoeba keratitis. PMID:22950898

  14. Acanthamoeba castellanii STAT protein.

    PubMed

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  15. Photochemotherapeutic Strategy against Acanthamoeba Infections

    PubMed Central

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Anwar, Ayaz; Shah, Muhammad Raza; Khoja, Shahrukh

    2015-01-01

    Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is an important adhesin expressed on the surface membranes of pathogenic Acanthamoeba organisms. To specifically target Acanthamoeba, the overall aim of this study was to synthesize a photosensitizing compound (porphyrin) conjugated with mannose and test its efficacy in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde and pyrrole, yielding tetraphenylporphyrin. Tetraphenylporphyrin was then converted into mono-nitrophenylporphyrin by selectively nitrating the para position of the phenyl rings, as confirmed by nuclear magnetic resonance (NMR) spectroscopy. The mono-nitrophenylporphyrin was reduced to mono-aminophenylporphyrin in the presence of tin dichloride and confirmed by a peak at m/z 629. Finally, mono-aminoporphyrin was conjugated with mannose, resulting in the formation of an imine bond. Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it absorbed light of wavelengths ranging from 425 to 475 nm. To determine the antiacanthamoebic effects of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h and washed 3 times to remove extracellular compound. Next, the amoebae were exposed to light of the appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin produced potent trophicidal effects and blocked excystation. In contrast, Acanthamoeba castellanii incubated with mannose alone and porphyrin alone did not exhibit an antiamoebic effect

  16. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage.

    PubMed

    Levasseur, Anthony; Bekliz, Meriem; Chabrière, Eric; Pontarotti, Pierre; La Scola, Bernard; Raoult, Didier

    2016-03-10

    Since their discovery, giant viruses have revealed several unique features that challenge the conventional definition of a virus, such as their large and complex genomes, their infection by virophages and their presence of transferable short element transpovirons. Here we investigate the sensitivity of mimivirus to virophage infection in a collection of 59 viral strains and demonstrate lineage specificity in the resistance of mimivirus to Zamilon, a unique virophage that can infect lineages B and C of mimivirus but not lineage A. We hypothesized that mimiviruses harbour a defence mechanism resembling the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system that is widely present in bacteria and archaea. We performed de novo sequencing of 45 new mimivirus strains and searched for sequences specific to Zamilon in a total of 60 mimivirus genomes. We found that lineage A strains are resistant to Zamilon and contain the insertion of a repeated Zamilon sequence within an operon, here named the 'mimivirus virophage resistance element' (MIMIVIRE). Further analyses of the surrounding sequences showed that this locus is reminiscent of a defence mechanism related to the CRISPR-Cas system. Silencing the repeated sequence and the MIMIVIRE genes restores mimivirus susceptibility to Zamilon. The MIMIVIRE proteins possess the typical functions (nuclease and helicase) involved in the degradation of foreign nucleic acids. The viral defence system, MIMIVIRE, represents a nucleic-acid-based immunity against virophage infection. PMID:26934229

  17. Mimivirus shows dramatic genome reduction after intraamoebal culture.

    PubMed

    Boyer, Mickaël; Azza, Saïd; Barrassi, Lina; Klose, Thomas; Campocasso, Angélique; Pagnier, Isabelle; Fournous, Ghislain; Borg, Audrey; Robert, Catherine; Zhang, Xinzheng; Desnues, Christelle; Henrissat, Bernard; Rossmann, Michael G; La Scola, Bernard; Raoult, Didier

    2011-06-21

    Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-μm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors. PMID:21646533

  18. Acanthamoeba castellanii: cellular changes induced by chlorination.

    PubMed

    Mogoa, Emerancienne; Bodet, Charles; Legube, Bernard; Héchard, Yann

    2010-09-01

    Chlorination is a well-known disinfection method, used in water treatment to inactivate various microorganisms, it induces numerous cellular changes. Even though Acanthamoebae are frequently found in water, the cellular changes induced in Acanthamoebae have not been described in the literature. Acanthamoebae are pathogenic amoebae and may provide a reservoir for pathogenic bacteria such as Legionellapneumophila; it is consequently important to understand the response of this amoeba to chlorination, and our study was indeed aimed at examining cellular changes in Acanthamoebae following chlorination. Acanthamoeba trophozoites were treated at various chlorine concentrations (1-5mg/L). A 3-log reduction in Acanthamoebae population was achieved with 5mg/L of free chlorine. Confocal microscopy and flow cytometry experiments indicated that chlorination induced cell permeabilization, size reduction and likely intracellular thiol concentration. Our data show that among the non-cultivable cells some remained impermeabilized (negative staining with propidium iodide), thereby suggesting that these cells might remained viable. A similar state is described in other microorganisms as a VBNC (viable but not cultivable) state. Electron microscopy observations illustrate drastic morphological changes: the pseudopods disappeared and subcellular components, such as mitochondrion, were pronouncedly affected. In conclusion, depending on the concentration used, chlorination leads to many cellular effects on Acanthamoeba that could well arise in cell inactivation. PMID:20034490

  19. In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba

    PubMed Central

    Legendre, Matthieu; Lartigue, Audrey; Bertaux, Lionel; Jeudy, Sandra; Bartoli, Julia; Lescot, Magali; Alempic, Jean-Marie; Ramus, Claire; Bruley, Christophe; Labadie, Karine; Shmakova, Lyubov; Rivkina, Elizaveta; Couté, Yohann; Abergel, Chantal; Claverie, Jean-Michel

    2015-01-01

    Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of “omic” approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host’s ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses’ diversity remains to be fully explored. PMID:26351664

  20. In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba.

    PubMed

    Legendre, Matthieu; Lartigue, Audrey; Bertaux, Lionel; Jeudy, Sandra; Bartoli, Julia; Lescot, Magali; Alempic, Jean-Marie; Ramus, Claire; Bruley, Christophe; Labadie, Karine; Shmakova, Lyubov; Rivkina, Elizaveta; Couté, Yohann; Abergel, Chantal; Claverie, Jean-Michel

    2015-09-22

    Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of "omic" approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host's ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses' diversity remains to be fully explored. PMID:26351664

  1. Culture Negative Confoscan Positive Acanthamoeba Keratitis

    PubMed Central

    Al Kharousi, Nadia S; Wali, Upender K

    2009-01-01

    Acanthamoeba keratitis is a protozoal infection of the eye, mainly due to the use of non-sterile solutions, like saline for disinfecting contact lenses. We report a case where delay in the diagnosis of acanthamoeba keratitis due to inadequate laboratory investigations and clinical management led to an excruciatingly painful course of the disease. The importance of non-invasive imaging techniques of confocal microscopy in the diagnosis of acanthamoeba keratitis, in the absence of positive culture reports, is highlighted in this case. PMID:21509321

  2. Unexpected postmortem diagnosis of acanthamoeba meningoencephalitis in an immunocompetent child

    PubMed Central

    Binesh, Fariba; Karimi, Mehran; Navabii, Hossein

    2011-01-01

    Meningoencephalitis caused by Acanthamoeba spp. is a rare opportunistic infection, difficult to diagnose and treat, which causes death in almost all cases. Here, the authors report a 5-year-old Iranian immunocompetent girl who died of fulminant acanthamoeba meningoencephalitis. To the authors’ knowledge, this is the first case of acanthamoeba meningoencephalitis in Iran. PMID:22679147

  3. Occurrence of Naegleria and Acanthamoeba in aquaria.

    PubMed Central

    De Jonckheere, J F

    1979-01-01

    Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi. PMID:539818

  4. Atomic force microscopy investigation of the giant mimivirus

    SciTech Connect

    Kuznetsov, Yuri G.; Xiao Chuan; Sun Siyang; Raoult, Didier; Rossmann, Michael; McPherson, Alexander

    2010-08-15

    Mimivirus was investigated by atomic force microscopy in its native state following serial degradation by lysozyme and bromelain. The 750-nm diameter virus is coated with a forest of glycosylated protein fibers of lengths about 140 nm with diameters 1.4 nm. Fibers are capped with distinctive ellipsoidal protein heads of estimated Mr = 25 kDa. The surface fibers are attached to the particle through a layer of protein covering the capsid, which is in turn composed of the major capsid protein (MCP). The latter is organized as an open network of hexagonal rings with central depressions separated by 14 nm. The virion exhibits an elaborate apparatus at a unique vertex, visible as a star shaped depression on native particles, but on defibered virions as five arms of 50 nm width and 250 nm length rising above the capsid by 20 nm. The apparatus is integrated into the capsid and not applied atop the icosahedral lattice. Prior to DNA release, the arms of the star disengage from the virion and it opens by folding back five adjacent triangular faces. A membrane sac containing the DNA emerges from the capsid in preparation for fusion with a membrane of the host cell. Also observed from disrupted virions were masses of distinctive fibers of diameter about 1 nm, and having a 7-nm periodicity. These are probably contained within the capsid along with the DNA bearing sac. The fibers were occasionally observed associated with toroidal protein clusters interpreted as processive enzymes modifying the fibers.

  5. Medical interventions for acanthamoeba keratitis

    PubMed Central

    Alkharashi, Majed; Lindsley, Kristina; Law, Hua Andrew; Sikder, Shameema

    2016-01-01

    Background Acanthamoeba are microscopic, free-living, single-celled organisms which can infect the eye and lead to Acanthamoeba keratitis (AK). AK can result in loss of vision in the infected eye or loss of eye itself; however, there are no formal guidelines or standards of care for the treatment of AK. Objectives To evaluate the relative effectiveness and safety of medical therapy for the treatment of AK. Search methods We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (2015, Issue 1), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to January 2015), EMBASE (January 1980 to January 2015), PubMed (1948 to January 2015), Latin American and Caribbean Health Sciences Literature Database (LILACS) (1982 to January 2015), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic search for trials. We last searched the electronic databases on 9 January 2015. Selection criteria We included randomized controlled trials (RCTs) of medical therapy for AK, regardless of the participants' age, sex, or etiology of disease. We included studies that compared either anti-amoeba therapy (drugs used alone or in combination with other medical therapies) with no anti-amoeba therapy or one anti-amoeba therapy with another anti-amoeba therapy. Data collection and analysis Two authors independently screened search results and full-text reports, assessed risk of bias, and abstracted data. We used standard methodological procedures as set forth by the Cochrane Collaboration. Main results We included one RCT (56 eyes of 55 participants) in this review. The study compared two types of topical biguanides for the treatment of AK

  6. Autophagy inhibitors as a potential antiamoebic treatment for Acanthamoeba keratitis.

    PubMed

    Moon, Eun-Kyung; Kim, So-Hee; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung; Kong, Hyun-Hee

    2015-07-01

    Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis. PMID:25896709

  7. Twenty Years of Acanthamoeba Diagnostics in Austria

    PubMed Central

    Walochnik, Julia; Scheikl, Ute; Haller-Schober, Eva-Maria

    2015-01-01

    Acanthamoebae are the causative agents of an often seriously progressing keratitis (AK) occurring predominantly in contact lens wearers and can cause several disseminating infections potentially resulting in granulomatous amoebic encephalitis (GAE) in the immunocompromised host. Our institution is the Austrian reference laboratory for Acanthamoeba diagnostics and the aim of this study was to give an overview of proven cases of Acanthamoeba infections in Austria during the past 20 yr. All samples of patients with suspected AK or GAE were screened for Acanthamoeba spp. by culture and/or PCR and the detected amoebae were genotyped. Altogether, 154 cases of AK and three cases of GAE were diagnosed. Age of the AK patients ranged from 8 to 82 yr (mean 37.8) and 58% of the patients were female. Approximately 89% of the AK patients were contact lens wearers, almost all cases were unilateral and 19% of the patients required a keratoplasty. Age of the GAE patients ranged from 2 to 25 yr (mean 14.7), all were HIV-negative, but two were severely immunosuppressed at the time of diagnosis. The predominant genotype in the AK cases was T4, other genotypes found were T3, T5, T6, T10 and T11. The three GAE cases involved genotypes T2, T4 and T5. PMID:25047131

  8. The Value of Cytology Smears for Acanthamoeba Keratitis

    PubMed Central

    Schaefer, Jamie L.; Paterson, Joyce; Liu, Weiguo; Gonzalez-Fernandez, Federico

    2016-01-01

    Purpose. Acanthamoeba keratitis remains a difficult diagnosis despite advances in genetic and imaging technologies. The purpose of this paper is to highlight the utility of cytology smears for diagnosis of Acanthamoeba keratitis. Methods. This is a case study of the diagnostic course for a patient with suspected Acanthamoeba keratitis. Results. A 40-year-old male with poor contact lens hygiene presented with severe left eye pain. Slit lamp examination showed two peripheral ring infiltrates without an epithelial defect. The epithelium over both infiltrates was removed with a Kimura spatula. Half of the sample was smeared on a dry microscope slide and the other half was submitted for Acanthamoeba culture and PCR. Both culture and PCR were negative for Acanthamoeba, but hematoxylin and eosin stain of the smear revealed double-walled cysts. Conclusion. H&E staining of corneal cytology specimens is an efficient and readily available test for diagnosis of Acanthamoeba keratitis. PMID:27403362

  9. Single mimivirus particles intercepted and imaged with an X-ray laser (CXIDB ID 2)

    DOE Data Explorer

    Seibert, M. Marvin; Ekeberg, Tomas

    2011-02-02

    These are the files used to reconstruct the images in the paper "Single Mimivirus particles intercepted and imaged with an X-ray laser". Besides the diffracted intensities, the Hawk configuration files used for the reconstructions are also provided. The files from CXIDB ID 2 are the pattern and configuration files for the pattern showed in Figure 2b in the paper.

  10. Single mimivirus particles intercepted and imaged with an X-ray laser (CXIDB ID 1)

    DOE Data Explorer

    Seibert, M. Marvin; Ekeberg, Tomas; Maia, Filipe R.N.C.

    2011-02-02

    These are the files used to reconstruct the images in the paper "Single Mimivirus particles intercepted and imaged with an X-ray laser". Besides the diffracted intensities, the Hawk configuration files used for the reconstructions are also provided. The files from CXIDB ID 1 are the pattern and configuration files for the pattern showed in Figure 2a in the paper.

  11. Is there evidence of sexual reproduction (meiosis) in Acanthamoeba?

    PubMed Central

    Khan, Naveed A.; Siddiqui, Ruqaiyyah

    2015-01-01

    Evolution of independently breeding species into males and females (gametes) has remained a puzzle. Given the significant advantages of sexual reproduction over asexual reproduction as a long-term species survival strategy; here, we pose the question whether there is some form of meiosis in Acanthamoeba species, which represents our ancient lineage. The recently available Acanthamoeba genome revealed several genes implicated in meiosis in sexual eukaryotes such as Spo11, Mre11, Rad50, Rad51, Rad52, Mnd1, Dmc1, Msh, and Mlh, suggesting that Acanthamoeba is capable of some form of meiosis, inferring the presence of sexual reproduction in Acanthamoeba, and that meiosis evolved early in eukaryotic evolution. PMID:25800982

  12. Current Status of Acanthamoeba in Iran: A Narrative Review Article

    PubMed Central

    NIYYATI, Maryam; REZAEIAN, Mostafa

    2015-01-01

    Background: Free-living amoebae belonging to the genus Acanthamoeba have an environmental distribution. Amoebic keratitis due to these protozoan parasites continue to rise in Iran and worldwide. In Iran, there are various researches regarding both morphological and molecular identification of Acanthamoeba spp. in environmental and clinical samples. However, there is no thorough review about Acanthamoeba genotypes and their distribution in environmental sources such as water, dust and biofilm in Iran. Besides, according to increasing cases of Amoebic keratitis in the region awareness regarding the pathogenic potential of these sight-threatening amoebae is of utmost importance. Methods: We conducted a thorough review based on the database sources such as MEDLINE, PubMed and Google scholar. No restrictions were placed on study date, study design or language of publication. We searched all valuable and relevant information considering the occurrence of the Acanthamoeba in both environmental and clinical samples. Results: According to our thorough review Acanthamoeba belonging to T4 genotype is the most prevalent type strain in environmental and clinical samples in several regions in Iran and worldwide, however, there are reports regarding Acanthamoeba belonging to other genotypes such as T2, T3, T5, T6 and T11 and the mentioned point could leads us to more researches with the goal of presenting the real genotype dominance of Acanthamoeba and related disease in the country. Conclusion: Overall, the present review will focus on present status of genotypes of Acanthamoeba in Iran during recent years. PMID:26246812

  13. Cytotoxic effect of acriflavine against clinical isolates of Acanthamoeba spp.

    PubMed

    Polat, Zubeyda Akin; Karakus, Gulderen

    2013-02-01

    Acanthamoeba keratitis (AK) is a potentially devastating and sight-threatening infection of the cornea caused by the ubiquitous free-living amoebae, Acanthamoeba species. Its eradication is difficult because the amoebas encyst, making it highly resistant to anti-amoebic drugs. Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. The aim of our study was to evaluate the time-dependent cytotoxicities of ACF against Acanthamoeba spp. Trophozoites and cysts of three different strains (strain PAT06 Acanthamoeba castellanii, strain 2HH Acanthamoeba hatchetti, and strain 11DS A. hatchetti) of Acanthamoeba spp. were tested. All strains had been isolated from patients suffering from a severe AK. The effects of the ACF with the concentrations ranging from 15 to 500 mg mL(-1) on the cytotoxicity of Acanthamoeba strains were examined. ACF showed a time- and dose-dependent amebicidal action on the trophozoites and cysts. Pat06 (A. castellanii) was the most resistant, while strain 11DS (A. hatchetti) was the most sensitive. As a result, ACF could be concluded as a new agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect. PMID:23052789

  14. Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

    PubMed Central

    Choi, Seon Hee; Cho, Min Kyoung; Ahn, Soon Cheol; Lee, Ji Eun; Lee, Jong Soo; Kim, Dong-Hee; Xuan, Ying-Hua; Hong, Yeon Chul; Kong, Hyun Hee; Chung, Dong Il

    2009-01-01

    In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the α-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba. PMID:19967080

  15. Occurrence of Potentially Pathogenic Bacterial-Endosymbionts in Acanthamoeba Spp.

    PubMed Central

    NIYYATI, Maryam; MAFI, Mahyar; HAGHIGHI, Ali; HAKEMI VALA, Mojdeh

    2015-01-01

    Background: Acanthamoeba- bacteria interactions enable pathogenic bacteria to tolerate harsh conditions and lead to transmission to the susceptible host. The present study was aimed to address the presence of bacterial endosymbionts of Acanthamoeba isolated from recreational water sources of Tehran, Iran. To the best of our knowledge this is the first study regarding occurrence of bacteria in environmental Acanthamoeba spp. in Iran. Methods: A total of 75 samples of recreational water sources were collected. Samples were cultured on non- nutrient agar 1.5% plates. Positive Acanthamoeba spp. were axenically grown. DNA extraction and PCR reaction was performed using JDP1-2 primers. All positive samples of Acanthamoeba were examined for the presence of endosymbionts using staining and molecular methods. The PCR products were then sequenced in order to determine the genotypes of Acanthamoeba and bacteria genera. Results: Out of 75 samples, 16 (21.3%) plates were positive for Acanthamoeba according to the morphological criteria. Molecular analysis revealed that Acanthamoeba belonged to T4 and T5 genotypes. Five isolates (35.7%) were positive for bacterial endosymbionts using staining method and PCR test. Sequencing of PCR products confirmed the presence of Pseudomonas aeruginosa and Agrobacterium tumefasiens. Conclusion: The presence of Acanthamoeba bearing pathogenic endosymbionts in water sources leads us to public health issues including improved sanitation and decontamination measures in recreational water sources in order to prevent amoebae-related infection. To the best of our knowledge this is the first report regarding the isolation of A. tumefasiens from Acanthamoeba in Iran and worldwide. PMID:26246815

  16. Detection Of Bacterial Endosymbionts In Clinical Acanthamoeba Isolates

    PubMed Central

    Iovieno, Alfonso; Ledee, Dolena R.; Miller, Darlene; Alfonso, Eduardo C.

    2009-01-01

    Purpose To determine the presence of four clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK. Design Experimental study Participants Acanthamoeba isolates (N=37) recovered from cornea and contact lens paraphernalia of 23 patients with culture proven AK and 1 environmental isolate. Methods Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacteria and Chlamydia using molecular techniques (Polymerase chain reaction and sequence analysis, fluorescent in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay of human corneal epithelial cells in vitro. Initial visual acuity (VA), location and characteristics of the infiltrate, time to detection of the infection and symptoms duration at presentation were evaluated in all patients. Main Outcome Measures Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK. Results Twenty-two of the 38 (59.4%) cultures examined contained at least one bacterial endosymbiont. One isolate contained two endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoebae hosting endosymbionts compared to isolates without endosymbionts (p<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (p<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial VA (p>0.05), central location (p<0.05), absence of radial perineuritis (p<0.05), delayed time to detection (p>0.05) and longer symptoms duration at

  17. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    PubMed Central

    Thai, Vu; Renesto, Patricia; Fowler, C. Andrew; Brown, Darin J.; Davis, Tara; Gu, Wanjun; Pollock, David D.; Kern, Dorothee; Raoult, Didier; Eisenmesser, Elan Z.

    2010-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses. PMID:18342330

  18. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    SciTech Connect

    Thai,V.; Renesto, P.; Fowler, C.; Brown, D.; Davis, T.; Gu, W.; Pollock, D.; Kern, D.; Raoult, D.; Eisenmesser, E.

    2008-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (ie HIV-1 and SARS) or virally encoded (ie Mimivirus), are localized on viral surfaces for at least a subset of viruses.

  19. Identification of an l-Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses▿

    PubMed Central

    Parakkottil Chothi, Madhu; Duncan, Garry A.; Armirotti, Andrea; Abergel, Chantal; Gurnon, James R.; Van Etten, James L.; Bernardi, Cinzia; Damonte, Gianluca; Tonetti, Michela

    2010-01-01

    Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar l-rhamnose: two UDP-d-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-d-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection. PMID:20538863

  20. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

    PubMed Central

    Lorenzo-Morales, Jacob; Khan, Naveed A.; Walochnik, Julia

    2015-01-01

    Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections. PMID:25687209

  1. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment.

    PubMed

    Lorenzo-Morales, Jacob; Khan, Naveed A; Walochnik, Julia

    2015-01-01

    Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections. PMID:25687209

  2. Diversity and Seasonal Impact of Acanthamoeba Species in a Subtropical Rivershed

    PubMed Central

    Kao, Po-Min; Chou, Ming-Yuan; Tao, Chi-Wei; Huang, Wen-Chien; Hsu, Bing-Mu; Shen, Shu-Min; Fan, Cheng-Wei; Chiu, Yi-Chou

    2013-01-01

    This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7%) of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4%) were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2), Acanthamoeba sp. IS2/T4 (T4), Acanthamoeba lenticulata (T5), Acanthamoeba hatchetti (T11), Acanthamoeba healyi (T12), and Acanthamoeba jacobsi (T15). The most frequently identified Acanthamoeba genotype was T4 (68.7%). Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed. PMID:24490160

  3. Gene discovery in the Acanthamoeba castellanii genome

    SciTech Connect

    Anderson, Iain J.; Watkins, Russell F.; Samuelson, John; Spencer,David F.; Majoros, William H.; Gray, Michael W.; Loftus, Brendan J.

    2005-08-01

    Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit adversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptorserine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.

  4. Advances in the Diagnosis and Treatment of Acanthamoeba Keratitis

    PubMed Central

    Clarke, Benjamin; Sinha, Arti; Parmar, Dipak N.

    2012-01-01

    This paper aims to review the recent literature describing Acanthamoeba keratitis and outline current thoughts on pathogenesis, diagnosis, and treatment as well as currently emerging diagnostic and treatment modalities. PMID:23304449

  5. Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis

    PubMed Central

    Rusciano, Giulia; Capriglione, Paola; Pesce, Giuseppe; Del Prete, Salvatore; Cennamo, Gilda; Di Cave, David; Cerulli, Luciano; Sasso, Antonio

    2013-01-01

    Acanthamoeba keratitis is a rare but serious corneal disease, often observed in contact lens wearers. Clinical treatment of infected patients frequently involves the use of polyhexamethylene biguanide (PHMB), a polymer used as a disinfectant and antiseptic, which is toxic also for the epithelial cells of the cornea. Prompt and effective diagnostic tools are hence highly desiderable for both starting early therapy and timely suspension of the treatment. In this work we use Raman microspectroscopy to analyse in vitro a single Acanthamoeba cell in cystic phase. In particular, we investigate the effect of PHMB at the single-cell level, providing useful information on both the underlying biochemical mechanism and the time frame for Acanthamoeba eradication in ocular infections. Furthermore, we demonstrate that Raman spectroscopy, in conjunction with standard multivariate analysis methods, allows discriminating between live and dead Acanthamoebas, which is fundamental to optimizing patients’ treatment. PMID:23977228

  6. Several staining techniques to enhance the visibility of Acanthamoeba cysts.

    PubMed

    El-Sayed, Nagwa Mostafa; Hikal, Wafaa Mohamed

    2015-03-01

    Acanthamoeba is one of the most common free-living amoebae. It is widespread in the environment and can infect humans causing keratitis. Delayed diagnosis or misdiagnosis leads to extensive corneal inflammation and profound visual loss. Therefore, accurate and rapid diagnosis of Acanthamoeba keratitis is essential for successful treatment and good prognosis. This study was designed to use different staining techniques to facilitate the identification of Acanthamoeba cysts. Acanthamoeba cysts were isolated by cultivation of either corneal scraping specimens or tap water samples onto non-nutrient agar plates seeded with Escherichia coli. Subcultures were done from positive cultures until unique cysts were isolated. Acanthamoeba cysts were stained temporarily using iodine, eosin, methylene blue, and calcofluor white (CFW) stains and as permanent slides after processing for mounting using modified trichrome, Gimenez and Giemsa staining. These stains were compared on the basis of staining quality including clarity of morphological details, differentiation between cytoplasm and nuclei, color and contrast, and also other characteristics of the staining techniques, including ease of handling, time taken for the procedure, and cost effectiveness. The cysts of Acanthamoeba were recognized in the form of double-walled cysts: the outer wall (ectocyst) that was being differentiated from the variably stained surrounding background and the inner wall (endocyst) that was sometimes stellated, polygonal, round, or oval and visualized as separate from the spherical, sometimes irregular, outline of the ectocyst. Regarding the temporary stains, it was found that they were efficient for visualizing the morphological details of Acanthamoeba cysts. In CFW staining, Acanthamoeba cysts appeared as bluish-white or turquoise oval halos although the internal detail was not evident. On the other hand, the results of permanent-stained slides showed the most consistent stain for identification of

  7. Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of Acanthamoeba

    PubMed Central

    Khan, Naveed A.; Jarroll, Edward L.; Panjwani, Noorjahan; Cao, Zhiyi; Paget, Timothy A.

    2000-01-01

    Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5. PMID:10921939

  8. Statins and Voriconazole Induce Programmed Cell Death in Acanthamoeba castellanii

    PubMed Central

    López-Arencibia, Atteneri; Sifaoui, Ines; Reyes-Batlle, María; Valladares, Basilio; Martínez-Carretero, Enrique; Piñero, José E.; Maciver, Sutherland K.; Lorenzo-Morales, Jacob

    2015-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC50s) and IC90s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba. PMID:25733513

  9. Encystment in Acanthamoeba castellanii: a review.

    PubMed

    Lloyd, David

    2014-11-01

    Differentiation of Acanthamoeba castellanii trophozoites involves massive turnover of cellular components and remodelling of organelle structure and function so as to produce a cryptobiotic cell, resistant to desiccation, heat, freezing, and chemical treatments. This review presents a summary of a decade of research on the most studied aspects of the biochemistry of this process, with emphasis on problems of biocide and drug resistances, putative new targets, molecular and cell biology of the process of encystment, and the characteristics of the encysted state. As well as the intrinsic pathogenicity of the organism towards the cornea, and the ability of related species to invade the human brain, its propensity for harbouring and transmitting pathogenic bacteria and viruses is considerable and leads to increasing concerns. The long-term survival and resistance of cysts to drugs and biocides adds another layer of complexity to the problem of their elimination. PMID:24726698

  10. Fatal Acanthamoeba Encephalitis in a Patient With a Total Artificial Heart (Syncardia) Device

    PubMed Central

    Tan, Susanna K.; Gajurel, Kiran; Tung, Christie; Albers, Gregory; Deresinski, Stan; Montoya, Jose G.; Sheikh, Ahmad Y.; Banerjee, Dipanjan; Ha, Richard

    2014-01-01

    Acanthamoeba encephalitis is an uncommon but often fatal infection complication. Here we report the first case of Acanthamoeba encephalitis in a patient with a Total Artificial Heart device. PMID:25734127

  11. Fatal acanthamoeba encephalitis in a patient with a total artificial heart (syncardia) device.

    PubMed

    Tan, Susanna K; Gajurel, Kiran; Tung, Christie; Albers, Gregory; Deresinski, Stan; Montoya, Jose G; Sheikh, Ahmad Y; Banerjee, Dipanjan; Ha, Richard

    2014-09-01

    Acanthamoeba encephalitis is an uncommon but often fatal infection complication. Here we report the first case of Acanthamoeba encephalitis in a patient with a Total Artificial Heart device. PMID:25734127

  12. IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS IN ISOLATES OF Acanthamoeba

    PubMed Central

    Maschio, Vinicius José; Corção, Gertrudes; Rott, Marilise Brittes

    2015-01-01

    Acanthamoeba is a “Trojan horse” of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas. PMID:25651331

  13. Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser

    SciTech Connect

    Ekeberg, Tomas

    2015-05-26

    This dataset contains the diffraction patterns that were used for the first three-dimensional reconstruction of a virus using FEL data. The sample was the giant mimivirus particle, which is one of the largest known viruses with a diameter of 450 nm. The dataset consists of the 198 diffraction patterns that were used in the analysis.

  14. Characterization of a Trifunctional Mimivirus mRNA Capping Enzyme and Crystal Structure of the RNA Triphosphatase Domain

    SciTech Connect

    Benarroch,D.; Smith, P.; Shuman, S.

    2008-01-01

    The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.

  15. Acanthamoebae bind to glycolipids of rabbit corneal epithelium.

    PubMed Central

    Panjwani, N; Zhao, Z; Baum, J; Pereira, M; Zaidi, T

    1992-01-01

    By use of a thin-layer chromatogram (TLC) overlay procedure, 35S-labeled acanthamoebae were shown to bind to seven glycolipids of rabbit corneal epithelium. Corneal epithelial cells were grown in culture and were subjected to Folch extraction to isolate a chloroform-rich lower phase containing neutral glycosphingolipids (NGSL) and an aqueous upper phase containing gangliosides, i.e., sialic acid-containing glycolipids. Thin-layer chromatography of the upper phase revealed the presence of 10 ganglioside components. Acanthamoebae were shown to bind to four of these components, referred to as 2, 3, 6, and 7. On TLC plates, ganglioside components 2 and 3 migrated slightly ahead of the glycolipid standard GD1a, component 7 comigrated with standard GM3, and component 6 migrated a little more slowly than GM3. Likewise, of the 10 NGSL known to be present in the lower phase, acanthamoebae bound to components 1, 5, and 6. NGSL components 1, 5, and 6 migrated on TLC plates with relative mobilities similar to those of standards asialo GM1, asialo GM2, and ceramidetrihexoside, respectively. We propose that one or more of the Acanthamoeba-reactive glycolipids of corneal epithelium identified in this study may play a role in the pathogenesis of Acanthamoeba keratitis by mediating the adherence of the parasites to the cornea. Images PMID:1639517

  16. Mimivirus inaugurated in the 21st century the beginning of a reclassification of viruses.

    PubMed

    Sharma, Vikas; Colson, Philippe; Pontarotti, Pierre; Raoult, Didier

    2016-06-01

    Mimivirus and other giant viruses are visible by light microscopy and bona fide microbes that differ from other viruses and from cells that have a ribosome. They can be defined by: giant virion and genome sizes; their complexity, with the presence of DNA and mRNAs and dozens or hundreds of proteins in virions; the presence of translation-associated components; a mobilome including (pro)virophages (and a defence mechanism, named MIMIVIRE, against them) and transpovirons; their monophyly; the presence of the most archaic protein motifs they share with cellular organisms but not other viruses; a broader host range than other viruses. These features show that giant viruses are specific, autonomous, biological entities that warrant the creation of a new branch of microbes. PMID:26829206

  17. Colonization of broilers by Campylobacter jejuni internalized within Acanthamoeba castellanii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present the first report that Campylobacter jejuni, internalized within Acanthamoeba castellanii, colonized broilers. After 1, 3, 7 and 14 days post challenge none of the broilers challenged with negative controls were colonized, but were with internalized C. jejuni. The biology of protozoa-Cam...

  18. Updating strategies for isolating and discovering giant viruses.

    PubMed

    Khalil, Jacques Yaacoub Bou; Andreani, Julien; La Scola, Bernard

    2016-06-01

    Almost fifteen years ago, the discovery of Acanthamoeba polyphaga mimivirus, the first giant virus, changed how we define a virus. It was discovered incidentally in a process of isolating Legionella sp. from environmental samples in the context of pneumonia epidemics using a co-culture system with Acanthamoeba. Since then, much effort and improvement has been put into the original technique. In addition to the known families of Mimiviridae and Marseilleviridae, four new proposed families of giant viruses have been isolated: Pandoravirus, Pithovirus, Faustovirus and Mollivirus. Major improvements were based on enrichment systems, targeted use of antibiotics and high-throughput methods. The most recent development, using flow cytometry for isolation and presumptive identification systems, opens a path to large environmental surveys that may discover new giant virus families in new protozoa supports used for culture support. PMID:27039269

  19. Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism

    PubMed Central

    Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

    2005-01-01

    Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase. PMID:15845472

  20. Niemeyer Virus: A New Mimivirus Group A Isolate Harboring a Set of Duplicated Aminoacyl-tRNA Synthetase Genes

    PubMed Central

    Boratto, Paulo V. M.; Arantes, Thalita S.; Silva, Lorena C. F.; Assis, Felipe L.; Kroon, Erna G.; La Scola, Bernard; Abrahão, Jônatas S.

    2015-01-01

    It is well recognized that gene duplication/acquisition is a key factor for molecular evolution, being directly related to the emergence of new genetic variants. The importance of such phenomena can also be expanded to the viral world, with impacts on viral fitness and environmental adaptations. In this work we describe the isolation and characterization of Niemeyer virus, a new mimivirus isolate obtained from water samples of an urban lake in Brazil. Genomic data showed that Niemeyer harbors duplicated copies of three of its four aminoacyl-tRNA synthetase genes (cysteinyl, methionyl, and tyrosyl RS). Gene expression analysis showed that such duplications allowed significantly increased expression of methionyl and tyrosyl aaRS mRNA by Niemeyer in comparison to APMV. Remarkably, phylogenetic data revealed that Niemeyer duplicated gene pairs are different, each one clustering with a different group of mimivirus strains. Taken together, our results raise new questions about the origins and selective pressures involving events of aaRS gain and loss among mimiviruses. PMID:26635738

  1. Isolation of Acanthamoeba-Specific Antibodies from a Bacteriophage Display Library

    PubMed Central

    Khan, Naveed A.; Greenman, John; Topping, Katherine P.; Hough, Victoria C.; Temple, Graham S.; Paget, Timothy A.

    2000-01-01

    Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness. Current methods for identifying this organism rely on culture and microscopy. In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba. A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba. Individual clones were studied by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Four antibody clones that specifically bind to Acanthamoeba spp. were identified. PMID:10835006

  2. Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan.

    PubMed

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Liu, Jorn-Hon; Chang, Hsiang-Yu; Ji, Wen-Tsai; Tzeng, Kai-Jiun; Huang, Shih-Wei; Huang, Yu-Li

    2015-03-01

    In order to detect the presence/absence of Acanthamoeba along with geographical variations, water quality variations and seasonal change of Acanthamoeba in Taiwan was investigated by 18S ribosomal RNA (rRNA) gene TaqMan quantitative real-time PCR. Samples were collected quarterly at 19 drinking water reservoir sites from November 2012 to August 2013. Acanthamoeba was detected in 39.5 % (30/76) of the water sample, and the detection rate was 63.2 % (12/19) from samples collected in autumn. The average concentration of Acanthamoeba was 3.59 × 10(4) copies/L. For geographic distribution, the detection rate for Acanthamoeba at the northern region was higher than the central and southern regions in all seasons. Results of Spearman rank test revealed that heterotrophic plate count (HPC) had a negative correlation (R = -0.502), while dissolved oxygen (DO) had a positive correlation (R = 0.463) in summer. Significant differences were found only between the presence/absence of Acanthamoeba and HPC in summer (Mann-Whitney U test, P < 0.05). T2 and T4 genotypes of Acanthamoeba were identified, and T4 was the most commonly identified Acanthamoeba genotypes. The presence of Acanthamoeba in reservoirs presented a potential public health threat and should be further examined. PMID:25263419

  3. A Checklist of Iranian Cockroaches (Blattodea) with Description of Polyphaga sp as a New Species in Iran

    PubMed Central

    Hashemi-Aghdam, Saedeh Sadat; Oshaghi, Mohammad Ali

    2015-01-01

    Background: Cockroaches are of vital importance medically and hygienically. They are able to contaminate foods and act as vectors of pathogenic agents such as bacteria, protozoa, and parasites to human environment either mechanically or through their digestive system. Cockroaches belong to the phylum Arthropoda, class Insecta, and order Blattodea or Blattaria. To date, over 4,500 cockroach species have been reported from different parts of the world. We overviewed the documents involved Iranian cockroaches to up-to-date checklist of cockroach species distributed in various provinces of Iran. Methods: An extensive literature review was performed in 2013 on Iranian handbooks, reports and published data available since 1986 to obtain a comprehensive list of Iranian cockroaches. Furthermore, in an entomological survey in Tehran, cockroach specimens were collected and identified based on morphological and the DNA sequences of the mitochondrial cytochrome oxidase subunit II (COII) gene (mt-DNA COII) characteristics. Results: Morphological characterization revealed presence of an un-described species very similar to Polyphaga aegyptiaca, P. indica and somehow to Pycnoscelus surinamensis, however, supplementary molecular analysis revealed the species was associated with Polyphaga of Corydiidae (Polyphagidae). With regards to the report of the un-described species, the cockroach fauna of Iran includes three families, 14 genera, and 26 species. Conclusion: Some species has not been collected or reported recently and also many geographical regions of the country have not been studied yet, hence a systematic research is required to reveal the real cockroach list of the country. Geographical distributions, nomination changes, and synonyms of cockroach species are presented. PMID:26623428

  4. Gene repertoire of amoeba-associated giant viruses.

    PubMed

    Colson, Philippe; Raoult, Didier

    2010-01-01

    Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. PMID:20551685

  5. Detection of glycoproteins in the Acanthamoeba plasma membrane

    SciTech Connect

    Paatero, G.I.L. ); Gahmberg, C.G. )

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  6. Anti-Acanthamoeba activity of contact lens solutions

    PubMed Central

    Niszl, I.; Markus, M.

    1998-01-01

    AIMS—This study was undertaken to investigate the effects of contact lens disinfecting solutions on strains of Acanthamoeba from the United Kingdom and southern Africa and to compare the results with those of other researchers. No information was previously available for southern African isolates.
METHODS—11 contact lens solutions were tested on cysts of 10 strains of Acanthamoeba.
RESULTS—Not all solutions used in the study were effective, with some for hard and gas permeable contact lenses being more satisfactory than those for soft contact lenses. The most effective of the gas permeable and hard contact lens solutions tested was Transoak (0.01% (wt/vol) benzalkonium chloride), which killed cysts of all strains within 4 hours of exposure. Oxysept 1 (31 mg hydrogen peroxide/ml) was the best soft contact lens solution tested. It eliminated cysts of certain strains within 4 hours, whereas cysts of other strains were only inactivated within either 8 or 72 hours.
CONCLUSIONS—Manufacturers should be aware of the killing time for Acanthamoeba by contact lens solutions and should provide appropriate guidelines for the use thereof. The killing time for cysts of the African and UK isolates studied is, in general, similar. Therefore, it must in the present state of knowledge be assumed that usage guidelines suggested in the UK are also appropriate for travellers to South Africa and for local residents in South Africa.

 Keywords: contact lenses; Acanthamoeba; keratitis PMID:9893594

  7. Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba.

    PubMed

    Dupuy, Mathieu; Mazoua, Stéphane; Berne, Florence; Bodet, Charles; Garrec, Nathalie; Herbelin, Pascaline; Ménard-Szczebara, Florence; Oberti, Sandrine; Rodier, Marie-Hélène; Soreau, Sylvie; Wallet, France; Héchard, Yann

    2011-01-01

    Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chlorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems. PMID:21093012

  8. Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser

    NASA Astrophysics Data System (ADS)

    Ekeberg, Tomas; Svenda, Martin; Abergel, Chantal; Maia, Filipe R. N. C.; Seltzer, Virginie; Claverie, Jean-Michel; Hantke, Max; Jönsson, Olof; Nettelblad, Carl; van der Schot, Gijs; Liang, Mengning; DePonte, Daniel P.; Barty, Anton; Seibert, M. Marvin; Iwan, Bianca; Andersson, Inger; Loh, N. Duane; Martin, Andrew V.; Chapman, Henry; Bostedt, Christoph; Bozek, John D.; Ferguson, Ken R.; Krzywinski, Jacek; Epp, Sascha W.; Rolles, Daniel; Rudenko, Artem; Hartmann, Robert; Kimmel, Nils; Hajdu, Janos

    2015-03-01

    We present a proof-of-concept three-dimensional reconstruction of the giant mimivirus particle from experimentally measured diffraction patterns from an x-ray free-electron laser. Three-dimensional imaging requires the assembly of many two-dimensional patterns into an internally consistent Fourier volume. Since each particle is randomly oriented when exposed to the x-ray pulse, relative orientations have to be retrieved from the diffraction data alone. We achieve this with a modified version of the expand, maximize and compress algorithm and validate our result using new methods.

  9. Identifying differentially expressed genes in trophozoites and cysts of Acanthamoeba T4 genotype: Implications for developing new treatments for Acanthamoeba keratitis.

    PubMed

    Abedkhojasteh, Hoda; Niyyati, Maryam; Rezaei, Sasan; Mohebali, Mehdi; Farnia, Shohreh; Kazemi-Rad, Elham; Roozafzoon, Reza; Sianati, Hamed; Rezaeian, Mostafa; Heidari, Mansour

    2015-02-01

    Acanthamoeba T4 genotype is the most prevalent genotype associated with amoebic keratitis. Acanthamoeba keratitis therapy is difficult due to transformation of trophozoite to cyst stage, which hinders the treatment of the disease. Although encystation assists the organism to survive against the chemotherapeutic compounds, the precise mechanism of encystation remains poorly understood. The purpose of this work was to identify differentially expressed genes in Acanthamoeba T4 genotype which might be useful for understanding of the encystment process and may thus help develop more efficient treatment. The mRNA profile of trophozoite and cyst of Acanthamoeba T4 genotype isolated from a soft contact lens wearer were analyzed using a cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. Subsequently, a real time reverse transcriptase-PCR was performed to validate the cDNA-AFLP results. Three genes, heat shock protein70 (hsp70), actin-I and elongation factor-1alpha (EF-1α) were differentially expressed during Acanthamoeba differentiation. An in silico result predicted that transformation of trophozoite to cyst could be mediated through their cooperation with the protein partners interaction. Taken together, our experimental and bioinformatics findings suggested potential functions of hsp70, EF-1α and actin-I in differentiation of Acanthamoeba T4 genotype which may be useful in the design of an efficient therapeutic strategy in AK. PMID:25543551

  10. Susceptibility of Acanthamoeba castellanii to contact lens disinfecting solutions.

    PubMed Central

    Zanetti, S; Fiori, P L; Pinna, A; Usai, S; Carta, F; Fadda, G

    1995-01-01

    A corneal isolate of Acanthamoeba castellanii was exposed to commercial contact lens disinfecting solutions containing hydrogen peroxide, benzalkonium chloride, polyaminopropyl biguanide, polyquaternium 1, and chlorhexidine-thimerosal. The minimum trophozoite amebicidal concentration and exposure times required to kill trophozoites and cysts were determined. Solutions containing hydrogen peroxide or chlorhexidine-thimerosal were active against both trophozoites and cysts. The benzalkonium chloride-based solution was effective only against trophozoites. Solutions containing polyaminopropyl biguanide or polyquaternium 1 were completely ineffective. The need for adequate exposure times must be stressed. PMID:7492111

  11. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway

    PubMed Central

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei

    2015-01-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections. PMID:26014935

  12. Acanthamoeba spp. in Contact Lenses from Healthy Individuals from Madrid, Spain

    PubMed Central

    Gomes, Thiago dos Santos; Magnet, Angela; Izquierdo, Fernando; Vaccaro, Lucianna; Redondo, Fernando; Bueno, Sara; Sánchez, Maria Luisa; Angulo, Santiago; Fenoy, Soledad; Hurtado, Carolina; del Aguila, Carmen

    2016-01-01

    Purpose Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus. Methods One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba. Results Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of “not cleaning the CL case” presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples. Conclusions The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis. PMID

  13. Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa

    PubMed Central

    Dornas, Fábio P.; Khalil, Jacques Y. B.; Pagnier, Isabelle; Raoult, Didier; Abrahão, Jônatas; La Scola, Bernard

    2015-01-01

    The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation. PMID:26500630

  14. Pathogenic and nonpathogenic Acanthamoeba spp. in thermally polluted discharges and surface waters

    SciTech Connect

    de Jonckheere, J.F.

    1981-02-01

    During spring and autumn, the total number of amoebae and the number of acanthamoeba species able to grow at 37 degrees C were determined in six thermally polluted factory discharges and the surrounding surface waters. The isolated Acanthamoeba strains were studied for growth in axenic medium, cytopathic effect in Vito cell cultures, and virulence in mice. Although more amoebae were isolated in autumn, the number of Acanthamoeba species was lower than in spring, when the percent of pathogenic strains among the isolates was highest. Higher concentrations of amoebae were found in warm discharges, and more virulent strains occurred in thermal discharges than in surface waters.

  15. Acanthamoeba and Fusarium interactions: A possible problem in keratitis.

    PubMed

    Nunes, Thais Esther Teixeira; Brazil, Nathalya Tesch; Fuentefria, Alexandre Meneghello; Rott, Marilise Brittes

    2016-05-01

    The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoebae and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of three Acanthamoeba castellanii strains with Fusarium conidia and the possible implications on keratitis. F. conidia were internalized by A. castellanii strains and were able to germinate inside the amoebae. The co-culture with the live amoebae, as well as the amoebal culture supernatant and lysate, increased the fungal growth significantly. Moreover, live F. solani and its culture supernatant enhanced the survival of amoebae, but in a different way in each amoebal strain. The encystment of the A. castellanii strain re-isolated from rat lung was increased by the fungus. These results show that A. castellanii and F. solani interaction may have an important influence on survival of both, and specially indicate a possible effect on virulence characteristics of these microorganisms. These data suggest that the A. castellanii-F. solani interaction may cause severe impacts on keratitis. PMID:26851515

  16. Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts.

    PubMed

    Rivière, Delphine; Szczebara, Florence Ménard; Berjeaud, Jean-Marc; Frère, Jacques; Héchard, Yann

    2006-01-01

    Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms. PMID:15923051

  17. Acanthamoeba adherence to contact lenses, removal by rinsing procedures, and survival to some ophthalmic products.

    PubMed

    Cancrini, G; Iori, A; Mancino, R

    1998-09-01

    Unworn soft and rigid gas-permeable contact lenses were inoculated with an Acanthamoeba keratitis strain to study the protozoon's ability to adhere. Furthermore, the efficacy of the rinsing in saline on acanthamoeba removal was evaluated, as well as the amebicidal activity of five commercial cleaning/disinfecting products: hydrogen peroxide, chlorhexidine, polyaminopropyl biguanide-poloxamine, thimerosal-polyquaternium, and thimerosal-chlorhexidine. Microscopic count of cells showed that Acanthamoeba trophozoites and cysts adhered to all types of contact lenses. A significantly greater adherence of trophozoites than cysts was recorded. The rinsing in saline using a flow-method was significantly more effective than the immersion-method, particularly in removing trophozoites from rigid gas-permeable lenses. The cleaning/disinfecting solutions tested were ineffective in removing or in affecting the viability of all Acanthamoeba trophozoites or cysts in the 17 hours allotted for the experiment. The need for a better care in mechanical and physical hygiene procedures is stressed. PMID:10376283

  18. Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species.

    PubMed

    Jha, Bijay Kumar; Jung, Hui-Jung; Seo, Incheol; Suh, Seong-Il; Suh, Min-Ho; Baek, Won-Ki

    2015-12-01

    Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has not been defined yet. The aim of this study was to investigate the effect of juglone on Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba castellanii at 3-5 μM concentrations. Juglone increased the production of reactive oxygen species (ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-l-cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a significant role in the juglone-induced cell death of Acanthamoeba. PMID:26358271

  19. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    PubMed Central

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum. PMID:16825391

  20. Reevaluation of an Acanthamoeba Molecular Diagnostic Algorithm following an Atypical Case of Amoebic Keratitis

    PubMed Central

    Lau, Rachel; Cunanan, Marlou; Jackson, Jonathan; Ali, Ibne Karim M.; Chong-Kit, Ann; Gasgas, Jason; Tian, Jinfang; Ralevski, Filip

    2015-01-01

    Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement initiative with respect to the molecular detection of acanthamoebae in our laboratory because of an unusual case of discordance. Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked, surplus corneal scraping specimens were analyzed for the presence of acanthamoebae with four separate real-time PCR assays. The assay used by the Free-Living and Intestinal Amebas Laboratory of the CDC was considered the reference standard, and the performance characteristics of each individual assay and pairs of assays were calculated. Outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. The sensitivities of the individual assays ranged from 64.3% to 92.9%, compared to the gold standard, while the specificities ranged from 88.6% to 91.4%. The PPVs and NPVs ranged from 69.2% to 78.6% and from 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance, with sensitivities ranging from 92.9% to 100% and specificities ranging from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed to be detected by certain individual assays included Acanthamoeba castellanii, Acanthamoeba culbertsoni, and Acanthamoeba lenticulata. For three clinical specimens, false negativity of the gold standard assay could not be excluded. Molecular diagnostic approaches, especially combinations of highly sensitive and specific assays, offer a reasonably performing, operator-independent, rapid strategy for the detection of acanthamoebae in clinical specimens and are likely to be more practical than either culture or direct microscopic detection. PMID:26202123

  1. In the case of transmission of Mycobacterium ulcerans in buruli ulcer disease Acanthamoeba species stand accused.

    PubMed

    Wilson, M D; Boakye, D A; Mosi, L; Asiedu, K

    2011-03-01

    Buruli ulcer disease caused by Mycobacterium ulcerans results in extensive destruction of skin and soft tissue and long-term functional disabilities that ultimately require surgery and rehabilitation. The disease is associated with aquatic and swampy environments with the mycobacterium occurring in biofilms, soil, aquatic insects, fish and wildlife however, the mode of transmission to humans remains an enigma. Current transmission ideas including bites from predatory water bugs and mosquitoes, do not explain satisfactorily the spasmodic disease distribution in human populations. Here we argue that Acanthamoeba species are the natural hosts of M. ulcerans and are mainly responsible for disease transmission because; (i) Acanthamoebae are known natural hosts of several microbial pathogens including M. marinum, M. avium and Legionella pneumophila, (ii) culture of slow-to-grow microbial pathogens hosted in nature by Acanthamoeba spp is enhanced when the media is seeded with the protozoa, (iii) acanthamoebae and M. ulcerans share similar bio-ecological and epidemiological settings, (iv) documented evidence that prior growth of L. pneumophila and M. avium in acanthamoebae influences entry mechanisms, intracellular growth and virulence in human monocytes, (v) Acanthamoeba spp also infect humans and cause diseases via routes of openings including broken skin and sites of trauma similar to M. ulcerans and (vi) M. ulcerans is rather a fastidious intracellular organism as recent analysis of the genome indicate. We argue further that temperature plays a significant role in transmission determining the fate of either the intracellular microbe or the host cells. Also, Acanthamoeba-pathogen association has a long evolutionary history because the same set of bacterial genes and gene products e.g. in L. pneumophila are required for survival in both mammalian and protozoan host cells. We suggest that the involvement of Acanthamoeba in the transmission of M. ulcerans to humans better

  2. Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

    PubMed

    Magnet, A; Peralta, R H S; Gomes, T S; Izquierdo, F; Fernandez-Vadillo, C; Galvan, A L; Pozuelo, M J; Pelaz, C; Fenoy, S; Del Águila, C

    2015-02-01

    Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown. PMID:25461091

  3. Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

    PubMed Central

    MAHMOUDI, Mohammad Reza; KAZEMI, Bahram; HAGHIGHI, Ali; KARANIS, Panagiotis

    2015-01-01

    Background: Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples. Methods: A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively. Results: Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites. Conclusion: The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran. PMID:26246823

  4. Evaluation of Ozone Application in Dental Unit Water Lines Contaminated with Pathogenic Acanthamoeba

    PubMed Central

    HIKAL, Wafaa; ZAKI, Basma; SABRY, Hany

    2015-01-01

    Background: In this study morphological and molecular characterization of Acanthamoeba strains, isolated from dental unit waterlines (DUWLs) were surveyed and the levels of disinfection achievable in vitro by the application of ozone disinfectant to DUWLs were evaluate. Methods: Water samples were collected from air-water syringes, cup fillers and tap water before and at the end of the working day. They were cultured on non-nutrient agar (NNA) plates. Species identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene. The cellular response to ozone was tested on Acanthamoeba cyst with different doses at different contact time in vitro twice. Results: Prevalence rates for Acanthamoeba contamination were 100, 100 and 72% for air-water syringes, cup fillers and tap water, respectively. The morphological analysis revealed the presence of A. castellanii, A. griffin, A. hatchitti and A. lenticulata. Phylogenetic analysis of the sequences showed the four strains to be closely related to a sequence type (T3, T4, T5 and T11). Acanthamoeba cells were stained with trypan blue, which revealed killed of Acanthamoeba instantaneously after 10 minutes in ozonized water. There was no growth of Acanthamoeba occurred after ozone treatment in water bottles for 5 minutes with a flow rate of 500 mg/hour. Conclusion : Ozone can play an important role in controlling the problem of contamination of DUWLs as a potent disinfectant. PMID:26622296

  5. Arcobacter butzleri survives within trophozoite of Acanthamoeba castellanii.

    PubMed

    Villanueva, María P; Medina, Gustavo; Fernández, Heriberto

    2016-01-01

    The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri. PMID:26972277

  6. First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

    PubMed Central

    Boughalmi, Mondher; Pagnier, Isabelle; Aherfi, Sarah; Colson, Philippe; Raoult, Didier; La Scola, Bernard

    2013-01-01

    Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection. PMID:24287596

  7. Acanthamoeba and bacteria produce antimicrobials to target their counterpart

    PubMed Central

    2014-01-01

    Background In the microbial ecosystem, microbes compete for space and nutrients. Consequently, some have developed the ability to kill or inhibit the growth of other competing microbes by producing antimicrobial substances. As the ‘producer’ species are generally immune to these substances, their compounds act on the competing microbial species and give the producer more space and access to nutrients for growth. Many currently used antibiotics were developed by exploiting this potential of certain microbes. Findings Here, the free-living amoeba, Acanthamoeba castellanii, was investigated for its antibacterial activity against representative Gram positive and Gram negative bacteria, while bacterial isolates were tested for their anti-amoebic properties. Conditioned medium from A. castellanii showed remarkable bactericidal properties against methicillin-resistant Staphylococcus aureus (MRSA) exhibiting almost 100% kill rate, but had limited effect against Acinetobacter sp., Pseudomonas aeruginosa and vancomycin-resistant Enterococcus faecalis (VRE). Similarly, the conditioned medium of E. coli K1 and Enterobacter sp., exhibited potent anti-Acanthamoebic effects in a concentration-dependent manner. Conditioned media of Acanthamoeba, E. coli K1 and Enterobacter sp. showed no cytotoxicity in vitro when tested against human brain microvascular endothelial cells. Active molecule/s in aforementioned amoebic and two bacterial conditioned media were 5 – 10 kDa, and <5 kDa respectively. Conclusions A. castellanii conditioned medium showed potent bactericidal properties against MRSA. The active molecule(s) are heat- and pronase-resistant, and in the 5 to 10 kDa molecular mass range. Contrary to this, E. coli K1 and Enterobacter sp., conditioned medium showed anti-amoebic effects that are <5 kDa in molecular mass, suggestive of active metabolites. PMID:24479709

  8. [Use of the presence of cellulose in cellular wall of Acanthamoeba cysts for diagnostic purposes].

    PubMed

    Derda, Monika; Hadaś, Edward

    2009-01-01

    Species identification within the genus Acanthamoeba is based predominantly on morphological and biochemical features. It is labor-intensive and requires cloning and axenization. We described a novel immunocytochemical method for the identification of Acanthamoeba spp. based on selective binding of Clostridium cellulovorans cellulase to protozoan cyst wall cellulose. Free-living amoebae isolated from different water sources by filtration and subsequent cultivation on non-nutrient agar were assigned to genera Acanthamoeba, Naegleria or Hartmannella using morphological taxonomic criteria. Tissues samples from experimentally infected mice were fixed in formalin and for sectioning embedded in paraffin or snap frozen. The Cellulose-Binding Domain of C. cellulovorans cellulase (CBD) obtained as a recombinant protein, were coupled to the fluorescent dye using Alexa Fluor350, 488, 568 - Protein Labelling Kit or labelled with the biotin using EZ-Link Sulfo-NHS-Biotin. All coupling procedures were performed according to the methods provided by manufacturers. For staining with CBD conjugate, slides containing cysts collected from the agar plates or tissue sections were immersed with PBS and incubated with CBD for 30 min at room temperature, washed 3 times with PBS. For staining with CBD-biotin slides containing cysts were incubated with biotinylated CBD for 30 min at room temperature. Subsequent washings in changes of PBS were followed by the incubation with Strept ABComplex/HRP, for 30 min at room temperature, than 3,3 diaminobenzidine tetrahydrochloride was added for 15 min. Slides were rinsed with water, dried and examined in the light microscope. We showed that cellulose could be easily detected by immunofluorescence using conjugated CBD in the inner cyst wall of Acanthamoeba spp. The reference strains of Acanthamoeba spp. and all Acanthamoeba strains isolated from water and from tissues of infected animals gave positive reaction. CBD prepared as a biotynylated protein

  9. "Marseilleviridae", a new family of giant viruses infecting amoebae.

    PubMed

    Colson, Philippe; Pagnier, Isabelle; Yoosuf, Niyaz; Fournous, Ghislain; La Scola, Bernard; Raoult, Didier

    2013-04-01

    The family "Marseilleviridae" is a new proposed taxon for giant viruses that infect amoebae. Its first member, Acanthamoeba polyphaga marseillevirus (APMaV), was isolated in 2007 by culturing on amoebae a water sample collected from a cooling tower in Paris, France. APMaV has an icosahedral shape with a diameter of ≈250 nm. Its genome is a double-stranded circular DNA that is 368,454 base pairs (bp) in length. The genome has a GC content of 44.7 % and is predicted to encode 457 proteins. Phylogenetic reconstructions showed that APMaV belongs to a new viral family among nucleocytoplasmic large DNA viruses, a group of viruses that also includes Acanthamoeba polyphaga mimivirus (APMV) and the other members of the family Mimiviridae as well as the members of the families Poxviridae, Phycodnaviridae, Iridoviridae, Ascoviridae, and Asfarviridae. In 2011, Acanthamoeba castellanii lausannevirus (ACLaV), another close relative of APMaV, was isolated from river water in France. The ACLaV genome is 346,754 bp in size and encodes 450 genes, among which 320 have an APMaV protein as the closest homolog. Two other giant viruses closely related to APMaV and ACLaV have been recovered in our laboratory from a freshwater sample and a human stool sample using an amoebal co-culture method. The only currently identified hosts for "marseilleviruses" are Acanthamoeba spp. The prevalence of these viruses in the environment and in animals and humans remains to be determined. PMID:23188494

  10. Chloroquine Has a Cytotoxic Effect on Acanthamoeba Encystation through Modulation of Autophagy

    PubMed Central

    Jha, Bijay Kumar; Jung, Hui-Jung; Seo, Incheol; Kim, Hyun Ah; Suh, Seong-Il; Suh, Min-Ho

    2014-01-01

    Encystation of Acanthamoeba castellanii is associated with resistance to chemotherapeutic agents. Blocking the encystation process could potentiate the efficacy of chemotherapeutic agents and biocides. During encystation, autophagy is highly stimulated and required for proper encystation of Acanthamoeba. In this study, the cytotoxic effect of chloroquine, a well-known autophagy-inhibitory drug, was tested in A. castellanii. Chloroquine was able to selectively reduce cell survival during the encystation of A. castellanii. However, A. castellanii trophozoites and mature cysts were resistant to chloroquine. Chloroquine treatment led to an increase in the number and size of lysosomes in encysting cells. Moreover, chloroquine inhibited the degradation of long-lived proteins in the encysting cells. Decreased autophagic flux, indicated by an increased number of lysosomes and decreased degradation of long-lived proteins, may be the mechanism by which cell death is induced by chloroquine in encysting Acanthamoeba. These results suggest a potential novel therapeutic application of chloroquine as an anti-Acanthamoeba drug. Our findings also suggest that targeting autophagy could be a therapeutic strategy against Acanthamoeba infection. PMID:25114131

  11. A multisystemic Acanthamoeba infection in a dog in Tenerife, Canary Islands, Spain.

    PubMed

    Valladares, María; Reyes-Batlle, María; Mora-Peces, Inmaculada; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Dorta-Gorrín, Alexis; Comyn-Afonso, Estefanía; Martínez-Carretero, Enrique; Maciver, Sutherland K; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2014-10-15

    A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24h, the dog was found to have a high fever (39.5 °C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 °C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain. PMID:25193180

  12. Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

    PubMed

    Omaña-Molina, Maritza; Vanzzini-Zago, Virginia; Hernandez-Martinez, Dolores; Gonzalez-Robles, Arturo; Salazar-Villatoro, Lizbeth; Ramirez-Flores, Elizabeth; Oregon-Miranda, Eric; Lorenzo-Morales, Jacob; Martinez-Palomo, Adolfo

    2016-02-01

    Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection. PMID:26581373

  13. Failure of chemotherapy in the first reported cases of Acanthamoeba keratitis in Pakistan

    PubMed Central

    Siddiqui, Ruqaiyyah; Chaudhry, Tanveer; Lakhundi, Sahreena; Ahmad, Khabir; Khan, Naveed Ahmed

    2014-01-01

    Acanthamoeba keratitis is a painful and progressive infection of the cornea that can result in loss of vision. Here, for the first time in Pakistan, we report two cases of Acanthamoeba keratitis. The first patient was a 37-year-old female who presented with severe itching, redness, pain, along with loss of vision. The patient was a regular soft contact lens wearer. The second patient was a 25-year-old female who had been using soft contact lenses for the past two years. She presented with a burning sensation and extreme pain, along with loss of vision. Both patients were treated for a possible microbial keratitis with topical moxifloxacin hydrochloride drops, vancomycin drops, propamidine isethionate ointment, amphotericin B drops, and amikacin drops. However, the response was inadequate and both patients were referred for corneal transplant. Acanthamoeba castellanii was isolated by placing contact lenses and contact lens cases on non-nutrient agar plates containing a lawn of non-invasive Escherichia coli K-12 HB101 bacteria. The polymerase chain reaction (PCR) using genus-specific probes confirmed the identity of Acanthamoeba spp., whereas the morphological characteristics of trophozoites and cysts were suggestive of A. castellanii in both cases. With growing use of contact lenses for vision correction/cosmetic use coupled with sub-standard lens care in this region and the possibility of non-contact lens-associated Acanthamoeba keratitis, a need for increased awareness of this sight-threatening infection is discussed further. PMID:24548160

  14. Viability and morphological changes of Acanthamoeba spp. cysts after treatment with Effective microorganisms (EM).

    PubMed

    Sampaotong, Tanitta; Lek-Uthai, Usa; Roongruangchai, Jantima; Roongruangchai, Kosol

    2016-06-01

    Acanthamoeba is a free-living opportunistic protozoan parasite that is found in diverse environments. It can cause keratitis, mostly related to inappropriate use of contact lenses, as well as life threatening diseases including encephalitis, disseminated sinusitis, and skin ulcers. This study investigated morphological changes and fine structures of the cyst form of Acanthamoeba spp. after treatment with effective microorganisms (EM™) using light and scanning electron microscopies. Acanthamoeba cysts treated with 1:2, 1:4, 1:6, and undiluted EM™ showed higher percentages of non-viable cysts than those treated with 1:8, 1:10, 1:100, 1:200, and 1:400 EM™ and at 5 days post-treatment developed from cystic stage to trophozoite stage. Acanthamoeba cysts treated at concentrations of 1:2, 1:4, 1:6, and undiluted EM™ exhibited cytoplasmic clumping and shrinkage of amoeba cells away from cyst walls. The effective EM™ concentration lethal to Acanthamoeba spp. cyst could provide information to monitor the environmental control system. PMID:27413306

  15. Chloroquine has a cytotoxic effect on Acanthamoeba encystation through modulation of autophagy.

    PubMed

    Jha, Bijay Kumar; Jung, Hui-Jung; Seo, Incheol; Kim, Hyun Ah; Suh, Seong-Il; Suh, Min-Ho; Baek, Won-Ki

    2014-10-01

    Encystation of Acanthamoeba castellanii is associated with resistance to chemotherapeutic agents. Blocking the encystation process could potentiate the efficacy of chemotherapeutic agents and biocides. During encystation, autophagy is highly stimulated and required for proper encystation of Acanthamoeba. In this study, the cytotoxic effect of chloroquine, a well-known autophagy-inhibitory drug, was tested in A. castellanii. Chloroquine was able to selectively reduce cell survival during the encystation of A. castellanii. However, A. castellanii trophozoites and mature cysts were resistant to chloroquine. Chloroquine treatment led to an increase in the number and size of lysosomes in encysting cells. Moreover, chloroquine inhibited the degradation of long-lived proteins in the encysting cells. Decreased autophagic flux, indicated by an increased number of lysosomes and decreased degradation of long-lived proteins, may be the mechanism by which cell death is induced by chloroquine in encysting Acanthamoeba. These results suggest a potential novel therapeutic application of chloroquine as an anti-Acanthamoeba drug. Our findings also suggest that targeting autophagy could be a therapeutic strategy against Acanthamoeba infection. PMID:25114131

  16. Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools.

    PubMed

    Staggemeier, Rodrigo; Arantes, Thalita; Caumo, Karin S; Rott, Marilise B; Spilki, Fernando R

    2016-01-01

    Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water. PMID:27142544

  17. Acanthamoeba keratitis presenting as dendritic keratitis in a soft contact lens wearer.

    PubMed

    Yeung, Edward Yip; Huang, Samuel Chao-Ming; Tsai, Ray Jui-Fang

    2002-03-01

    Acanthamoeba keratitis is a rare cause of corneal infection in Taiwan, which can result in devastating visual outcomes. A 37-year-old woman, who wore soft contact lenses, suffered from severe pain in her left eye. Biomicroscopy revealed dendritic keratitis, radial keratoneuritis, and fine keratic precipitates on her cornea. Culture, using non-nutrient agar plate seeded with Escherichia coli, resulted in heavy growth of Acanthamoeba. The inpatient treatment, including topical neomycin-polymyxin B and metronidazole (0.5%) eyedrops, oral ketoconazole, and then oral prednisolone, successfully controlled the corneal infection. The best-corrected visual acuity was 0.9 without any evidence of recurrence of infection after 21 months of follow up. Acanthamoeba keratitis can present as dendritic keratitis, which mimics herpes simplex infection, thus, delays appropriate treatment. Early diagnosis and judicious treatment are essential for restoring the vision and avoiding the subsequent need of penetrating keratoplasty. PMID:12022742

  18. Lethal Effects of Helianthemum lippii (L.) on Acanthamoeba castellanii Cysts in Vitro

    PubMed Central

    Badria, F.A.; Hetta, M.H.; Sarhan, Rania M.; Ezz El-Din, M.H.

    2014-01-01

    Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role. PMID:25031463

  19. Temperature-induced membrane-lipid adaptation in Acanthamoeba castellanii.

    PubMed

    Jones, A L; Hann, A C; Harwood, J L; Lloyd, D

    1993-02-15

    A method has been developed for the separation of the major membrane fractions of Acanthamoeba castellanii after growth at different temperatures. The acyl-lipid compositions of individual membrane fractions, microsomal membranes, plasma membrane and mitochondria were analysed after a shift in culture temperature from 30 degrees C to 15 degrees C. The major change in lipid composition observed was an alteration in the relative proportions of oleate and linoleate. This reciprocal change was seen in all the membrane fractions, but occurred most rapidly in the phosphatidylcholine of the microsomal fraction. Thus, there appears to be a rapid induction of delta 12-desaturase activity in A. castellanii after a downward shift in growth temperature. Changes were also seen in the proportions of the n-6 C20 fatty acids, with a decrease in the proportions of icosadienoate and increases of icosatrienoate and arachidonate. However, unlike the alteration in oleate/linoleate ratios, this change was not seen in all the individual lipids of each membrane fraction. PMID:8439295

  20. Acanthamoeba royreba: morphological features and in vitro cytopathic effect.

    PubMed

    González-Robles, Arturo; Salazar-Villatoro, Lizbeth; Omaña-Molina, Maritza; Lorenzo-Morales, Jacob; Martínez-Palomo, Adolfo

    2013-04-01

    Observations on cultured Acanthamoeba royreba trophozoites and in vitro cytopathogenicity of this amoeba are described. In culture, amoebae were active, pleomorphic and moved on the substrate by producing endocytic structures and emitting slight cytoplasmic microprojections from the cell surface. These projections were formed by hyaline cytoplasm and they were related to motion structures such as acanthopodia and lamellipodia, in which actin provides a framework that allows rapid changes in morphology. In the cytoplasm abundant vacuoles of different size and content were seen. By means of electron microscopy, it was possible to observe the compact fibrogranular appearance of the cytoplasm, along with the main cellular organelles such as the Golgi complex, the endoplasmic reticulum, digestive vacuoles, mitochondria and contractile vacuoles. Incubation of MDCK epithelial cell monolayers with conditioned medium did not produce a significant structural damage to the monolayer, even after 24h of incubation. When the trophozoites were incubated with the target cells the monolayer exhibited a clear injury created by the amoebae, which produced focal damage. Nevertheless, the rest of the monolayer appeared to remain intact, suggesting that a contact-dependent interaction is necessary to damage the target cells. These observations demonstrate the low invasive capacity of this amoeba. PMID:23357648

  1. Survival of taylorellae in the environmental amoeba Acanthamoeba castellanii

    PubMed Central

    2014-01-01

    Background Taylorella equigenitalis is the causative agent of contagious equine metritis, a sexually-transmitted infection of Equidae characterised in infected mares by abundant mucopurulent vaginal discharge and a variable degree of vaginitis, cervicitis or endometritis, usually resulting in temporary infertility. The second species of the Taylorella genus, Taylorella asinigenitalis, is considered non-pathogenic, although mares experimentally infected with this bacterium can develop clinical signs of endometritis. To date, little is understood about the basic molecular virulence and persistence mechanisms employed by the Taylorella species. To clarify these points, we investigated whether the host-pathogen interaction model Acanthamoeba castellanii was a suitable model for studying taylorellae. Results We herein demonstrate that both species of the Taylorella genus are internalised by a mechanism involving the phagocytic capacity of the amoeba and are able to survive for at least one week inside the amoeba. During this one-week incubation period, taylorellae concentrations remain strikingly constant and no overt toxicity to amoeba cells was observed. Conclusions This study provides the first evidence of the capacity of taylorellae to survive in a natural environment other than the mammalian genital tract, and shows that the alternative infection model, A. castellanii, constitutes a relevant alternative system to assess host-pathogen interactions of taylorellae. The survival of taylorellae inside the potential environmental reservoir A. castellanii brings new insight, fostering a broader understanding of taylorellae biology and its potential natural ecological niche. PMID:24641089

  2. Risk factors for acanthamoeba keratitis in contact lens users: a case-control study.

    PubMed Central

    Radford, C. F.; Bacon, A. S.; Dart, J. K.; Minassian, D. C.

    1995-01-01

    OBJECTIVE--To investigate reasons for an increase in cases of Acanthamoeba keratitis related to contact lenses. DESIGN--Case-control study. Cases were contact lens related acanthamoeba keratitis patients treated between 1 September 1989 and 31 August 1992. Controls were lens users without lens related disease who presented as new patients to the casualty department from 1 March 1992 to 31 August 1992. All subjects completed a questionnaire detailing lens use and hygiene practices. SETTING--Eye hospital. SUBJECTS--35 cases with acanthamoeba keratitis and 378 controls. MAIN OUTCOME MEASURES--Relative risks comparing different contact lens types, socioeconomic classification, age, sex, lens use, lens wearing experience, hygiene compliance, and hygiene systems. RESULTS--The crude relative risk for developing acanthamoeba keratitis with the use of daily wear disposable lenses was 49.45 (95% confidence interval 6.53 to 2227; P < 0.001) compared with conventional soft lenses (the referent). Multivariable analysis showed that this increased risk could be largely attributed to lack of disinfection (relative risk 55.86 (10 to 302); P < 0.001) and use of chlorine based disinfection (14.63 (2.8 to 76); P = 0.001) compared with other chemical systems (the referent). None of the other outcome measures showed a significant association. CONCLUSIONS--Both failure to disinfect daily wear soft contact lenses and the use of chlorine release lens disinfection systems, which have little protective effect against the organism, are major risk factors for acanthamoeba keratitis. These risks have been particularly common in disposable lens use. Over 80% of acanthamoeba keratitis could be avoided by the use of lens disinfection systems that are effective against the organism. PMID:7787645

  3. Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser.

    PubMed

    Ekeberg, Tomas; Svenda, Martin; Seibert, M Marvin; Abergel, Chantal; Maia, Filipe R N C; Seltzer, Virginie; DePonte, Daniel P; Aquila, Andrew; Andreasson, Jakob; Iwan, Bianca; Jönsson, Olof; Westphal, Daniel; Odić, Duško; Andersson, Inger; Barty, Anton; Liang, Meng; Martin, Andrew V; Gumprecht, Lars; Fleckenstein, Holger; Bajt, Saša; Barthelmess, Miriam; Coppola, Nicola; Claverie, Jean-Michel; Loh, N Duane; Bostedt, Christoph; Bozek, John D; Krzywinski, Jacek; Messerschmidt, Marc; Bogan, Michael J; Hampton, Christina Y; Sierra, Raymond G; Frank, Matthias; Shoeman, Robert L; Lomb, Lukas; Foucar, Lutz; Epp, Sascha W; Rolles, Daniel; Rudenko, Artem; Hartmann, Robert; Hartmann, Andreas; Kimmel, Nils; Holl, Peter; Weidenspointner, Georg; Rudek, Benedikt; Erk, Benjamin; Kassemeyer, Stephan; Schlichting, Ilme; Strüder, Lothar; Ullrich, Joachim; Schmidt, Carlo; Krasniqi, Faton; Hauser, Günter; Reich, Christian; Soltau, Heike; Schorb, Sebastian; Hirsemann, Helmut; Wunderer, Cornelia; Graafsma, Heinz; Chapman, Henry; Hajdu, Janos

    2016-01-01

    Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms. PMID:27479754

  4. Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser

    PubMed Central

    Ekeberg, Tomas; Svenda, Martin; Seibert, M. Marvin; Abergel, Chantal; Maia, Filipe R.N.C.; Seltzer, Virginie; DePonte, Daniel P.; Aquila, Andrew; Andreasson, Jakob; Iwan, Bianca; Jönsson, Olof; Westphal, Daniel; Odić, Duško; Andersson, Inger; Barty, Anton; Liang, Meng; Martin, Andrew V.; Gumprecht, Lars; Fleckenstein, Holger; Bajt, Saša; Barthelmess, Miriam; Coppola, Nicola; Claverie, Jean-Michel; Loh, N. Duane; Bostedt, Christoph; Bozek, John D.; Krzywinski, Jacek; Messerschmidt, Marc; Bogan, Michael J.; Hampton, Christina Y.; Sierra, Raymond G.; Frank, Matthias; Shoeman, Robert L.; Lomb, Lukas; Foucar, Lutz; Epp, Sascha W.; Rolles, Daniel; Rudenko, Artem; Hartmann, Robert; Hartmann, Andreas; Kimmel, Nils; Holl, Peter; Weidenspointner, Georg; Rudek, Benedikt; Erk, Benjamin; Kassemeyer, Stephan; Schlichting, Ilme; Strüder, Lothar; Ullrich, Joachim; Schmidt, Carlo; Krasniqi, Faton; Hauser, Günter; Reich, Christian; Soltau, Heike; Schorb, Sebastian; Hirsemann, Helmut; Wunderer, Cornelia; Graafsma, Heinz; Chapman, Henry; Hajdu, Janos

    2016-01-01

    Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms. PMID:27479754

  5. Acanthamoeba Encephalitis: Isolation of Genotype T1 in Mycobacterial Liquid Culture Medium

    PubMed Central

    Azzam, Rula; Badenoch, Paul R.; Francis, Michelle J.; Fernandez, Charles; Adamson, Penelope J.; Dendle, Claire; Woolley, Ian; Robson, Jenny; Korman, Tony M.

    2014-01-01

    We report a case of Acanthamoeba encephalitis diagnosed from an antemortem brain biopsy specimen, where the organism was first isolated in mycobacterial liquid medium and first identified by using a sequence generated by a commercial panfungal sequencing assay. We correlate susceptibility results with clinical outcome. PMID:25502534

  6. Molecular Characterization of Pathogenic Acanthamoeba Isolated from Drinking and Recreational water in East Azerbaijan, Northwest Iran

    PubMed Central

    Behniafar, Hamed; Niyyati, Maryam; Lasjerdi, Zohreh

    2015-01-01

    Acanthamoeba- related infections, such as amoebic keratitis and granulomatous amoebic encephalitis, can develop in high-risk population through contaminated water sources. Thus, surveying water resources, particularly those available for human use, is of the utmost importance. In the present study, 67 water samples were collected from water resources in East Azerbaijan, a province in northwestern Iran. Samples were cultured on enriched non-nutrient agar plates, and sequencing-based approaches were used for genotyping. The pathogenic potential of the isolates was determined using thermo- and osmo-tolerance tests. Acanthamoeba were detected in 17 (25.4%) of the 67 collected samples. Sequencing analysis revealed that the isolates belonged to the T3 (23.52%), mixed T3/T4 (5.88%), T4 (58.82%), T5 (5.88%), and T13 (5.88%) genotypes. Through thermo- and osmo-tolerance tests, 88.23% of isolates were resistant to 37 °C, 40 °C temperature, and 0.5 M and 1 M osmolarity; thus, these isolates had the potential for pathogenicity. These findings point toa serious public health concern in the studied region. This study is the first to report Acanthamoeba isolated from drinking and recreational water sources in East Azerbaijan and Acanthamoeba T13 isolated from tap water in Iran. PMID:26157334

  7. Isolation and identification of Acanthamoeba spp. from thermal swimming pools and spas in Southern Brazil.

    PubMed

    Fabres, Laura Fuhrich; Rosa Dos Santos, Sayonara Peixoto; Benitez, Lisianne Brittes; Rott, Marilise Brittes

    2016-03-01

    Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Species of Acanthamoeba can cause keratitis and brain infections. In this study, 72 water samples were taken from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. When the isolates were submitted to PCR reaction only 8 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compared to the sequences in the GenBank, showed genotype distribution in group T3 (12,5%), T5 (12,5%), T4 (25%) and T15 (50%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health. PMID:27078644

  8. Microarray and KOG analysis of Acanthamoeba healyi genes up-regulated by mouse-brain passage.

    PubMed

    Moon, Eun-Kyung; Xuan, Ying-Hua; Kong, Hyun-Hee

    2014-08-01

    Long-term cultivation in a laboratory could reduce the virulence of Acanthamoeba. To identify virulence factors of Acanthamoeba, the authors compared the transcription profiles of long-term cultivated Acanthamoeba healyi (OLD) and three times mouse-brain passaged A. healyi (MBP) using microarray analysis and eukaryotic orthologous group (KOG) assignments. Microarray analysis revealed that 601 genes were up-regulated by mouse-brain passage. The results of real-time PCR of 8 randomly selected genes up-regulated in the MBP strain confirmed microarray analysis findings. KOG assignments showed relatively higher percentages of the MBP strain up-regulated genes in T article (signal transduction mechanism), O article (posttranslational modification, protein turnover, chaperones), C article (energy production and conversion), and J article (translation, ribosomal structure and biogenesis). In particular, the MBP strain showed higher expressions of cysteine protease and metalloprotease. A comparison of KOG assignments by microarray analysis and previous EST (expressed sequence tags) analysis showed similar populations of up-regulated genes. These results provide important information regarding the identification of virulence factors of pathogenic Acanthamoeba. PMID:24859526

  9. Fatal Granulomatous Amoebic Encephalitis Caused by Acanthamoeba in a Patient With Kidney Transplant: A Case Report

    PubMed Central

    Salameh, Ahmad; Bello, Nancy; Becker, Jennifer; Zangeneh, Tirdad

    2015-01-01

    Granulomatous amoebic encephalitis (GAE) due to Acanthamoeba is almost a uniformly fatal infection in immune-compromised hosts despite multidrug combination therapy. We report a case of GAE in a female who received a deceased donor kidney graft. She was treated with a combination of miltefosine, pentamidine, sulfadiazine, fluconazole, flucytosine, and azithromycin. PMID:26280011

  10. Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

    PubMed Central

    Lee, Yu-Ran; Na, Byoung-Kuk; Moon, Eun-Kyung; Song, Su-Min; Joo, So-Young; Kong, Hyun-Hee; Goo, Youn-Kyoung; Chung, Dong-Il; Hong, Yeonchul

    2015-01-01

    Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba. PMID:26075721

  11. Status of free-living amoebae (Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris) in drinking water supplies in Karachi, Pakistan.

    PubMed

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Subhani, Faysal; Khan, Naveed Ahmed

    2013-06-01

    The ability of pathogenic free-living amoebae to produce infections is a growing concern. In this study, we investigated the presence of free-living amoebae (Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris) in drinking water supplies in Karachi, Pakistan. Fifty-two domestic tap water samples were examined. Amoebae were identified by morphological characteristics and polymerase chain reaction. Thirty percent of the examined samples were positive for Acanthamoeba spp., 8% for N. fowleri while B. mandrillaris were not recovered. Additionally we examined secretory IgA antibody to Acanthamoeba and B. mandrillaris. Acanthamoeba antibody prevalence rate was 100% in both males and females, while B. mandrillaris antibody prevalence rate was 5.5% in males only (females were negative). Our findings suggest that free-living amoebae are a potential health hazard in domestic water supplies in Karachi, Pakistan. PMID:23708583

  12. Acanthamoeba genotypes T2, T4, and T11 in soil sources from El Hierro island, Canary Islands, Spain.

    PubMed

    Reyes-Batlle, María; Zamora-Herrera, Jonadab; Vargas-Mesa, Alejandro; Valerón-Tejera, Marco Antonio; Wagner, Carolina; Martín-Navarro, Carmen Ma; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Valladares, Basilio; Piñero, José E; Lorenzo-Morales, Jacob

    2016-08-01

    The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island. PMID:27075307

  13. Necrotizing Meningoencephalitis in a Captive Black and White Ruffed Lemur (Varecia variegata variegata) Caused by Acanthamoeba T4 Genotype.

    PubMed

    Gaide, N; Pelandakis, M; Robveille, C; Albaric, O; Jouvion, G; Souchon, M; Risler, A; Abadie, J

    2015-11-01

    A mature male, black and white ruffed lemur (Varecia variegata variegata) died in a zoological garden after a 4-day history of lethargy and non-responsive convulsions. Necropsy and histopathological examinations revealed acute necrotizing and haemorrhagic meningoencephalitis with intralesional amoebas confirmed by immunohistochemistry. Acanthamoeba T4 genotype was identified as the causative agent of the brain lesion, based on amplification and sequencing of 18S ribosomal RNA genes. The presence of free-living amoebas in water and mud from the lemur's environment was investigated by morphological and molecular analyses. The two predominant genera, representing 80% of isolated amoebas, were Naegleria spp. and Acanthamoeba spp. All Acanthamoeba isolates belonged to the T4 genotype. To the author's knowledge, this is the first report of a meningoencephalitis due to Acanthamoeba T4 genotype in Lemuridae with concurrent analysis of pathological tissues and environment. PMID:26297109

  14. Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City.

    PubMed

    Bonilla-Lemus, Patricia; Ramírez-Bautista, Gerardo A; Zamora-Muñoz, Claudia; Ibarra-Montes, María Del Rocío; Ramírez-Flores, Elizabeth; Hernández-Martínez, María Dolores

    2010-09-01

    A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis. PMID:19995560

  15. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    SciTech Connect

    Pan, Hong; Wu, Xinyi

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88

  16. Mechanisms of karyotype differentiation in Cassidinae sensu lato (Coleoptera, Polyphaga, Chrysomelidae) based on seven species of the Brazilian fauna and an overview of the cytogenetic data.

    PubMed

    de Julio, Milena; Fernandes, Flávia Rodrigues; Costa, Cleide; Almeida, Mara Cristina; Cella, Doralice Maria

    2010-01-01

    Among the subfamilies of Chrysomelidae, Cassidinae sensu lato (s.l.) includes 6000 species distributed in 43 tribes. Approximately 100 of these species were cytogenetically analyzed and most of them presented 2n=18=16+Xy(p), which was smaller than 2n=20=18+Xy(p) considered basal for Polyphaga. However, some groups of species presented maintenance of the basal diploid number and others showed increase in this number. Certain species of the latter group also exhibited variation in the type of sex chromosome system (SCS). Considering the recent taxonomic revision accomplished for the Cassidinae s.l. species, the existence of phylogenetic relationship for some species of this subfamily, the high diversity of species of this group in the Neotropical region, and the low number of Cassidinae s.l. species karyotyped so far, the aim of the present work was to establish the main mechanisms involved in the karyotype evolution of this subfamily through the study of seven species of the Brazilian fauna and overview of the cytogenetic data. The individuals were collected in southeast and south of Brazil. The chromosomal preparations obtained from embryo and testes of adult males were stained with Giemsa solution. The species Agroiconota inedita (2n=42=40+Xy(p)), Charidotella (s.str.) immaculata (2n=22=20+Xy(p)), Charidotella (s.str.) sexpunctata (2n=22=20+Xy(p)), and Stolas chalybaea (2n=24=22+Xy(p)) revealed diploid number higher than that established as basal for Polyphaga and biarmed chromosomes. The karyotype of Cteisella confusa, Deloyala cruciata, and Metriona elatior showed the chromosomal formulae 2n=18=16+Xy(p) considered modal for Cassidinae s.l. and biarmed chromosomes. The seven species exhibited easily identified sex chromosomes due to their size and/or morphology. The analysis of meiotic cells of all the species showed pachytenes with a positively heteropycnotic block probably corresponding to the sex chromosomes; diplotenes with a high number of bivalents with two

  17. Acanthamoeba produces disseminated infection in locusts and traverses the locust blood-brain barrier to invade the central nervous system

    PubMed Central

    2010-01-01

    Background Many aspects of Acanthamoeba granulomatous encephalitis remain poorly understood, including host susceptibility and chronic colonization which represent important features of the spectrum of host-pathogen interactions. Previous studies have suggested locusts as a tractable model in which to study Acanthamoeba pathogenesis. Here we determined the mode of parasite invasion of the central nervous system (CNS). Results Using Acanthamoeba isolates belonging to the T1 and T4 genotypes, the findings revealed that amoebae induced sickness behaviour in locusts, as evidenced by reduced faecal output and weight loss and, eventually, leading to 100% mortality. Significant degenerative changes of various tissues were observed by histological sectioning. Both isolates produced disseminated infection, with viable amoebae being recovered from various tissues. Histological examination of the CNS showed that Acanthamoeba invaded the locust CNS, and this is associated with disruption of the perineurium cell/glial cell complex, which constitutes the locust blood-brain barrier. Conclusions This is the first study to demonstrate that Acanthamoeba invades locust brain by modulating the integrity of the insect's blood-brain barrier, a finding that is consistent with the human infection. These observations support the idea that locusts provide a tractable model to study Acanthamoeba encephalitis in vivo. In this way the locust model may generate potentially useful leads that can be tested subsequently in mammalian systems, thus replacing the use of vertebrates at an early stage, and reducing the numbers of mammals required overall. PMID:20615207

  18. Isolation and molecular characterization of Acanthamoeba genotypes in recreational and domestic water sources from Jamaica, West Indies.

    PubMed

    Todd, Cheridah D; Reyes-Batlle, María; Piñero, José E; Martínez-Carretero, Enrique; Valladares, Basilio; Streete, Don; Lorenzo-Morales, Jacob; Lindo, John F

    2015-09-01

    Free living amoebae (FLA) are amphizoic protozoa that are ubiquitous in nature. Infection with FLA may result in neurological, ocular and skin infections. Exposure to Acanthamoeba occurs frequently through water contact and knowledge of the presence of the organisms in water sources is important in understanding transmission dynamics. The distribution of Acanthamoeba was studied in recreational and domestic water samples collected from across Jamaica. Morphological assessment and polymerase chain reaction revealed Acanthamoeba spp. isolates in 50.6% (42/83) and 17.3% (14/81) of recreational and domestic water, respectively. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T3, T4, T5, T10 and T11 corresponding to Acanthamoeba spp: A. griffini, A. triangularis, A. lenticulata, A. culbertsoni and A. hatchetti. Moreover, T4 was the most frequently isolated genotype in both recreational and domestic water. Thermotolerance and osmotolerance assays indicated that most isolates were potentially pathogenic. This is the first report of T3 and T10 genotypes in the Caribbean and the first report of these Acanthamoeba spp. in Jamaican waters. The study shows that there is potential risk of infection to contact wearers who practise poor lens care. Further, Acanthamoeba should be considered as a cause of neurological infections in Jamaica. PMID:26322776

  19. Acanthamoeba infection in lungs of mice expressed by toll-like receptors (TLR2 and TLR4).

    PubMed

    Derda, Monika; Wojtkowiak-Giera, Agnieszka; Kolasa-Wołosiuk, Agnieszka; Kosik-Bogacka, Danuta; Hadaś, Edward; Jagodziński, Paweł P; Wandurska-Nowak, Elżbieta

    2016-06-01

    Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznań, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP). PMID:26940205

  20. Isolation and genotyping of free-living environmental isolates of Acanthamoeba spp. from bromeliads in Southern Brazil.

    PubMed

    Landell, Melissa Fontes; Salton, Juliana; Caumo, Karin; Broetto, Leonardo; Rott, Marilise B

    2013-07-01

    Species of Acanthamoeba are frequently isolated from distinct environmental sources such as water, soil, dust and air. They are responsible to cause infections and disease in humans and animals. In addition, Acanthamoeba sp. are considered an important reservoir of bacteria, virus and fungi, which act as "Trojan horses" to protect these microorganisms of harsh environmental conditions. In this study, nine Acanthamoeba isolates from bromeliads phylloplane were identified based on the morphology of cyst and trophozoite forms. The genotype level was accessed by the sequence analysis of Acanthamoeba small-subunit rRNA gene. Genotypic characterization grouped five isolates in the genotype T2/T6, three in the T4 genotype and one in the genotype T16. The results obtained indicate that the genotype T2/T6 is common on phylloplane. To predict the pathogenic potential of the Acanthamoeba isolates, thermo and osmotolerance assays were employed, although all isolates were capable of surviving at temperatures of 37°C, other tests will be conducted in the future to determine the potential pathogenic of the isolates. Altogether, our results revealed the importance of the presence of Acanthamoeba associated with bromeliads in Rio Grande do Sul, Brazil, and the necessity for further studies to determine the environmental distribution and the role of these species. PMID:23562883

  1. Acanthamoeba misidentification and multiple labels: redefining genotypes T16, T19, and T20 and proposal for Acanthamoeba micheli sp. nov. (genotype T19).

    PubMed

    Corsaro, Daniele; Walochnik, Julia; Köhsler, Martina; Rott, Marilise B

    2015-07-01

    Acanthamoeba species are ubiquitous amoebae able to cause important infections in humans and other vertebrates. The full/near-full sequences (>2000 bp) of the small subunit ribosomal RNA gene (SSU rDNA or 18S rDNA) are used to cluster Acanthamoeba as genotypes, labeled T1 to T20. Genotype T15 remains an exception, being described only partially on a 1500-bp fragment. Strains are thus usually identified based on their 18S identity matches with reference strains, often using shorter (<500 bp) diagnostic fragments of the gene. Nevertheless, short fragments (<1000 bp) have been used to propose genotypes. This has been criticized, and doubts arise therefore on possible confusion leading to classify distinct partial sequences with a same label(s). We demonstrate herein that several partial sequences misassigned either to T16 or to T4, actually belong to at least two separate and distinct genotypes. We obtained the full 18S rDNA of a strain previously typed as T16 on the basis of a small fragment and demonstrated that it actually belongs to the recently described T19. We propose the name Acanthamoeba micheli sp. nov., for this strain. Furthermore, partial molecular phylogenies were performed to show that several other misassigned T16 partial sequences belong to a new genotype. This latter includes also misassigned T4 partial sequences, only recently available as full sequences and labeled as T20. We thus reassign these partial sequences to the genotype T20. Longer sequences, ideally at least 90 % of the total gene length, should be obtained from strains to ensure reliable diagnostic and phylogenetic results. PMID:25869957

  2. The mitochondrial genome of the multicolored Asian lady beetle Harmonia axyridis (Pallas) and a phylogenetic analysis of the Polyphaga (Insecta: Coleoptera).

    PubMed

    Niu, Fang-Fang; Zhu, Liang; Wang, Su; Wei, Shu-Jun

    2016-07-01

    Here, we report the mitochondrial genome sequence of the multicolored Asian lady beetle Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) (GenBank accession No. KR108208). This is the first species with sequenced mitochondrial genome from the genus Harmonia. The current length with partitial A + T-rich region of this mitochondrial genome is 16,387 bp. All the typical genes were sequenced except the trnI and trnQ. As in most other sequenced mitochondrial genomes of Coleoptera, there is no re-arrangement in the sequenced region compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN codons. Five, five and three protein-coding genes stop with termination codon TAA, TA and T, respectively. Phylogenetic analysis using Bayesian method based on the first and second codon positions of the protein-coding genes supported that the Scirtidae is a basal lineage of Polyphaga. The Harmonia and the Coccinella form a sister lineage. The monophyly of Staphyliniformia, Scarabaeiformia and Cucujiformia was supported. The Buprestidae was found to be a sister group to the Bostrichiformia. PMID:26057015

  3. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran

    PubMed Central

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; Lasjerdi, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran. PMID:27127409

  4. Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis

    PubMed Central

    Peretz, Avi; Geffen, Yuval; Socea, Soergiu D.; Pastukh, Nina; Graffi, Shmuel

    2015-01-01

    Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media—non-nutrient agar (NNA), culture on liquid growth media—peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. PMID:25962772

  5. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran.

    PubMed

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; Lasjerdi, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran. PMID:27127409

  6. Isolation of Acanthamoeba Genotype T4 from a Non-Contact Lens Wearer from the Philippines

    PubMed Central

    Buerano, Corazon C.; Trinidad, Abigail D.; Fajardo, Lindsay Sydney N.; Cua, Irwin Y.; Baclig, Michael O.; Natividad, Filipinas F.

    2014-01-01

    We report the case of a 76-year old Filipino male who presented with pain, redness, and blurring of vision of the right eye. Corneal scraping was done and sent to the St. Luke’s Research and Biotechnology Group for detection and identification of the infectious agent. Morphological detection was performed by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA.S1 confirmed the morphological identification. Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present in the eye of keratitis patients. PMID:25589879

  7. Provirophages and transpovirons as the diverse mobilome of giant viruses.

    PubMed

    Desnues, Christelle; La Scola, Bernard; Yutin, Natalya; Fournous, Ghislain; Robert, Catherine; Azza, Saïd; Jardot, Priscilla; Monteil, Sonia; Campocasso, Angélique; Koonin, Eugene V; Raoult, Didier

    2012-10-30

    A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ~7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer. PMID:23071316

  8. Acanthamoeba keratitis in Tennessee: a growing problem in patients wearing contact lenses.

    PubMed

    Johns, K J; Parrish, C M; Seal, M R; Jerkins, G W; Berrie, W R; Litchford, D W; Sullivan, W R; Boone, J E; Elliott, J H; O'Day, D M

    1989-11-01

    All physicians evaluating a painful red eye in a contact lens wearer should consider the diagnosis of Acanthamoeba keratitis. Although it remains relatively rare, the incidence of this infection is on the rise. Clues to the correct diagnosis include a corneal abrasion that fails to heal appropriately, pain out of proportion to the clinical findings, and a history of poor contact lens hygiene. Prompt recognition and appropriate ophthalmologic intervention can improve the visual outcome for patients with this devastating corneal infection. PMID:2622154

  9. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    PubMed Central

    Sissons, James; Alsam, Selwa; Goldsworthy, Graham; Lightfoot, Mary; Jarroll, Edward L; Khan, Naveed Ahmed

    2006-01-01

    Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae. PMID:16672059

  10. Effect of Parachlamydia acanthamoebae on pulmonary function parameters in a bovine respiratory model.

    PubMed

    Lohr, M; Prohl, A; Ostermann, C; Diller, R; Greub, G; Reinhold, P

    2016-07-01

    The aim of this study was to evaluate pulmonary dysfunction induced by experimental infection with Parachlamydia acanthamoebae in calves. Intrabronchial inoculation with P. acanthamoebae was performed in 31 calves aged 2-3 months old at two different challenge doses of 10(8) and 10(10) inclusion-forming units (IFU) per animal. Control animals received heat inactivated bacteria. The effects on pulmonary gas exchange were determined by arterial blood gas analysis and haemoximetry during the 7 days post inoculation (DPI). For pulmonary function testing (PFT), impulse oscillometry, capnography, and measurement of O2 uptake were undertaken in spontaneously breathing animals 7 and 3 days before inoculation and were repeated until 10 DPI. In the early phase after challenge (1-3 DPI), mild hypoxaemia occurred, which was accompanied by a significant reduction in both tidal and alveolar volumes (each related to bodyweight, BW). In parallel, expiratory flow rate and specific ventilation (i.e. minute ventilation related to O2 uptake) were significantly increased. Minute and alveolar ventilations (each related to metabolic BW) increased significantly due to higher respiratory rates, lasting until 4 and 5 DPI, respectively. Oxygen uptake was slightly reduced during the first 2 days after challenge, but increased significantly during the recovery phase, from 4 to 8 DPI. No deterioration in respiratory mechanics or acid-base balance was observed. Respiratory infection with 10(10) IFU P. acanthamoebae per calf induced mild respiratory dysfunction, mainly characterised by hypoxaemia. The study's findings do not indicate severe pathophysiological consequences of P. acanthamoebae infection on pulmonary function in the bovine host. PMID:27240907

  11. Experimental infection of T4 Acanthamoeba genotype determines the pathogenic potential.

    PubMed

    Alves, Daniella de Sousa Mendes Moreira; Moraes, Aline Silva; Alves, Luciano Moreira; Gurgel-Gonçalves, Rodrigo; Lino Junior, Ruy de Souza; Cuba-Cuba, César Augusto; Vinaud, Marina Clare

    2016-09-01

    T4 is the Acanthamoeba genotype most related to cases of granulomatous amoebic encephalitis (GAE) in immunocompromised patients and of keratitis in contact lens wearers. The determination of the pathogenic potential of Acanthamoeba clinical and environmental isolates using experimental models is extremely important to elucidate the capacity of free-living organisms to establish and cause disease in hosts. The aim of this study was to compare and evaluate the histopathology and culture between two different routes of experimental infection of T4 Acanthamoeba isolated from environmental and clinical source in mice (intracranial and intraperitoneal). Swiss isogenic healthy mice were inoculated with 10(4) trophozoites by intracranial (IC) and intraperitoneal (IP) routes and observed during 21 days. The brains from animals inoculated by the IC route were collected and from the animals of the IP inoculation group, the brains, livers, kidneys, spleens, and lungs were removed. The organs were prepared and appropriately divided to be evaluated with histopathology and culture. There was no significant difference between the inoculation routes in terms of isolates recovery (χ(2) = 0.09; p = 0.76). In the IC group, isolate recovery rate was significantly higher in histopathology than the one achieved by culture (χ(2) = 6.45; p < 0.01). Experimental infection revealed that all isolates inoculated could be considered invasive because it was possible to recover evolutive forms of Acanthamoeba in both routes. This work represents the first in vivo pathogenicity assay of primary isolation source in Central region of Brazil showing in vivo pathogenicity and hematogenous spread capacity of these protozoa, improving the knowledge on free-living amoebae isolates. PMID:27164833

  12. Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water.

    PubMed

    Shoff, M E; Rogerson, A; Kessler, K; Schatz, S; Seal, D V

    2008-03-01

    The purpose was to identify the prevalence of naked amoebae in tap water in south Florida to ascertain the risk of amoebal infections of the cornea in contact lens wearers. Over the course of a 2-year period, water samples were collected from sites throughout Broward, Palm Beach, and Dade counties, Florida. The presence of amoebae in samples was based on an enrichment cultivation method appropriate for Acanthamoeba. Amoebae were identified using diagnostic features discernable by light microscopy. A total of 283 water samples were processed and amoebae were noted in 80 of these. Acanthamoeba were found on 8 occasions (2.8%). The genera Hartmannella and Vahlkampfia, rarely involved in keratitis cases, were found in 3.5% and 2.8% of samples, respectively. A total of 19 different naked amoebae were recorded and amoebae (regardless of genus) were present in 19.4% of all samples. Previous surveys in England and Korea have shown that acanthamoebae are found in 15 to 30% of tap water samples in the home and have been associated with corneal infection in contact lens wearers. The incidence of acanthamoebae infection in the USA (2.8%) has been found to be lower than that in the UK and it has been postulated that this is related to the lack of a storage water tank in the roof loft space. However, the level of treatment of municipal water is clearly not effective at killing amoebal cysts (or trophozoites) as evidenced by the high occurrence of amoebae (19.4%) in this study. PMID:17998610

  13. Anaerobic respiration: In vitro efficacy of Nitazoxanide against mitochondriate Acanthamoeba castellanii of the T4 genotype.

    PubMed

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Farooq, Maria; Khan, Naveed Ahmed

    2015-10-01

    Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba. PMID:26297676

  14. Prevalence of acanthamoeba from tap water in rio grande do Sul, Brazil.

    PubMed

    Winck, Mari Aline Todero; Caumo, Karin; Rott, Marilise Brittes

    2011-11-01

    A total of 136 samples of tap water were collected from state and municipal schools between March and November 2009. The samples were filtered through cellulose nitrate membranes that were seeded at non-nutrient agar 1.5% containing an overlayer of Escherichia coli suspension. Thirty-one (22.79%) tap water samples investigated were found positive for free-living amoebae (FLA). From these, 13 presented as FLA that seems to belong to the genus Acanthamoeba. All samples of FLA were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites and by PCR using genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Physiological tests of thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. The sequencing analysis by comparing the sequences submitted to GenBank, showed genotype distribution into groups T2, T2/T6, T6, and T4. In tests of thermotolerance and osmotolerance, 50% of the isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in Rio Grande do Sul, Brazil, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential. PMID:21882008

  15. Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

    PubMed Central

    Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E.; Valladares, Basilio; Maciver, Sutherland K.

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

  16. Detection of Acanthamoeba on the ocular surface in a Spanish population using the Schirmer strip test: pathogenic potential, molecular classification and evaluation of the sensitivity to chlorhexidine and voriconazole of the isolated Acanthamoeba strains.

    PubMed

    Rocha-Cabrera, Pedro; Reyes-Batlle, María; Martín-Navarro, Carmen María; Dorta-Gorrín, Alexis; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Piñero, José E; Martín-Barrera, Fernando; Valladares, Basilio; Lorenzo-Morales, Jacob

    2015-08-01

    Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis, which is often associated with the misuse of contact lenses. However, there is still a question remaining to be answered, which is whether these micro-organisms are present on the ocular surface of healthy individuals. Therefore, the aim of this study was to determine the presence of Acanthamoeba on the ocular surface in healthy patients and also in those with other ocular surface infections. Sterile Schirmer test strips were used to collect samples from a group of patients who attended an ophthalmology consultation at the Hospital del Norte, Icod de los Vinos, Tenerife, Canary Islands. Most of the patients (46 individuals, 79.31  %) presented ocular surface pathologies such as blepharitis or conjunctivitis; the rest did not present any pathology. None of the patients included in the study wore contact lenses. The collected samples were cultured in 2  % non-nutrient agar plates and positive plates were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains as belonging to Acanthamoeba genotype tbl4, and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. Furthermore, all strains were assayed for sensitivity against voriconazole and chlorhexidine. Assays showed that both drugs were active against the tested strains. In conclusion, the Schirmer strip test is proposed as an effective tool for the detection of Acanthamoeba on the ocular surface. PMID:26293786

  17. Synthesis, characterization and amoebicidal potential of locally synthesized TiO2 nanoparticles against pathogenic Acanthamoeba trophozoites in vitro.

    PubMed

    Imran, Muhammad; Muazzam, Ambreen Gul; Habib, Amir; Matin, Abdul

    2016-06-01

    Acanthamoeba is an opportunistic protozoan pathogen that plays a pivotal role in the ecosystem. It may cause blinding keratitis and fatal encephalitis involving the central nervous system. Here we synthesized pure and Zn doped TiO2 nanoparticles (~10-30nm) via sol-gel and sol-hydrothermal methods and demonstrated its impact on the biological characteristics of pathogenic Acanthamoeba castellanii. Our results revealed that pure and Zn doped TiO2 nanoparticles synthesized by sol-hydrothermal methods (ranging 5, 10, 25 and 50μg/ml) exhibited amoebicidal effects i.e., >60% of trophozoites executed under normal light at maximum dose (50μg/ml) within 1h incubation. In contrast pure/doped TiO2 obtained via sol gel method showed ~40% amoeba damage. Furthermore, amoebae growth assay demonstrated that Zn doped TiO2 also inhibited Acanthamoeba numbers up to 7days in dose dependent manner. It was interesting to note that all the tested TiO2 nanoparticles have shown maximum amoebicidal effects at pH7 which is quite relevant to amoebic growth favorable conditions. Our results confirmed that TiO2 has inhibitory effects on Acanthamoeba growth and viability. Overall, we reported the amoebicidal and amoebic growth inhibition potential of pure and Zn doped TiO2 nanoparticles against Acanthamoeba due to attached OH(-) groups, reduced size and decreased band gap of sol hydrothermally synthesized TiO2 nanoparticles. PMID:27054875

  18. Characterization of a New Pathogenic Acanthamoeba Species, A. byersi n. sp., Isolated from a Human with Fatal Amoebic Encephalitis

    PubMed Central

    Qvarnstrom, Yvonne; Nerad, Thomas A.; Visvesvara, Govinda S.

    2015-01-01

    Acanthamoeba spp. are free-living amoebae that are ubiquitous in natural environments. They can cause cutaneous, nasopharyngeal and disseminated infection, leading to granulomatous amebic encephalitis (GAE) in immunocompromised individuals. In addition, they can cause amoebic keratitis in contact lens wearers. Acanthamoeba GAE is almost always fatal because of difficulty and delay in diagnosis and lack of optimal antimicrobial therapy. Here we report the description of an unusual strain isolated from skin and brain of a GAE patient. The amoebae displayed large trophozoites and star-shaped cysts, characteristics for acanthamoebas belonging to morphology Group 1. However, its unique morphology and growth characteristics differentiated this new strain from other Group 1 species. DNA sequence analysis, secondary structure prediction and phylogenetic analysis of the 18S rRNA gene confirmed that this new strain belonged to Group 1 but that it was distinct from the other sequence types within that group. Thus, we hereby propose the establishment of a new species, Acanthamoeba byersi n. sp. as well as a new sequence type, T18, for this new strain. To our knowledge, this is the first report of a Group 1 Acanthamoeba that is indisputably pathogenic in humans. PMID:23879685

  19. High occurrence of Acanthamoeba genotype T4 in soil sources from Bolívar State, Venezuela.

    PubMed

    Wagner, Carolina; Reyes-Batlle, María; Hernán, Aurora; Rojas, Elsy; Pérez, Gladymar; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2016-09-01

    Pathogenic strains of Acanthamoeba are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, twenty-seven soil samples were collected in the Bolivar State in Venezuela and checked for the presence of Acanthamoeba. Samples were cultivated onto 2% non-nutrient agar plates seeded with a layer of heat killed E. coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 51.8% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotype T4 in all samples. To the best of our knowledge, this is the first report of genotype T4 in soil sources from Venezuela. Further studies should be carried out in this State and in the country in order to determine the current occurrence of Acanthamoeba in Venezuelan environments. PMID:27447209

  20. Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts.

    PubMed

    Abjani, Farhat; Khan, Naveed Ahmed; Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah

    2016-06-01

    Acanthamoeba cysts are highly resistant to contact lens disinfecting solutions. Acanthamoeba cyst wall is partially made of 1,4 β-glucan (i.e., cellulose) and other complex polysaccharides making it a hardy shell that protects the resident amoeba. Here, we hypothesize that targeting the cyst wall structure in addition to antiamoebic compound would improve the efficacy of marketed contact lens disinfecting solutions. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents abolished viability of Acanthamoeba castellanii cysts and trophozoites. When tested alone, none of the agents nor contact lens disinfecting solutions completely destroyed A. castellanii cysts and trophozoites. The absence of cyst wall-degrading enzymes in marketed contact lens disinfecting solutions render them ineffective against Acanthamoeba cysts. It is concluded that the addition of cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy in decreasing the incidence of Acanthamoeba effectively. PMID:26675112

  1. Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections

    PubMed Central

    Lorenzo-Morales, Jacob; Machin, Rubén P.; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K.; Valladares, Basilio; Piñero, José E.

    2013-01-01

    Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease. PMID:23114753

  2. Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan (Tragopan temminckii).

    PubMed

    Visvesvara, Govinda S; Shoff, Megan E; Sriram, Rama; Booton, Gregory C; Crary, Monica; Fuerst, Paul A; Hanley, Christopher S; Garner, Michael M

    2010-06-24

    Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) (Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene. PMID:20347228

  3. Isolation and molecular characterization of Acanthamoeba strains isolated from the oral cavity of immunosuppressed individuals in Tehran, Iran.

    PubMed

    Memari, Fatemeh; Niyyati, Maryam; Lorenzo-Morales, Jacob; Jonaydi, Zaynab

    2016-09-01

    Acanthamoeba spp. is an opportunistic protozoan parasite which is the causative agent of granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). GAE usually occurs in immunocompromised patients which in most cases is fatal. The present study was conducted to determine the genotypes of Acanthamoeba isolated from patients with compromised immunological status. For this purpose, 90 samples from the oral cavity of these individuals were collected in different hospitals of Tehran, Iran using sterile cotton swabs. Samples were cultured in 2% Non-Nutrient Agar (NNA) plates in order to check for the presence of amoebae. Identification of isolates was carried out using both morphological and molecular tools. The pathogenic potential of the obtained strains was assessed by performing osmo- and thermotolerance assays as previously described. Genotyping of the isolates was carried out by PCR/sequencing of the DF3 region of the 18S rDNA gene of Acanthamoeba. From the 90 collected samples, 11 (13.4%) were positive for Acanthamoeba genus. Molecular analysis revealed the presence of genotypes T3, T4 and T11, although most of the isolates belonged to genotype T4. Only 3 of the isolates genotyped as T4 were positive for the pathogenic potential assays. To this end if the immunological status is considered as one of the key factors for the development of GAE due to Acanthamoeba in the previous reported cases, individuals suffering from the conditions mentioned in this study should be considered as a high risk group of population in Iran and worldwide. PMID:27447206

  4. A Brazilian Marseillevirus Is the Founding Member of a Lineage in Family Marseilleviridae

    PubMed Central

    Dornas, Fábio P.; Assis, Felipe L.; Aherfi, Sarah; Arantes, Thalita; Abrahão, Jônatas S.; Colson, Philippe; La Scola, Bernard

    2016-01-01

    In 2003, Acanthamoeba polyphaga mimivirus (APMV) was discovered as parasitizing Acanthamoeba. It was revealed to exhibit remarkable features, especially odd genomic characteristics, and founded viral family Mimiviridae. Subsequently, a second family of giant amoebal viruses was described, Marseilleviridae, whose prototype member is Marseillevirus, discovered in 2009. Currently, the genomes of seven different members of this family have been fully sequenced. Previous phylogenetic analysis suggested the existence of three Marseilleviridae lineages: A, B and C. Here, we describe a new member of this family, Brazilian Marseillevirus (BrMV), which was isolated from a Brazilian sample and whose genome was fully sequenced and analyzed. Surprisingly, data from phylogenetic analyses and comparative genomics, including mean amino acid identity between BrMV and other Marseilleviridae members and the analyses of the core genome and pan-genome of marseilleviruses, indicated that this virus can be assigned to a new Marseilleviridae lineage. Even if the BrMV genome is one of the smallest among Marseilleviridae members, it harbors the second largest gene content into this family. In addition, the BrMV genome encodes 29 ORFans. Here, we describe the isolation and genome analyses of the BrMV strain, and propose its classification as the prototype virus of a new lineage D within the family Marseilleviridae. PMID:26978387

  5. Acanthamoeba encystment: multifactorial effects of buffers, biocides, and demulcents present in contact lens care solutions

    PubMed Central

    Kovacs, Christopher J; Lynch, Shawn C; Rah, Marjorie J; Millard, Kimberly A; Morris, Timothy W

    2015-01-01

    Purpose To determine whether agents which are purportedly capable of inducing encystment of Acanthamoeba can recapitulate the signal when tested in differing formulations. Methods In accordance with the International Standard ISO 19045, Acanthamoeba castellanii ATCC 50370 trophozoites were cultured in antibiotic-free axenic medium, treated with test solutions, and encystment rates plus viability were measured via bright field and fluorescent microscopy. Test solutions included phosphate-buffered saline (PBS), borate-buffered saline, biguanide- and hydrogen peroxide (H2O2)-based biocides, propylene glycol (PG) and povidone (POV) ophthalmic demulcents, and one-step H2O2-based contact lens disinfection systems. Results Only PBS solutions with 0.25 ppm polyaminopropyl biguanide (PAPB) and increasing concentrations of PG and POV stimulated A. castellanii encystment in a dose-dependent manner, whereas PBS solutions containing 3% H2O2 and increasing concentrations of PG and POV did not stimulate encystment. Borate-buffered saline and PBS/citrate solutions containing PG also did not stimulate encystment. In addition, no encystment was observed after 24 hours, 7 days, or 14 days of exposures of trophozoites to one-step H2O2 contact lens disinfection products or related solutions. Conclusion The lack of any encystment observed when trophozoites were treated with existing or new one-step H2O2 contact lens care products, as well as when trophozoites were exposed to various related test solutions, confirms that Acanthamoeba encystment is a complex process which depends upon simultaneous contributions of multiple factors including buffers, biocides, and demulcents. PMID:26508829

  6. Meningoencephalitis due to Acanthamoeba SP. Pathogenesis and clinico-pathological study.

    PubMed

    Martínez, A J; Sotelo-Avila, C; Garcia-Tamayo, J; Morón, J T; Willaert, E; Stamm, W P

    1977-03-31

    Amebic Meningoencephalitis (AM) and Primary Amebic Meningoencephalitis (PAM) are infectious diseases essentially confined to the Central Nervous System (CNS) and caused by free-living amebas of the genus Acanthamoeba (A.) and Naegleria (N.) respectively. AM due to A. sp. (Acanthamoeba castellanii and Acanthamoeba culbertsoni) have been reported in chronically ill debilitated individuals, some of them under immunosuppressive therapy, or in immunologically impaired patients without a history of recent swimming in contrast to cases due to N. sp. which usually occurs in healthy, young individuals with a recent history of swimming in man-made lakes or heated swimming pools. AM due to A.sp. is characterized by a subacute or chronic granulomatous meningoencephalitis involving mainly the midbrain, basal areas of the temporal and occipital lobes and posterior fossa structures. CNS lesions in AM are perhaps secondary and the portal of entry in humans is probably from the lower respiratory tract, genitourinary system or skin reaching the CNS by hematogenous spread. The predominant host reaction is usually composed of lymphocytes, plasma cells, monocytes and multinucleated foreign body giant cells. Necrosis is moderate and hemorrhage scant or absent. Cysts as well as trophozoites may be seen within the CNS lesions. PAM is due to Naegleria fowleri and is characterized by an hemorrhagic necrotizing meningoencephalities with an acute inflammatory response. Only trophozoites are found in lesions. The portal of entry is through the olfactory neuroepithelium. CNS tissues fixed in formalin may be used for further identification and taxonomical classification of the causative protoza using immunofluorescent antibody techniques (IFAT) and electron microscopic methods. PMID:857580

  7. A novel antiamoebic agent against Acanthamoeba sp. - A causative agent for eye keratitis infection

    NASA Astrophysics Data System (ADS)

    Kusrini, Eny; Hashim, Fatimah; Azmi, Wan Nor Nadhirah Wan Noor; Amin, Nakisah Mat; Estuningtyas, Ari

    2016-01-01

    The terbium trinitrate.trihydrate.18-crown ether-6, Tb(NO3)3(OH2)3.(18C6) complex has been characterized by elemental analysis, photoluminescence and single X-ray diffraction. The IC50 values were determined based on MTT assay while light and fluorescence microscopy imaging were employed to evaluate the cellular morphological changes. Alkaline comet assay was performed to analyze the DNA damage. The photoluminescence spectrum of the Tb complex excited at 325 nm displayed seven luminescence peaks corresponding to the 5D4 → 7F0, 1, 2, 3, 4, 5, 6 transitions. The cytotoxicity and genotoxicity studies indicated that the Tb(NO3)3(OH2)3.(18C6) complex and its salt form as well as the 18C6 molecule have excellent anti-amoebic activity with very low IC50 values are 7, 2.6 and 1.2 μg/mL, respectively, with significant decrease (p < 0.05) in Acanthamoeba viability when the concentration was increased from 0 to 30 μg/mL. The mode of cell death in Acanthamoeba cells following treatment with the Tb complex was apoptosis. This is in contrast to the Tb(NO3)3.6H2O salt- and 18C6 molecule-treated Acanthamoeba, which exhibited necrotic type cells. The percentage of DNA damage following treatment with all the compounds at the IC25 values showed high percentage of type 1 with the % nuclei damage are 14.15 ± 2.4; 46.00 ± 4.2; 36.36 ± 2.4; 45.16 ± 0.6%, respectively for untreated, treated with Tb complex, Tb salt and 18C6 molecule. The work features promising potential of Tb(NO3)3(OH2)3.(18C6) complex as anti-amoebic agent, representing a therapeutic option for Acanthamoeba keratitis infection.

  8. A novel antiamoebic agent against Acanthamoeba sp.--A causative agent for eye keratitis infection.

    PubMed

    Kusrini, Eny; Hashim, Fatimah; Azmi, Wan Nor Nadhirah Wan Noor; Amin, Nakisah Mat; Estuningtyas, Ari

    2016-01-15

    The terbium trinitrate.trihydrate.18-crown ether-6, Tb(NO3)3(OH2)3.(18C6) complex has been characterized by elemental analysis, photoluminescence and single X-ray diffraction. The IC50 values were determined based on MTT assay while light and fluorescence microscopy imaging were employed to evaluate the cellular morphological changes. Alkaline comet assay was performed to analyze the DNA damage. The photoluminescence spectrum of the Tb complex excited at 325 nm displayed seven luminescence peaks corresponding to the (5)D4→(7)F(0, 1, 2, 3, 4, 5, 6) transitions. The cytotoxicity and genotoxicity studies indicated that the Tb(NO3)3(OH2)3.(18C6) complex and its salt form as well as the 18C6 molecule have excellent anti-amoebic activity with very low IC50 values are 7, 2.6 and 1.2 μg/mL, respectively, with significant decrease (p<0.05) in Acanthamoeba viability when the concentration was increased from 0 to 30 μg/mL. The mode of cell death in Acanthamoeba cells following treatment with the Tb complex was apoptosis. This is in contrast to the Tb(NO3)3.6H2O salt- and 18C6 molecule-treated Acanthamoeba, which exhibited necrotic type cells. The percentage of DNA damage following treatment with all the compounds at the IC25 values showed high percentage of type 1 with the % nuclei damage are 14.15±2.4; 46.00±4.2; 36.36±2.4; 45.16±0.6%, respectively for untreated, treated with Tb complex, Tb salt and 18C6 molecule. The work features promising potential of Tb(NO3)3(OH2)3.(18C6) complex as anti-amoebic agent, representing a therapeutic option for Acanthamoeba keratitis infection. PMID:26474244

  9. Isolation and genotyping of Acanthamoeba strains (T4, T9, and T11) from amoebic keratitis patients in Iran.

    PubMed

    Hajialilo, Elham; Behnia, Massoud; Tarighi, Fatemeh; Niyyati, Maryam; Rezaeian, Mostafa

    2016-08-01

    The continuous increase in Acanthamoeba keratitis, a severe corneal infection, worldwide is mainly due to the increase in the number of soft contact lens users. In the present study, which involves a 5-year study, a total of 138 corneal scraps and contact lenses together with their paraphernalias were obtained from suspected amoebic keratitis patients. All samples were cultured using culture-enrichment method. Pathogenic assay, using thermotolerance and osmotolerance tests were also performed on the positive strains. Sequencing of the isolated strains was done by targeting the DF3 region of 18s rRNA gene. The results revealed that 18 (13 %) of patients were infected with Acanthamoeba spp. As expected, T4 genotype was the most common genotype among the clinical samples; however, in three cases, Acanthamoeba belonging to T11 and T9 were detected. Interestingly, T9 genotype, commonly classified as non-pathogenic amoebae, was identified as a causal agent of a patient with amoebic keratitis. From the pathogenic assay, four strains belonging to T4 genotypes were highly pathogenic. This is the first report of Acanthamoeba T9 genotypes isolated in Iran and the first report of T9 type occurring in amoebic keratitis patients worldwide. Due to the increasing trend of amoebic keratitis (AK) and the identification of new genotypes, such as T9 as the causative agent of AK, more researches in this field are necessary in the region and the world at large. PMID:27102637

  10. Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

    PubMed Central

    Moon, Eun-Kyung; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung; Kong, Hyun-Hee

    2016-01-01

    Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa. PMID:27180570

  11. Temperature limitation may explain the containment of the trophozoites in the cornea during Acanthamoeba castellanii keratitis.

    PubMed

    Nielsen, Mattias Kiel; Nielsen, Kim; Hjortdal, Jesper; Sørensen, Uffe B Skov

    2014-12-01

    Acanthamoeba keratitis is a serious sight-threatening disease. The relatively low temperature of the cornea may explain why amoebic infections usually are localized in this tissue and rarely spread to other parts of the eye. In this study, the growth rate of the amoeba Acanthamoeba castellanii was examined at different temperatures. The aim was to establish the optimal growth temperature for A. castellanii and to examine the growth within the vicinity of the core body temperature. The growth rates of four clinical and two environmental strains of A. castellanii were estimated at different temperatures, and temperature limitations for the trophozoite stage was established. Movements influenced by temperature gradients were monitored for two clinical strains of A. castellanii. The highest growth rate for each of the six amoebic strains tested was found to be close to 32 °C. The growth of the trophozoites of all examined strains was greatly reduced or completely halted at temperatures above 36 °C and encysted at the elevated temperature. Thus, the optimal growth temperature for the four strains of A. castellanii is close to the surface temperature of the human cornea, while the higher body core-temperature induced encysting of the amoebae. This may explain why most amoebic eye infections are confined to the cornea. PMID:25204727

  12. Glycogen Phosphorylase in Acanthamoeba spp.: Determining the Role of the Enzyme during the Encystment Process Using RNA Interference▿

    PubMed Central

    Lorenzo-Morales, Jacob; Kliescikova, Jarmila; Martinez-Carretero, Enrique; De Pablos, Luis Miguel; Profotova, Bronislava; Nohynkova, Eva; Osuna, Antonio; Valladares, Basilio

    2008-01-01

    Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer. PMID:18223117

  13. Occurrence and molecular characterization of free-living amoeba species (Acanthamoeba, Hartmannella, and Saccamoeba limax) in various surface water resources of Iran.

    PubMed

    Mahmoudi, Mohammad Reza; Rahmati, Behnaz; Seyedpour, Seyed Hosssen; Karanis, Panagiotis

    2015-12-01

    This study was conducted to determine the presence and molecular identity of Acanthamoeba species in the surface water resources of four provinces in Iran, namely Guilan, Mazandaran (North of Iran), Alborz, and Tehran (capital city), using culture- and molecular-based methods. During March to November 2014, 49 surface water samples were collected from environmental water sources-the distinct surface waters of Guilan, Mazandaran, Alborz, and Tehran provinces, in Iran. For the isolation of Acanthamoeba species, approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 μ. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The presence of Acanthamoeba was confirmed by the genus-specific primer pair JDP1 and 2, and/or NA primers were used to identify Acanthamoeba and certain other free-living amoebae. In total, 38 out of 49 samples were positive by culture and/or PCR for Acanthamoeba and other free-living amoebae from all three provinces. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba (16 isolates belonged to T4 and 2 isolates belonged to T5), Hartmannella vermiformis (3/24), and Saccamoeba limax (2/24). The T4 and T5 genotypes were detected in Guilan and Mazandaran provinces. Two isolates from Guilan and Tehran provinces belonged to S. limax, and H. vermiformis was detected in Guilan province. The results of this study highlight the need to pay more attention to free-living amoebae, as human activity was observed in all of the localities from which these samples were taken. These surface waters can be potential sources for the distribution and transmission of pathogenic Acanthamoeba in the study areas, and free-living amoebas (FLA) (particularly the Acanthamoeba species) can serve as hosts for and vehicles of various microorganisms. PMID:26346454

  14. The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

    PubMed Central

    2012-01-01

    Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine). Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells. Results Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine) but not its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d] pyrimidine), had detrimental effects on the growth of A. castellanii (keratitis isolate, belonging to the T4 genotype). Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier. Conclusions For the first

  15. Oxygen induces fatty acid (n-6)-desaturation independently of temperature in Acanthamoeba castellanii.

    PubMed

    Thomas, K; Rutter, A; Suller, M; Harwood, J; Lloyd, D

    1998-03-20

    Induction of a microsomal oleate delta12 (n-6) desaturase which is mainly responsible for an increase in membrane lipid unsaturation at low temperature has been observed in the free-living amoeba Acanthamoeba castellanii. In this study we show that the enzyme can also be regulated by oxygen independently of temperature in batch cultures grown to O2-limitation. Raising the oxygen concentration from below the lower limit of detection (< 0.1 microM) to approximately air-saturation (230 microM), whilst maintaining the growth temperature constant (30 degrees C), increased lipid unsaturation and elevated n-6-desaturase activity 2.3-fold. Addition of the protein synthesis inhibitor, anisomycin, showed that increased desaturase activity was due to new protein synthesis rather than activation of pre-existing enzyme. These observations are important for future studies of the mechanism of temperature adaptation in poikilotherms. PMID:9541030

  16. Rapid induction of microsomal delta 12(omega 6)-desaturase activity in chilled Acanthamoeba castellanii.

    PubMed

    Jones, A L; Lloyd, D; Harwood, J L

    1993-11-15

    The activity of microsomal delta 12-desaturase in Acanthamoeba castellanii was increased after growing cultures were chilled from the optimal growth temperature (30 degrees C) to 15 degrees C. This increase was detectable in microsomes isolated from organisms subjected to only 10 min chilling. The mechanism of induction was investigated. The increase in activity on chilling was greatly reduced when protein synthesis was blocked before the temperature shift. Thus the major mechanism for the induction of delta 12-desaturase is increased protein synthesis. delta 12-Desaturase activity was higher when assayed at 20 degrees C than when assayed at 30 degrees C, but these changes were not due to the increased solubility of O2 at 20 degrees C. The major substrate of delta 12-desaturase was found to be 1-acyl-2-oleoyl phosphatidylcholine. PMID:8250841

  17. Acanthamoeba castellanii: proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation.

    PubMed

    Bouyer, Sabrina; Rodier, Marie-Hélène; Guillot, Alain; Héchard, Yann

    2009-09-01

    Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis. PMID:19523468

  18. An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii.

    PubMed

    Van der Henst, Charles; Scrignari, Tiziana; Maclachlan, Catherine; Blokesch, Melanie

    2016-04-01

    Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host-pathogen interaction. PMID:26394005

  19. Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment.

    PubMed

    Seal, D V

    2003-11-01

    A reliable figure for the expected incidence of Acanthamoeba keratitis of one per 30000 contact lens wearers per year has now been obtained from a combination of three cohort and three Questionnaire Reporting Surveys; 88% of cases wore hydrogel lenses and 12% wore rigid lenses. This figure now provides a basis for the expected number of cases against which to judge either epidemic outbreaks or effects of prevention with disinfecting solutions, better hygiene, or the use of disposable lenses. Molecular biology of Acanthamoeba has advanced considerably in the last 10 years with new automated sequencing technology. This has allowed the construction of a genotype identification scheme with 13 different genotypes against which to compare clinical isolates for epidemiological investigations or pathogenicity markers. So far, only four genotypes have been associated with keratitis of which the majority have been T4 but T3, T6, and T11 have each caused individual cases. Each genotype is heterogenous and can be further subdivided by comparison of sequences of diagnostic fragments of 18S rDNA, riboprinting by PCR-RFLP of 18S rDNA, or by mitochondrial DNA RFLP. Drug therapy has been revolutionised with the introduction of the biguanides-chlorhexidine or polyhexamethylene biguanide-with most but not all infections quickly resolving. Failure can still occur occasionally and further research is needed on more effective combination chemotherapy. A number of guanidines have been identified in this paper that could be usefully pursued as part of combination chemotherapy along with the alkylphosphocholines. PMID:14631394

  20. Resistance of Acanthamoeba cysts to disinfection treatments used in health care settings.

    PubMed

    Coulon, Céline; Collignon, Anne; McDonnell, Gerald; Thomas, Vincent

    2010-08-01

    Free-living amoebae that belong to the genus Acanthamoeba are widespread in the environment, including water. They are responsible for human infections and can host pathogenic microorganisms. Under unfavorable conditions, they form cysts with high levels of resistance to disinfection methods, thus potentially representing a threat to public health. In the present study we evaluated the efficacies of various biocides against trophozoites and cysts of several Acanthamoeba strains. We demonstrated that disinfectant efficacy varied depending on the strains tested, with environmental strains demonstrating greater resistance than collection strains. Trophozoites were inactivated by all treatments except those using glutaraldehyde as an active compound: for these treatments, we observed resistance even after 30 min exposure. Cysts resisted many treatments, including certain conditions with glutaraldehyde and other biocides. Moist heat at 55 degrees C was not efficient against cysts, whereas exposure at 65 degrees C was. Several chemical formulations containing peracetic acid, hydrogen peroxide, or ortho-phthalaldehyde presented greater efficacy than glutaraldehyde, as did ethanol and sodium hypochlorite; however, some of these treatments required relatively long incubation times to achieve cyst inactivation. Amoebal cysts can be highly resistant to some high-level disinfectants, which has implications for clinical practice. These results highlight the need to consider the effective disinfection of protozoa in their vegetative and resistant forms due to their intrinsic resistance. This is important not only to prevent the transmission of protozoa themselves but also due to the risks associated with a range of microbial pathogens that are found to be associated intracellularly with these microorganisms. PMID:20519477

  1. Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks.

    PubMed

    Hamilton, K A; Ahmed, W; Palmer, A; Sidhu, J P S; Hodgers, L; Toze, S; Haas, C N

    2016-10-01

    A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendall's tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data. PMID:27336236

  2. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  3. Role of phospholipase A₂ (PLA₂) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis.

    PubMed

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-08-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P < 0.05). Additionally, cPLA2α inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P < 0.05). Subconjunctival injection of purified MIP-133 in Chinese hamster eyes induced cytopathic effects resulting in corneal ulceration. Animals infected with A. castellanii-laden contact lenses and treated with AACOCF3 and CAY10650, showed significantly less severe keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. PMID:23792108

  4. Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain.

    PubMed

    Huang, Jian-Ming; Lin, Wei-Chen; Li, Sung-Chou; Shih, Min-Hsiu; Chan, Wen-Ching; Shin, Jyh-Wei; Huang, Fu-Chin

    2016-07-01

    Acanthamoeba keratitis (AK) is a serious ocular disease caused by pathogenic Acanthamoeba gaining entry through wounds in the corneal injury; generally, patients at risk for contracting AK wear contact lenses, usually over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequences: it makes the cornea turbid and difficult to operate on, including procedures such as enucleation of the eyeball. At present, diagnosis of this disease is not straightforward, and treatment is very demanding. We have established the comparative transcriptome and extracellular secreted proteomic database according to the non-pathogenic strain ATCC 30010 and the pathogenic strains NCKU_B and NCKU_D. We identified 44 secreted proteins successfully, 10 consensus secreted proteins and 34 strain-specific secreted proteins. These proteins may provide targets for therapy and immuno-diagnosis of Acanthamoeba infections. This study shows a suitable approach to identify secreted proteins in Acanthamoeba and provides new perspectives for the study of molecules potentially involved in the AK. PMID:26995533

  5. In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan (Ramphastos sulfuratus).

    PubMed

    Visvesvara, Govinda S; Booton, Gregory C; Kelley, Darryl J; Fuerst, Paul; Sriram, Rama; Finkelstein, Ariana; Garner, Michael M

    2007-01-19

    Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene. PMID:16962705

  6. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    PubMed Central

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria. PMID:27118300

  7. Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

    PubMed

    Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

    2012-11-01

    The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits. PMID:22709906

  8. Cellular response of the amoeba Acanthamoeba castellanii to chlorine, chlorine dioxide, and monochloramine treatments.

    PubMed

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-07-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii. PMID:21602398

  9. Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments ▿

    PubMed Central

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-01-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii. PMID:21602398

  10. Yersinia pseudotuberculosis IP32953 survives and replicates in trophozoites and persists in cysts of Acanthamoeba castellanii

    PubMed Central

    Santos-Montañez, Jennifer; Benavides-Montaño, Javier A.; Hinz, Angela K.; Vadyvaloo, Viveka

    2015-01-01

    Yersinia pseudotuberculosis is a foodborne enteric pathogen that causes a mild self-limiting diarrhea in humans. Yersinia pseudotuberculosis is able to persist in soil and water and in association with fresh produce, but the mechanism by which it persists is unknown. It has been shown that Y. pseudotuberculosis co-occurs with protozoans in these environments; therefore, this study investigates if bacterivorous free-living amoeba (FLA) are able to support persistence of Y. pseudotuberculosis. Coculture studies of Y. pseudotuberculosis and the prototype FLA, Acanthamoeba castellanii revealed that bacteria had an enhanced capacity to survive in association with amoeba and in the absence of any cytotoxic effects. Yersinia pseudotuberculosis is able to survive and replicate in trophozoites specifically localized within vacuoles, and persists within cysts over a period of at least a week. These data present the first evidence that Y. pseudotuberculosis is able to resist the bacterivorous nature of FLA and instead exhibits an enhanced ability to replicate and persist in coculture with amoeba. This study sheds light on the potential role of FLA in the ecology of Y. pseudotuberculosis which may have implications for food safety. PMID:26025069

  11. Increased Persistence of Salmonella enterica Serovar Typhi in the Presence of Acanthamoeba castellanii▿

    PubMed Central

    Douesnard-Malo, Frédéric; Daigle, France

    2011-01-01

    Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of the systemic disease typhoid fever. Transmission occurs via ingestion of contaminated food or water. S. Typhi is specific to humans, and no animal or environmental reservoirs are known. As the free-living amoeba Acanthamoeba castellanii is an environmental host for many pathogenic bacteria, this study investigates interactions between S. Typhi and A. castellanii by using cocultures. Growth of both organisms was estimated by cell count, viable count, flow cytometry, and fluorescence microscopy. Results indicate that S. Typhi can survive at least 3 weeks when grown with A. castellanii, as opposed to less than 10 days when grown as singly cultured bacteria under the same conditions. Interestingly, growth rates of amoebae after 14 days were similar in cocultures or when amoebae were singly cultured, suggesting that S. Typhi is not cytotoxic to A. castellanii. Bacteria surviving in coculture were not intracellular and did not require a physical contact with amoebae for their survival. These results suggest that S. Typhi may have a selective advantage when it is associated with A. castellanii and that amoebae may contribute to S. Typhi persistence in the environment. PMID:21926221

  12. Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

    PubMed

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

    2015-01-01

    Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. PMID:25041405

  13. Exploring the Unique N-Glycome of the Opportunistic Human Pathogen Acanthamoeba*

    PubMed Central

    Schiller, Birgit; Makrypidi, Georgia; Razzazi-Fazeli, Ebrahim; Paschinger, Katharina; Walochnik, Julia; Wilson, Iain B. H.

    2012-01-01

    Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex8–9HexNAc2Pnt0–1 tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets. PMID:23139421

  14. Acanthamoeba and other free-living amoebae in bat guano, an extreme habitat.

    PubMed

    Mulec, Janez; Dietersdorfer, Elisabeth; Üstüntürk-Onan, Miray; Walochnik, Julia

    2016-04-01

    Several representatives of the so-called free-living amoebae (FLA) are of medical relevance, not only as facultative pathogens but also as vehicles for pathogenic bacteria. Some FLA can survive and even grow under extreme environmental conditions. Bat guano is an exceptional habitat, the conditions becoming gradually more extreme with aging. In the current study, samples of bat guano of different ages from five caves in Slovenia were screened for the presence of FLA. FLA were isolated from almost all guano samples, including guano with a pH of 3.5. Only the two samples that had been drawn from >20-year-old guano were negative for FLA. Generally, FLA diversity correlated to high concentrations of cultivable bacteria (∼10(8) CFU/g) and fungi (∼10(5) CFU/g). Interestingly, the absence of FLA in seasoned guanos was mirrored by the presence of dictyostelid slime moulds. The isolated amoebae were identified as belonging to the genera Acanthamoeba, Copromyxa, Naegleria, Sappinia, Tetramitus, Thecamoeba, Vahlkampfia, Vannella and Vermamoeba. To the best of our knowledge, this is the first study on the diversity of FLA in guano. PMID:26678653

  15. Evidence for a Hydrogenosomal-Type Anaerobic ATP Generation Pathway in Acanthamoeba castellanii

    PubMed Central

    Gray, Michael W.; Roger, Andrew J.

    2013-01-01

    Diverse, distantly-related eukaryotic lineages have adapted to low-oxygen environments, and possess mitochondrion-related organelles that have lost the capacity to generate adenosine triphosphate (ATP) through oxidative phosphorylation. A subset of these organelles, hydrogenosomes, has acquired a set of characteristic ATP generation enzymes commonly found in anaerobic bacteria. The recipient of these enzymes could not have survived prior to their acquisition had it not still possessed the electron transport chain present in the ancestral mitochondrion. In the divergence of modern hydrogenosomes from mitochondria, a transitional organelle must therefore have existed that possessed both an electron transport chain and an anaerobic ATP generation pathway. Here, we report a modern analog of this organelle in the habitually aerobic opportunistic pathogen, Acanthamoeba castellanii. This organism possesses a complete set of enzymes comprising a hydrogenosome-like ATP generation pathway, each of which is predicted to be targeted to mitochondria. We have experimentally confirmed the mitochondrial localizations of key components of this pathway using tandem mass spectrometry. This evidence is the first supported by localization and proteome data of a mitochondrion possessing both an electron transport chain and hydrogenosome-like energy metabolism enzymes. Our work provides insight into the first steps that might have occurred in the course of the emergence of modern hydrogenosomes. PMID:24086244

  16. Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling

    PubMed Central

    2013-01-01

    Background The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Results Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host. PMID:23375108

  17. Apoptosis-like cell death induced by Salmonella in Acanthamoeba rhysodes.

    PubMed

    Feng, Ye; Hsiao, Yi-Hsing; Chen, Hsiu-Ling; Chu, Chishih; Tang, Petrus; Chiu, Cheng-Hsun

    2009-08-01

    Free-living amoebae act as environmental hosts of several intracellular pathogens. We examined the interaction between Acanthamoeba rhysodes and Salmonella, a human intracellular pathogen. There was no difference among three different serovars of Salmonella in terms of their growth within A. rhysodes over time. The number of intracellular bacteria increased at 6 h post-infection, and the viability of A. rhysodes was significantly reduced at 24 h post-infection. Amoebic cell death was characterized by TUNEL and Annexin V assay, without DNA ladder identified, indicating an apoptosis-like cell death in Salmonella-infected A. rhysodes. Global gene expression screening between intracellular and extracellular Salmonella by microarray and quantitative PCR showed that genes from Salmonella pathogenicity islands and virulence plasmid were up-regulated within A. rhysodes. The phase-dependent expression pattern suggests their distinct roles in the pathogenesis. A. rhysodes and Salmonella provide a model to study transient symbiosis between bacterial pathogens and protozoa in an aquatic ecosystem. PMID:19446019

  18. Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

    PubMed

    Rutter, Andrew J; Thomas, Katie L; Herbert, Derek; Henderson, R James; Lloyd, David; Harwood, John L

    2002-11-15

    Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Delta(10)-nonadecenoate showed that the enzyme is an n -6 desaturase rather than a Delta(12)-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b (5) as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b (5)) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n -6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n -6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms. PMID:12153399

  19. Giant virus with a remarkable complement of genes infects marine zooplankton

    PubMed Central

    Fischer, Matthias G.; Allen, Michael J.; Wilson, William H.; Suttle, Curtis A.

    2010-01-01

    As major consumers of heterotrophic bacteria and phytoplankton, microzooplankton are a critical link in aquatic foodwebs. Here, we show that a major marine microflagellate grazer is infected by a giant virus, Cafeteria roenbergensis virus (CroV), which has the largest genome of any described marine virus (≈730 kb of double-stranded DNA). The central 618-kb coding part of this AT-rich genome contains 544 predicted protein-coding genes; putative early and late promoter motifs have been detected and assigned to 191 and 72 of them, respectively, and at least 274 genes were expressed during infection. The diverse coding potential of CroV includes predicted translation factors, DNA repair enzymes such as DNA mismatch repair protein MutS and two photolyases, multiple ubiquitin pathway components, four intein elements, and 22 tRNAs. Many genes including isoleucyl-tRNA synthetase, eIF-2γ, and an Elp3-like histone acetyltransferase are usually not found in viruses. We also discovered a 38-kb genomic region of putative bacterial origin, which encodes several predicted carbohydrate metabolizing enzymes, including an entire pathway for the biosynthesis of 3-deoxy-d-manno-octulosonate, a key component of the outer membrane in Gram-negative bacteria. Phylogenetic analysis indicates that CroV is a nucleocytoplasmic large DNA virus, with Acanthamoeba polyphaga mimivirus as its closest relative, although less than one-third of the genes of CroV have homologs in Mimivirus. CroV is a highly complex marine virus and the only virus studied in genetic detail that infects one of the major groups of predators in the oceans. PMID:20974979

  20. Giant virus with a remarkable complement of genes infects marine zooplankton.

    PubMed

    Fischer, Matthias G; Allen, Michael J; Wilson, William H; Suttle, Curtis A

    2010-11-01

    As major consumers of heterotrophic bacteria and phytoplankton, microzooplankton are a critical link in aquatic foodwebs. Here, we show that a major marine microflagellate grazer is infected by a giant virus, Cafeteria roenbergensis virus (CroV), which has the largest genome of any described marine virus (≈730 kb of double-stranded DNA). The central 618-kb coding part of this AT-rich genome contains 544 predicted protein-coding genes; putative early and late promoter motifs have been detected and assigned to 191 and 72 of them, respectively, and at least 274 genes were expressed during infection. The diverse coding potential of CroV includes predicted translation factors, DNA repair enzymes such as DNA mismatch repair protein MutS and two photolyases, multiple ubiquitin pathway components, four intein elements, and 22 tRNAs. Many genes including isoleucyl-tRNA synthetase, eIF-2γ, and an Elp3-like histone acetyltransferase are usually not found in viruses. We also discovered a 38-kb genomic region of putative bacterial origin, which encodes several predicted carbohydrate metabolizing enzymes, including an entire pathway for the biosynthesis of 3-deoxy-d-manno-octulosonate, a key component of the outer membrane in Gram-negative bacteria. Phylogenetic analysis indicates that CroV is a nucleocytoplasmic large DNA virus, with Acanthamoeba polyphaga mimivirus as its closest relative, although less than one-third of the genes of CroV have homologs in Mimivirus. CroV is a highly complex marine virus and the only virus studied in genetic detail that infects one of the major groups of predators in the oceans. PMID:20974979

  1. Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging.

    PubMed

    Kolar, Satya Sree N; Manarang, Joseph C; Burns, Alan R; Miller, William L; McDermott, Alison M; Bergmanson, Jan P G

    2015-12-01

    In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba. PMID:26208952

  2. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    PubMed Central

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  3. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

    PubMed

    Khalil, Jacques Y B; Robert, Stephane; Reteno, Dorine G; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  4. Free-living Acanthamoeba and Naegleria spp. amebae in water sources of León, Nicaragua.

    PubMed

    Leiva, Byron; Clasdotter, Emma; Linder, Ewert; Winiecka-Krusnell, Jadwiga

    2008-06-01

    Free-living amebae (FLA) are known to occur worldwide in water-related biotopes, but only limited information is available on these organisms in developing countries and so far no information on their presence is available from Nicaragua. The aims of this study were to evaluate the prevalence of potentially pathogenic Acanthamoeba spp. and Naegleria spp. in different water sources to which the population of Le6n municipality is exposed. Since pathogenic amebae are thermotolerant, we were especially interested in the occurrence of FLA in geothermal areas. Water samples were collected from Le6n area in Nicaragua: 88 samples were from rivers and springs, 111 from wells, 74 from water taps and 21 from water tanks in urban and suburban Le6n and from three nearby geothermal areas of San Jacinto, Posoltega and Tipitapa. Amebae were identified using morphological and physiological criteria, immunohistochemical staining procedures and molecular methods. Indirect immunofluorescent test was performed on cysts and trophozoites fixed on microscopical slides and incubated for 30 min at room temperature in separate experiments with the following antibodies: rabbit-anti N fowleri/N lovanensis (Nf-Pab), mouse monoclonal antibody anti N. fowleri (Nf-5D12u), rabbit antibodies against Acanthamoeba spp. And fluorescent in situ hybridization (FISH) was performed using 18S rRNA-targeted fluorescent oligonucleotide probes. Probes: GSP for the detection of Acanthamoeba and NAEG1088 for the detection of Naegleria. Free-living amebae were recovered from approximately 43 % of the samples. Acanthamoeba spp was found in 21% of samples from León municipality and in 2% of samples from geothermal areas. Amoeboflagellates were found in 10 % of samples from Le6n and in 19% in geothermal areas. Fifty three percent of tested wells in the geothermal area contained thermotolerant amoeboflagellates. Naegleria spp. was identified in 24 out of 39 (61.5 %) of isolated amoeboflagellates. Twelve of them were

  5. An Acanthamoeba castellanii metacaspase associates with the contractile vacuole and functions in osmoregulation.

    PubMed

    Saheb, Entsar; Trzyna, Wendy; Bush, John

    2013-03-01

    Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection. PMID:23274641

  6. The effect of peptidic and non-peptidic proteasome inhibitors on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Siddiqui, Ruqaiyyah; Saleem, Sahreena; Khan, Naveed Ahmed

    2016-09-01

    The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections. PMID:27327524

  7. Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi.

    PubMed

    Hong, Yeon-Chul; Hwang, Mi-Yul; Yun, Ho-Cheol; Yu, Hak-Sun; Kong, Hyun-Hee; Yong, Tai-Soon; Chung, Dong-Il

    2002-03-01

    We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues. Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue. PMID:11949209

  8. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

    PubMed Central

    2013-01-01

    Background Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Methods Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. Results All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). Conclusions A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii

  9. Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis.

    PubMed

    Khairnar, Krishna; Tamber, Gurdip S; Ralevski, Filip; Pillai, Dylan R

    2011-08-01

    Acanthamoeba spp. are responsible for a significant annual number of keratitis (AK) cases leading to vision-threatening disease worldwide. Current methods rely on direct examination of specimens by microscopy and/or culture. The former lacks sensitivity and the latter suffers from a poor turnaround time. We undertook a comparison of all published molecular methods, evaluating performance characteristics such as analytical sensitivity, specificity, limit of detection (LOD), reproducibility, accuracy, and cost of test. The study population comprised 128 patients. Eligible specimens were tested prospectively between April 2007 and May 2010 by microscopy and/or culture. Eleven different specimen types were used including corneal scrapings (51.5%), corneal swab (17.9%), and contact lens material (10.9%). Results of 2 published gel-based polymerase chain reaction (PCR) and 2 published real-time quantitative (Q) PCR methods were compared in a blinded manner to direct microscopic examination and/or culture for the detection of Acanthamoeba in clinical specimens. QPCR (Riviere method) had the highest sensitivity at 89.3%, excellent accuracy using ROC analysis (AUC ∼0.90), lowest LOD down to 0.1 organism per microliter, and superior linear correlation with parasite density (R(2) = 0.9965) when compared with microscopy, culture, and other molecular methods. Phylogenetic analysis using a sequence-based typing method revealed that clinical isolates in this population with AK were genetically distinct from granulomatous amebic encephalitis or environmental isolates. The QPCR method was more expensive ($14.80) than traditional methods such as culture ($2.50) or microscopy ($2.50). However, 13 culture- and microscopy-negative specimens were positive by QPCR during the study period, suggesting that detection using QPCR may result in reduced complications and health care costs associated with misdiagnosed AK. PMID:21658877

  10. Selective Requirement of the Shikimate Pathway of Legionella pneumophila for Intravacuolar Growth within Human Macrophages but Not within Acanthamoeba

    PubMed Central

    Jones, Snake C.; Price, Christopher T. D.; Santic, Marina

    2015-01-01

    Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids. PMID:25847958

  11. Effect of non-steroidal anti-inflammatory drugs on biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Siddiqui, Ruqaiyyah; Lakhundi, Sahreena; Iqbal, Junaid; Khan, Naveed Ahmed

    2016-09-01

    Non-steroidal anti-inflammatory drug, Diclofenac, targeting COX have shown promise in the treatment of Acanthamoeba keratitis, but the underlying mechanisms remain unknown. Using various NSAIDs, Diclofenac sodium, Indomethacin, and Acetaminophen, here we determined the effects of NSAIDs on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype. Using amoebicidal assays, the results revealed that Diclofenac sodium, and Indomethacin affected growth of A. castellanii. In contrast, none of the compounds tested had any effect on the viability of A. castellanii. Importantly, all NSAIDs tested abolished A. castellanii encystation. This is a significant finding as the ability of amoebae to transform into the dormant cyst form presents a significant challenge in the successful treatment of infection. The NSAIDs inhibit production of cyclo-oxegenase, which regulates the synthesis of prostaglandins suggesting that cyclooxygenases (COX-1 and COX-2) and prostaglandins play significant role(s) in Acanthamoeba biology. As NSAIDs are routinely used in the clinical practice, these findings may help design improved preventative strategies and/or of therapeutic value to improve prognosis, when used in combination with other anti-amoebic drugs. PMID:27381503

  12. Efficacy of contact lens storage solutions against trophozoite and cyst of Acanthamoeba castellanii strain 1BU and their cytotoxic potential on corneal cells.

    PubMed

    Polat, Zubeyde Akin; Vural, Ayse; Cetin, Ali

    2007-09-01

    Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Contact lens wearers are most at risk and account for about 95% of cases. We investigated the in vitro effectiveness of 10 contact lens solutions against Acanthamoeba castellanii and their cytotoxicity on corneal cells in vitro. Contact lens solutions were evaluated for their amoebicidal activities using an inverted light microscope. To determine of their cytotoxicity on corneal cells, agar diffusion tests were performed. According to the results obtained from the tests, AVIZOR Aqua Soft Comfort and Elegance(R) showed the best amoebicidal effect on A. castellanii trophozoites. Cysts were still viable after overnight (8 h) exposure. In the case of the cyctotoxic acitivities, All In One Light, Astek, SOLO-Care Aqua, Maxima, and Horien showed no cytotoxicity on the corneal cells. ReNu MultiPlus, AVIZOR Aqua Soft Comfort, Carrera, and Elegance showed mild cytotoxicity on the corneal cells. Plurisol.M presented moderate cytotoxicity on the corneal cells. All commercial solutions examined in this study are the lack of efficacy against A. castellanii. Improvement or development of new contact lens disinfecting systems by the manufacturers is needed to prevent Acanthamoeba keratitis. PMID:17514482

  13. Emerging Threats for Human Health in Poland: Pathogenic Isolates from Drug Resistant Acanthamoeba Keratitis Monitored in terms of Their In Vitro Dynamics and Temperature Adaptability

    PubMed Central

    Chomicz, Lidia; Conn, David Bruce; Padzik, Marcin; Szaflik, Jacek P.; Walochnik, Julia; Zawadzki, Paweł J.; Pawłowski, Witold; Dybicz, Monika

    2015-01-01

    Amphizoic amoebae generate a serious human health threat due to their pathogenic potential as facultative parasites, causative agents of vision-threatening Acanthamoeba keratitis (AK). Recently, AK incidences have been reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, severe cases of AK in Poland and respective pathogenic isolates were assessed at clinical, morphological, and molecular levels. Misdiagnoses and the unsuccessful treatment in other ophthalmic units delayed suitable therapy, and resistance to applied chemicals resulted in severe courses and treatment difficulties. Molecular assessment indicated that all sequenced pathogenic corneal isolates deriving from Polish patients with AK examined by us showed 98–100% homology with Acanthamoeba genotype T4, the most prevalent genotype in this human ocular infection worldwide. In vitro assays revealed that the pathogenic strains are able to grow at elevated temperature and have a wide adaptive capability. This study is our subsequent in vitro investigation on pathogenic Acanthamoeba strains of AK originating from Polish patients. Further investigations designed to foster a better understanding of the factors leading to an increase of AK observed in the past years in Poland may help to prevent or at least better cope with future cases. PMID:26682216

  14. Antimicrobial Activity of Simulated Solar Disinfection against Bacterial, Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin▿

    PubMed Central

    Heaselgrave, Wayne; Kilvington, Simon

    2010-01-01

    Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m−2) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log10 after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log10 after 6 h) was obtained only in the presence of riboflavin and 250 W m−2 irradiance. PMID:20639371

  15. Antimicrobial activity of simulated solar disinfection against bacterial, fungal, and protozoan pathogens and its enhancement by riboflavin.

    PubMed

    Heaselgrave, Wayne; Kilvington, Simon

    2010-09-01

    Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m(-2)) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log(10) after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log(10) after 6 h) was obtained only in the presence of riboflavin and 250 W m(-2) irradiance. PMID:20639371

  16. Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

    PubMed Central

    Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

    1997-01-01

    The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

  17. DVC-FISH and PMA-qPCR techniques to assess the survival of Helicobacter pylori inside Acanthamoeba castellanii.

    PubMed

    Moreno-Mesonero, Laura; Moreno, Yolanda; Alonso, José Luis; Ferrús, M Antonia

    2016-01-01

    Free-living amoebae (FLA) are ubiquitous microorganisms commonly found in water. They can act as Trojan Horses for some amoeba-resistant bacteria (ARB). Helicobacter pylori is a pathogenic bacteria, suggested to be transmitted through water, which could belong to the ARB group. In this work, a co-culture assay of H. pylori and Acanthamoeba castellanii, one of the most common FLA, was carried out to identify the presence and survival of viable and potentially infective forms of the bacteria internalized by the amoeba. Molecular techniques including FISH, DVC-FISH, qPCR and PMA-qPCR were used to detect the presence of internalized and viable H. pylori. After 24 h in co-culture and disinfection treatment to kill extra-amoebic bacteria, viable H. pylori cells were observed inside A. castellanii. When PMA-qPCR was applied to the co-culture samples, only DNA from internalized H. pylori cells was detected, whereas qPCR amplified total DNA from the sample. By the combined DVC-FISH method, the viability of H. pylori cells in A. castellanii was observed. Both specific techniques provided evidence, for the first time, that the pathogen is able to survive chlorination treatment in occurrence with A. castellanii and could be very useful methods for performing further studies in environmental samples. PMID:26342651

  18. The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

    PubMed

    Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

    2015-07-01

    Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle. PMID:26074248

  19. Survival of enterohemorrhagic Escherichia coli in the presence of Acanthamoeba castellanii and its dependence on Pho regulon

    PubMed Central

    Chekabab, Samuel Mohammed; Daigle, France; Charette, Steve J; Dozois, Charles M; Harel, Josée

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are involved in outbreaks of food-borne illness and transmitted to humans through bovine products or water contaminated by cattle feces. Microbial interaction is one of the strategies used by pathogenic bacteria to survive in the environment. Among protozoa, the free-living amoebae are known to host and protect several water-borne pathogens. In this study, the interaction between EHEC and the predacious protozoa Acanthamoeba castellanii was investigated. Using monoculture and cocultures, growth of both organisms was estimated for 3 weeks by total and viable cell counts. The numbers of EHEC were significantly higher when cultured with amoebae than without, and less EHEC shifted into a viable but nonculturable state in the presence of amoebae. Using several mutants, we observed that the Pho regulon is required for EHEC growth when cocultured with amoebae. In contrast, the Shiga toxins (Stx) were not involved in this association phenotype. Cocultures monitored by electron microscopy revealed a loss of the regular rod shape of EHEC and the secretion of multilamellar vesicles by the amoebae, which did not contain bacteria. As the interaction between A. castellanii and EHEC appears beneficial for bacterial growth, this supports a potential role for protozoa in promoting the persistence of EHEC in the environment. PMID:23233434

  20. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    SciTech Connect

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  1. The effect of different environmental conditions on the encystation of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Iftikhar, Hira; Khan, Naveed Ahmed

    2013-09-01

    In this study, Acanthamoeba castellanii was cultivated under different stress conditions to induce possible encystation. The morphological and histological properties were analysed by light and electron microscopy as well as cyst-specific staining. The findings revealed that cysts prepared through liquid medium using higher osmolarity as a trigger (10% glucose with 50mM magnesium chloride for 72 h) are similar to cysts prepared using non-nutrient agar (nutrient deprivation as a trigger in plating assays for 14 days), as determined by SDS-resistance, cyst-specific Calcofluor white staining and transmission electron microscopy. Using liquid medium assay, A. castellanii encystation was studied by exposing trophozoites to media lacking growth ingredients (phosphate buffered saline or distilled water), inappropriate temperatures (4-45°C), pH (3-9), artificial light-dark cycles, 5% CO2, and microaerophilic conditions. Optimal encystation was observed when cells were incubated in PBS with 50mM MgCl2 and 10% glucose at 24-30°C at pH 7. Increasing temperature over 37°C or pH 9 adversely affected encystation, while light-dark cycles, 5% CO2 and microaerophilic conditions had no effect on encystation of A. castellanii. None of the aforementioned conditions had any effect on the viability of A. castellanii, as determined by Trypan blue exclusion assay. A complete knowledge of encystation in A. castellanii is crucial to our understanding of the biology of these ecologically and medically important organisms. PMID:23769934

  2. Tracking amino acid's uptake into the protozoan Acanthamoeba castellanii by stable-isotope labelling and Raman spectral imaging

    NASA Astrophysics Data System (ADS)

    Naemat, Abida; Elsheikha, Hany M.; Notingher, Ioan

    2016-04-01

    The capacity of pathogens to acquire nutrients from their host cells is one of the most fundamental aspects of infection biology. Hence, measuring the patterns of nutrients' uptake by pathogens is essential for understanding the interactions of pathogens with eukaryotic host cells. In this study, we optimized a technique that allows fast and non-destructive measurement of the amino acid Phenylalanine (Phe) acquired by the trophozoite stage of the protozoan Acanthamoeba castellanii (A. castellanii) as they engage with individual human retinal pigment epithelial cells (ARPE-19). ARPE-19 host cells were pre-saturated with Deuterated Phe (L-Phe(D8)) to replace the native substrate Phe (L-Phe). The uptake of L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy. This approach allowed us to characterize the uptake patterns of this essential amino acid into A. castellanii trophozoites at a single cell level. At 24 hours post infection (PI) A. castellanii trophozoites are capable of salvaging L-Phe(D8) from host cells. The uptake pattern was time-dependent during the first 24 hours of infection and complete substitution with L-Phe(D8) in all parasites was detected at 48 hours PI. On the other hand, isolated A. castellanii trachyzoites (grown without host cells) did not show significant uptake for L-Phe(D8) from the media; only achieved an uptake ratio of 16-18% of L-Phe(D8) from the culture medium after 24 hours. These findings demonstrate the potential of combining Raman microspectroscopy and stable isotope labelling approaches to elucidate the role of metabolism in mediating A. castellanii interaction with host cells.

  3. Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

    PubMed Central

    Ovrutsky, Alida R.; Kartalija, Marinka; Bai, Xiyuan; Jackson, Mary; Gibbs, Sara; Falkinham, Joseph O.; Iseman, Michael D.; Reynolds, Paul R.; McDonnell, Gerald

    2013-01-01

    The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species. PMID:23475613

  4. Amoebicidal activity of a preserved contact lens multipurpose disinfecting solution compared to a disinfection/neutralisation peroxide system.

    PubMed

    Buck, S L; Rosenthal, R A; Abshire, R L

    1998-01-01

    The amoebicidal activity of a contact lens multipurpose disinfecting solution (MPDS) containing polyquaternium-1 and myristamidopropyl dimethylamine was compared to a disinfection/neutralisation peroxide system against Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites and cysts. A quantitative microtitre method was used to evaluate the solutions. The MPDS showed similar amoebicidal activity to the disinfection/neutralisation peroxide system against the trophozoites of both species and equal or more rapid activity against the cysts of both species. PMID:16303382

  5. Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

    EPA Science Inventory

    The potential effect of in-premise plumbing temperatures (24, 32, 37 and 41 °C) on the growth of five different L. pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Compared to controls only fed E...

  6. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  7. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    SciTech Connect

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  8. A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii.

    PubMed

    Iwamoto, M; Pi, M; Kurihara, M; Morio, T; Tanaka, Y

    1998-04-01

    We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+ ++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. PMID:9560439

  9. In vitro efficacies of clinically available drugs against growth and viability of an Acanthamoeba castellanii keratitis isolate belonging to the T4 genotype.

    PubMed

    Baig, Abdul Mannan; Iqbal, Junaid; Khan, Naveed Ahmed

    2013-08-01

    The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype. In vitro growth inhibition (amoebistatic) assays were performed by incubating A. castellanii with various concentrations of drugs in the growth medium for 48 h at 30°C. To determine amoebicidal effects, amoebae were incubated with drugs in phosphate-buffered saline for 24 h, and viability was determined using trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. Of the eight drugs tested, amlodipine, prochlorperazine, and loperamide showed potent amoebicidal effects, as no viable trophozoites were observed (>95% kill rate), while amiodarone, procyclidine, digoxin, and apomorphine exhibited up to 50% amoebicidal effects. In contrast, haloperidol did not affect viability, but all the drugs tested inhibited A. castellanii growth. Importantly, amlodipine, prochlorperazine, and loperamide showed compelling cysticidal effects. The cysticidal effects were irreversible, as cysts treated with the aforementioned drugs did not reemerge as viable amoebae upon inoculation in the growth medium. Except for apomorphine and haloperidol, all the tested drugs blocked trophozoite differentiation into cysts in encystation assays. Given the limited availability of effective drugs to treat amoebal infections, the clinically available drugs tested in this study represent potential agents for managing keratitis and granulomatous amoebic encephalitis caused by Acanthamoeba spp. and possibly against other meningoencephalitis-causing amoebae, such as Balamuthia mandrillaris and Naegleria fowleri. PMID:23669391

  10. In Vitro Efficacies of Clinically Available Drugs against Growth and Viability of an Acanthamoeba castellanii Keratitis Isolate Belonging to the T4 Genotype

    PubMed Central

    Baig, Abdul Mannan; Iqbal, Junaid

    2013-01-01

    The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype. In vitro growth inhibition (amoebistatic) assays were performed by incubating A. castellanii with various concentrations of drugs in the growth medium for 48 h at 30°C. To determine amoebicidal effects, amoebae were incubated with drugs in phosphate-buffered saline for 24 h, and viability was determined using trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. Of the eight drugs tested, amlodipine, prochlorperazine, and loperamide showed potent amoebicidal effects, as no viable trophozoites were observed (>95% kill rate), while amiodarone, procyclidine, digoxin, and apomorphine exhibited up to 50% amoebicidal effects. In contrast, haloperidol did not affect viability, but all the drugs tested inhibited A. castellanii growth. Importantly, amlodipine, prochlorperazine, and loperamide showed compelling cysticidal effects. The cysticidal effects were irreversible, as cysts treated with the aforementioned drugs did not reemerge as viable amoebae upon inoculation in the growth medium. Except for apomorphine and haloperidol, all the tested drugs blocked trophozoite differentiation into cysts in encystation assays. Given the limited availability of effective drugs to treat amoebal infections, the clinically available drugs tested in this study represent potential agents for managing keratitis and granulomatous amoebic encephalitis caused by Acanthamoeba spp. and possibly against other meningoencephalitis-causing amoebae, such as Balamuthia mandrillaris and Naegleria fowleri. PMID:23669391

  11. Acanthamoeba Keratitis FAQs

    MedlinePlus

    ... lenses improperly Disinfecting lenses improperly (such as using tap water or homemade solutions to clean the lenses) Swimming, ... rinsed with sterile contact lens solution (never use tap water), emptied, and left open to dry after each ...

  12. A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids.

    PubMed

    Sayanova, Olga; Haslam, Richard; Guschina, Irina; Lloyd, David; Christie, William W; Harwood, John L; Napier, Johnathan A

    2006-12-01

    The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist. PMID:16950768

  13. Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

    PubMed Central

    Kicka, Sébastien; Trofimov, Valentin; Harrison, Christopher; Ouertatani-Sakouhi, Hajer; McKinney, John; Scapozza, Leonardo; Hilbi, Hubert; Cosson, Pierre; Soldati, Thierry

    2014-01-01

    Tuberculosis is considered to be one of the world’s deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches. PMID:24498207

  14. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    SciTech Connect

    El-Etr, S H; Margolis, J; Monack, D; Robison, R; Cohen, M; Moore, E; Rasley, A

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP) is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.

  15. Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

    PubMed Central

    Lück, P. Christian; Schmitt, Jürgen W.; Hengerer, Arne; Helbig, Jürgen H.

    1998-01-01

    We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected. PMID:9797218

  16. Impact of non-Legionella bacteria on the uptake and intracellular replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis.

    PubMed

    Declerck, P; Behets, J; Delaedt, Y; Margineanu, A; Lammertyn, E; Ollevier, F

    2005-11-01

    In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health. PMID:16341636

  17. The Transcriptional Response of Cryptococcus neoformans to Ingestion by Acanthamoeba castellanii and Macrophages Provides Insights into the Evolutionary Adaptation to the Mammalian Host

    PubMed Central

    Paes, Hugo Costa; Albuquerque, Patrícia; Tavares, Aldo Henrique F. P.; Fernandes, Larissa; Silva-Pereira, Ildinete; Casadevall, Arturo

    2013-01-01

    Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host. PMID:23524994

  18. Effects of oakmoss and its components on Acanthamoeba castellanii ATCC 30234 and the uptake of Legionella pneumophila JCM 7571 (ATCC 33152) into A. castellanii.

    PubMed

    Nomura, Harue; Isshiki, Yasunori; Sakuda, Keisuke; Sakuma, Katsuya; Kondo, Seiichi

    2015-01-01

    Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a natural fragrance ingredient, and its components are antibacterial agents specifically against the genus Legionella. In the present study, oakmoss and its components were investigated for their amoebicidal activity against A. castellanii ATCC 30234 and the inhibitory effect on the uptake of L. pneumophila JCM 7571 (ATCC 33152) into A. castellanii. The oakmoss and its components 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate(5), and 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (12) exhibited high amoebicidal activity (IC50 values; 10.5 ± 2.3, 16.3 ± 4.0 and 17.5 ± 2.8 μg/mL, respectively) after 48 h of treatment, which were equivalent to that of the reference compound, chlorhexidine gluconate. Pretreatment of L. pneumophila with sub-minimal inhibitory concentration of oakmoss, compound 5, 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate (10) and 8-(2,4-dihydroxy-6-pentylphenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (14) obviously reduced the uptake of L. pneumophila into A.castellanii (p < 0.05).The inhibitory effect of compound 5 on the uptake of L. pneumophila was almost equivalent to that of ampicillin used as a reference. Thus, the oakmoss and its components were considered to be good candidates for disinfectants against not only genus Legionella but also A. castellanii. PMID:25817814

  19. Isolation and identification of free-living amoebae from tap water in Sivas, Turkey.

    PubMed

    Coşkun, Kübra Açıkalın; Ozçelik, Semra; Tutar, Lütfi; Elaldı, Nazif; Tutar, Yusuf

    2013-01-01

    The present work focuses on a local survey of free-living amoebae (FLA) that cause opportunistic and nonopportunistic infections in humans. Determining the prevalence of FLA in water sources can shine a light on the need to prevent FLA related illnesses. A total of 150 samples of tap water were collected from six districts of Sivas province. The samples were filtered and seeded on nonnutrient agar containing Escherichia coli spread. Thirty-three (22%) out of 150 samples were found to be positive for FLA. The FLA were identified by morphology and by PCR using 18S rDNA gene. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of three different species, Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. Naegleria fowleri, Balamuthia mandrillaris, or Sappinia sp. was not isolated during the study. All A. castellanii and A. polyphaga sequence types were found to be genotype T4 that contains most of the pathogenic Acanthamoeba strains. The results indicated the occurrence and distribution of FLA species in tap water in these localities of Sivas, Turkey. Furthermore, the presence of temperature tolerant Acanthamoeba genotype T4 in tap water in the region must be taken into account for health risks. PMID:23971043

  20. Preliminary crystallographic analysis of the Megavirus superoxide dismutase.

    PubMed

    Lartigue, Audrey; Philippe, Nadège; Jeudy, Sandra; Abergel, Chantal

    2012-12-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein. PMID:23192047

  1. Preliminary crystallographic analysis of the Megavirus superoxide dismutase

    PubMed Central

    Lartigue, Audrey; Philippe, Nadège; Jeudy, Sandra; Abergel, Chantal

    2012-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein. PMID:23192047

  2. Microbiological investigations into an outbreak of Pontiac fever due to Legionella micdadei associated with use of a whirlpool.

    PubMed Central

    Fallon, R J; Rowbotham, T J

    1990-01-01

    In the investigation of a large outbreak of non-pneumonic legionellosis at a leisure complex in Lochgoilhead, Scotland all direct cultures of environmental samples were initially negative for legionellae. Legionellae readily infect appropriate protozoa under suitable conditions, and following immunofluorescence to select specimens for special study, Legionella micdadei was isolated from whirlpool water via co-cultivation with Acanthamoeba polyphaga. L micdadei was also isolated, along with host amoebae, from the whirlpool filter. The use of amoebae in the isolation of legionellae from environmental (and other) sources can be of great value, especially if specimens shown by indirect immunofluorescence to contain legionellae fail to yield legionellae on routine culture. PMID:2199533

  3. The tetrahymena and acanthamoeba model systems.

    PubMed

    Berk, Sharon G; Garduño, Rafael A

    2013-01-01

    Although the study of protozoology has been active for centuries, very few current academic curricula incorporate requirements or even options for coursework on the study of protists; yet, protozoa are becoming widely recognized by investigators as organisms that play a significant role in the evolution, pathogenicity, protection and amplification of human pathogens in the environment. This is particularly true for the study of Legionella, as this accidental human pathogen has naturally evolved to infect protozoa in fresh water environments. Researchers have made great progress in the study of pathogenicity, evolution, and ecology of Legionella and its protozoan hosts, which include amoebae and ciliated protozoa. Our own collaboration in this field has been active for over a decade, and we have gained a valuable experience working with these protozoa, particularly aspects of their biology and the methods needed to address new experimental concepts. Therefore, in this chapter we provide the most effective procedures that we have developed or modified through our years of practice. We also offer notes on what procedures, in our opinion, should be avoided; and we provide the rationale for such precautions. PMID:23150411

  4. Altered host cell-bacteria interaction due to nanoparticle interaction with a bacterial biofilm.

    PubMed

    Raftery, Tara D; Lindler, Heidi; McNealy, Tamara L

    2013-02-01

    Nanoparticle (NP) use in everyday applications creates the potential for NPs to enter the environment where, in aquatic systems, they are likely to settle on substrates and interact with microbial communities. Legionella pneumophila biofilms are found as part of microbial communities in both natural and man-made environments, especially in man-made cooling systems. The bacterium is the causative agent of Legionnaires' disease. Legionella requires a host cell for replication in the environment, and amoebae commonly serve as this host cell. Our previous work demonstrated significant changes in Legionella biofilm morphology after exposure to 0.7 μg/L gold NPs (AuNPs). Here, we investigate how these morphology changes alter host-bacteria interactions using Acanthamoeba polyphaga as a model. Host-bacteria-NP interactions are affected by NP characteristics. Biofilms exposed to 4- and 18-nm, citrate-capped, spherical AuNPs significantly altered the grazing ability of A. polyphaga, which was not observed in biofilms exposed to 24-nm polystyrene beads. Uptake and replication of NP-exposed planktonic L. pneumophila within A. polyphaga were not altered regardless of NP size or core chemistry. Nanomaterial effects on the interaction of benthic organisms and bacteria may be directly or, as shown here, indirectly dependent on bacterial morphology. NP contamination therefore may alter interactions in a normal ecosystem function. PMID:23052925

  5. Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).

    PubMed

    Amaral, Danilo T; Mitani, Yasuo; Ohmiya, Yoshihiro; Viviani, Vadim R

    2016-07-25

    Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis. PMID:27060405

  6. The first elateroid beetles (Coleoptera: Polyphaga: Elateroidea) from the Upper Jurassic of Australia.

    PubMed

    Oberprieler, Rolf G; Ashman, Lauren G; Frese, Michael; Ślipiński, Adam

    2016-01-01

    The first elateroid fossils from the Upper Jurassic Talbragar Fish Bed in Australia are described and illustrated. Wongaroo amplipectorale gen. et sp. n., based on two specimens, is placed in the family Cerophytidae due to its convex, posteriorly weakly angled and laterally carinate pronotum obscuring the head in dorsal view, its relatively long, pointed elytra and slender legs, its 9-striate elytra with deep basal pits and the absence of metacoxal plates. Beattieellus jurassicus gen. et sp. n., described from one specimen, possesses the acutely angled pronotum without a carina on the posterolateral angles and the ventral click apparatus typical of Eucnemidae and is classified in this family. Assignment of it to a eucnemid subfamily is impossible because of the insufficient preservation of relevant characters in the fossil. Four other elateroid fossils, possibly representing eucnemids and elaterids, are illustrated and briefly described but not named, due to their insufficient preservation. These fossils represent the first of their kind in Australia and in the Southern Hemisphere, and Beattieellus is also the oldest eucnemid fossil known and extends the fossil record of Eucnemidae into the Upper Jurassic. The discovery of elateroid fossils in the Talbragar Fish Bed adds to the coleopteran diversity of this ancient lake ecosystem, indicating that it was well wooded and provided suitable habitats of rotten wood for the development of the larvae of these taxa. PMID:27515614

  7. Giant virus in the sea: Extending the realm of Megaviridae to Viridiplantae.

    PubMed

    Claverie, Jean-Michel

    2013-11-01

    The viral nature of the first "giant virus," Mimivirus, was realized in 2003, 10 y after its initial isolation from the water of a cooling tower in Bradford, UK. Soon after its genome was sequenced, the mining of the Global Ocean Sampling environmental sequence database revealed that the closest relatives of Mimivirus, only known to infect Acanthamoeba, were to be found in the sea. These predicted marine Mimivirus relatives remained elusive until 2010, with the first genomic characterization of a virus infecting a heterotrophic unicellular eukaryote, the microflagellate grazer Cafeteria roenbergensis. The genome analysis of a virus (PgV) infecting the common unicellular algae Phaeocystis globosa now shows that it is a bona fide member of the Mimivirus family (i.e., the Megaviridae), extending the realm of these giant viruses to abundant blooming phytoplankton species. Despite its smaller genome size (460 kb encoding 434 proteins), PgV exhibits the most intriguing feature of the previously characterized Megaviridae: an associated virophage. However, the 19-kb virophage genome, devoid of a capsid gene, is packaged in the PgV particle and propagated as a "viral plasmid," the first ever described. The PgV genome also exhibits the duplication of "core genes," normally present as single copies and a putative new type of mobile element. In a DNA polymerase phylogeny including representatives of the three cellular domains, PgV and the other Megaviridae cluster into their own clade deeply branching between domains Archaea and Eukarya domains, thus exhibiting the topology of a fourth domain in the Tree of Life. PMID:24563700

  8. Contact lens disinfection by ultraviolet light

    SciTech Connect

    Dolman, P.J.; Dobrogowski, M.J. )

    1989-12-15

    A 253.7-nm ultraviolet light with an intensity of 1,100 microW/cm2 was tested for its germicidal activity against contact lenses and storage solutions contaminated with various corneal pathogens. The exposure time necessary to reduce a concentration of organisms from 10(6)/ml to less than 10/ml was 30 seconds for Staphylococcus aureus, 60 seconds for Pseudomonas aeruginosa, and 84 seconds for Candida albicans. The time necessary to sterilize a suspension of 10(4)/ml Acanthamoeba polyphaga was less than three minutes with this technique. Four brands of soft contact lenses were exposed to ultraviolet light for over eight hours without changing their appearance, comfort, or refraction.

  9. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    PubMed

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

    2015-04-01

    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. PMID:25725384

  10. Relative importance of evolutionary dynamics depends on the composition of microbial predator-prey community.

    PubMed

    Friman, Ville-Petri; Dupont, Alessandra; Bass, David; Murrell, David J; Bell, Thomas

    2016-06-01

    Community dynamics are often studied in subsets of pairwise interactions. Scaling pairwise interactions back to the community level is, however, problematic because one given interaction might not reflect ecological and evolutionary outcomes of other functionally similar species interactions or capture the emergent eco-evolutionary dynamics arising only in more complex communities. Here we studied this experimentally by exposing Pseudomonas fluorescens SBW25 prey bacterium to four different protist predators (Tetrahymena pyriformis, Tetrahymena vorax, Chilomonas paramecium and Acanthamoeba polyphaga) in all possible single-predator, two-predator and four-predator communities for hundreds of prey generations covering both ecological and evolutionary timescales. We found that only T. pyriformis selected for prey defence in single-predator communities. Although T. pyriformis selection was constrained in the presence of the intraguild predator, T. vorax, T. pyriformis selection led to evolution of specialised prey defence strategies in the presence of C. paramecium or A. polyphaga. At the ecological level, adapted prey populations were phenotypically more diverse, less stable and less productive compared with non-adapted prey populations. These results suggest that predator community composition affects the relative importance of ecological and evolutionary processes and can crucially determine when rapid evolution has the potential to change ecological properties of microbial communities. PMID:26684728

  11. Giant viruses come of age.

    PubMed

    Fischer, Matthias G

    2016-06-01

    Viruses with genomes up to a few megabases in length are a common occurrence in nature, even though they have escaped our notice until recently. These giant viruses infect mainly single-celled eukaryotes and isolation efforts concentrating on amoebal hosts alone have spawned hundreds of viral isolates, featuring viruses with previously unseen virion morphologies and the largest known viral genomes and particles. One of the challenges that lie ahead is to analyze and categorize the available data and to establish an approved classification system that reflects the evolutionary relationships and biological properties of these viruses. Extensive sampling of Acanthamoeba-infecting mimiviruses and initial characterization of their virophage parasites have provided a first blueprint of the genetic diversity and composition of a giant virus clade that will facilitate the taxonomic grouping of these fascinating microorganisms. PMID:26999382

  12. A New Zamilon-like Virophage Partial Genome Assembled from a Bioreactor Metagenome

    PubMed Central

    Bekliz, Meriem; Verneau, Jonathan; Benamar, Samia; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2015-01-01

    Virophages replicate within viral factories inside the Acanthamoeba cytoplasm, and decrease the infectivity and replication of their associated giant viruses. Culture isolation and metagenome analyses have suggested that they are common in our environment. By screening metagenomic databases in search of amoebal viruses, we detected virophage-related sequences among sequences generated from the same non-aerated bioreactor metagenome as recently screened by another team for virophage capsid-encoding genes. We describe here the assembled partial genome of a virophage closely related to Zamilon, which infects Acanthamoeba with mimiviruses of lineages B and C but not A. Searches for sequences related to amoebal giant viruses, other Megavirales representatives and virophages were conducted using BLAST against this bioreactor metagenome (PRJNA73603). Comparative genomic and phylogenetic analyses were performed using sequences from previously identified virophages. A total of 72 metagenome contigs generated from the bioreactor were identified as best matching with sequences from Megavirales representatives, mostly Pithovirus sibericum, pandoraviruses and amoebal mimiviruses from three lineages A–C, as well as from virophages. In addition, a partial genome from a Zamilon-like virophage, we named Zamilon 2, was assembled. This genome has a size of 6716 base pairs, corresponding to 39% of the Zamilon genome, and comprises partial or full-length homologs for 15 Zamilon predicted open reading frames (ORFs). Mean nucleotide and amino acid identities for these 15 Zamilon 2 ORFs with their Zamilon counterparts were 89% (range, 81–96%) and 91% (range, 78–99%), respectively. Notably, these ORFs included two encoding a capsid protein and a packaging ATPase. Comparative genomics and phylogenetic analyses indicated that the partial genome was that of a new Zamilon-like virophage. Further studies are needed to gain better knowledge of the tropism and prevalence of virophages in

  13. Interactions of Bacterial and Amoebal Populations in Soil Microcosms with Fluctuating Moisture Content

    PubMed Central

    Bryant, R. J.; Woods, L. E.; Coleman, D. C.; Fairbanks, B. C.; McClellan, J. F.; Cole, C. V.

    1982-01-01

    Sterilized soil samples (20 g of soil per 50-ml flask), amended with 600 μg of glucose-carbon and 60 μg of NH4-N · g of dry soil−1, were inoculated with bacteria (Pseudomonas paucimobilis) alone or with bacteria and amoebae (Acanthamoeba polyphaga). We used wet-dry treatments, which involved air drying the samples to a moisture content of approximately 2% and remoistening the samples three times during the 83-day experiment. Control treatments were kept moist. In the absence of amoebae, bacterial populations were reduced by the first drying to about 60% of the moist control populations, but the third drying had no such effect. With amoebae present, bacterial numbers were not significantly affected by the dryings. Amoebal grazing reduced bacterial populations to 20 to 25% of the ungrazed bacterial populations in both moisture treatments. Encystment was an efficient survival mechanism for amoebae subjected to wet-dry cycles. The amoebal population was entirely encysted in dry soil, but the total number of amoebae was not affected by the three dryings. Growth efficiencies for amoebae feeding on bacteria were 0.33 and 0.39 for wet-dry and constantly moist treatments, respectively, results that compared well with those previously reported for Acanthamoeba spp. PMID:16345984

  14. Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.

    PubMed

    Wheat, William H; Casali, Amy L; Thomas, Vincent; Spencer, John S; Lahiri, Ramanuj; Williams, Diana L; McDonnell, Gerald E; Gonzalez-Juarrero, Mercedes; Brennan, Patrick J; Jackson, Mary

    2014-12-01

    Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs

  15. Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

    PubMed Central

    Lu, Jingrang; Ashbolt, Nicholas J.

    2014-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. PMID:25381242

  16. Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts

    PubMed Central

    Wheat, William H.; Casali, Amy L.; Thomas, Vincent; Spencer, John S.; Lahiri, Ramanuj; Williams, Diana L.; McDonnell, Gerald E.; Gonzalez-Juarrero, Mercedes; Brennan, Patrick J.; Jackson, Mary

    2014-01-01

    Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs

  17. Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages.

    PubMed

    Habyarimana, Fabien; Al-Khodor, Souhaila; Kalia, Awdhesh; Graham, James E; Price, Christopher T; Garcia, Maria Teresa; Kwaik, Yousef Abu

    2008-06-01

    Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic

  18. Hydraena (Hydraenopsis) ateneo, new species (Coleoptera, Hydraenidae) and other aquatic Polyphaga from a small habitat patch in a highly urbanized landscape of Metro Manila, Philippines.

    PubMed

    Freitag, Hendrik

    2013-01-01

    Seven species of Hydraenidae, Hydrophilidae and Elmidae are recorded from temporary freshwater habitats at the Ateneo de Manila University Campus in the metropolitan area of Manila, Philippines. They were identified as Enochrus (Lumetus) fragiloides d'Orchymont, Helochares (Hydrobaticus) lepidus d'Orchymont, Helochares (Helochares) pallens (MacLeay), Hydraena (Hydraenopsis) scabra d'Orchymont, Hydraena (Hydraenopsis) palawanensis Freitag & Jäch (new record for Luzon Island), Stenelmis sp. A further hydraenid species was unknown to science and is newly described: Hydraena (Hydraenopsis) ateneo Freitag, sp. n. Aedeagus, gonocoxite, spermatheca, and female tergite X are illustrated by computer-based line drawings. Habitus images of all three Hydraena Kugelann species recorded and a checklist of the Philippine Hydraena are provided. The presence of these seven species in the Ateneo campus is briefly discussed in regard to the area's history. Measures to maintain and extend semi-natural islands of biodiversity in urban areas are suggested. PMID:24146550

  19. A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability▿

    PubMed Central

    Dusserre, Eric; Ginevra, Christophe; Hallier-Soulier, Sylvie; Vandenesch, François; Festoc, Gabriel; Etienne, Jerome; Jarraud, Sophie; Molmeret, Maëlle

    2008-01-01

    Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 106 genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 105 and 102 metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations. PMID:18515476

  20. Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

    PubMed Central

    Gryseels, Sophie; Amissah, Diana; Durnez, Lies; Vandelannoote, Koen; Leirs, Herwig; De Jonckheere, Johan; Portaels, Françoise; Ablordey, Anthony; Eddyani, Miriam

    2012-01-01

    Background The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa. Methodology/Principal Findings We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities. Conclusions/Significance This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer. PMID:22880141

  1. The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model.

    PubMed

    Kahane, Simona; Dvoskin, Bella; Friedman, Maureen G

    2008-03-01

    Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo. PMID:18081846

  2. Complete genome sequence of Cannes 8 virus, a new member of the proposed family "Marseilleviridae".

    PubMed

    Aherfi, Sarah; Pagnier, Isabelle; Fournous, Ghislain; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2013-12-01

    Marseillevirus is a giant virus that was isolated in 2007 by culturing water collected from a cooling tower in Paris, France, on Acanthamoeba polyphaga. Since then, five other marseilleviruses have been detected in environmental or human samples. The genomes of two of the six marseilleviruses have been described in detail. We describe herein the genome of Cannes 8 virus, a new member of the proposed family "Marseilleviridae." Cannes 8 virus was isolated from water collected from a cooling tower in Cannes in southeastern France. Its genome is a circular double-stranded DNA molecule with 374,041 base pairs, larger than the Marseillevirus and Lausannevirus genomes. This genome harbors 484 open reading frames predicted to encode proteins with sizes ranging from 50 to 1,537 amino acids, among which 380 (79%) and 272 (56%) are bona fide orthologs of Marseillevirus and Lausannevirus proteins, respectively. In addition, 407 and 336 predicted proteins have significant hits against Marseillevirus and Lausannevirus proteins, respectively, and 294 proteins are shared by all three marseilleviruses. The Cannes 8 virus genome has a high level of collinearity (for 96% of orthologs) with the Marseillevirus genome. About two-thirds of the Cannes 8 virus gene repertoire is composed of family ORFans. The description and annotation of the genomes of new marseilleviruses that will undoubtedly be recovered from environmental or clinical samples will be helpful to increase our knowledge of the pan-genome of the family "Marseilleviridae." PMID:23912978

  3. Genome Sequence of Rickettsia bellii Illuminates the Role of Amoebae in Gene Exchanges between Intracellular Pathogens

    PubMed Central

    Ogata, Hiroyuki; La Scola, Bernard; Audic, Stéphane; Renesto, Patricia; Blanc, Guillaume; Robert, Catherine; Fournier, Pierre-Edouard; Claverie, Jean-Michel; Raoult, Didier

    2006-01-01

    The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells. PMID:16703114

  4. Giant Virus Megavirus chilensis Encodes the Biosynthetic Pathway for Uncommon Acetamido Sugars*

    PubMed Central

    Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G.

    2014-01-01

    Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. PMID:25035429

  5. Evolutionary dynamics of giant viruses and their virophages.

    PubMed

    Wodarz, Dominik

    2013-07-01

    Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested. PMID:23919155

  6. Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

    PubMed

    Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G

    2014-08-29

    Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. PMID:25035429

  7. PRODUCTION OF RESPIRABLE VESICLES CONTAINING LIVE LEGIONELLA PNEUMOPHILA CELLS BY TWO ACANTHAMOEBA SPP. (R825352)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  8. MOLECULAR AND PHYSIOLOGICAL EVALUATION OF SUBTROPICAL ENVIRONMENTAL ISOLATES OF ACANTHAMOEBA KERATITIS. (R828830)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  9. Relationship between Organic Carbon and Opportunistic Pathogens in Simulated Glass Water Heaters

    PubMed Central

    Williams, Krista; Pruden, Amy; Falkinham, Joseph O.; Edwards, Marc

    2015-01-01

    Controlling organic carbon levels in municipal water has been hypothesized to limit downstream growth of bacteria and opportunistic pathogens in premise plumbing (OPPPs). Here, the relationships between influent organic carbon (0–15,000 µg ozonated fulvic acid /L) and the number of total bacteria [16S rRNA genes and heterotrophic plate counts (HPCs)] and a wide range of OPPPs (gene copy numbers of Acanthamoeba polyphaga, Vermamoeba vermiformis, Legionella pneumophila, and Mycobacterium avium) were examined in the bulk water of 120-mL simulated glass water heaters (SGWHs). The SGWHs were operated at 32–37 °C, which is representative of conditions encountered at the bottom of electric water heaters, with water changes of 80% three times per week to simulate low use. This design presented advantages of controlled and replicated (triplicate) conditions and avoided other potential limitations to OPPP growth in order to isolate the variable of organic carbon. Over seventeen months, strong correlations were observed between total organic carbon (TOC) and both 16S rRNA gene copy numbers and HPC counts (avg. R2 > 0.89). Although M. avium gene copies were occasionally correlated with TOC (avg. R2 = 0.82 to 0.97, for 2 out of 4 time points) and over a limited TOC range (0–1000 µg/L), no other correlations were identified between other OPPPs and added TOC. These results suggest that reducing organic carbon in distributed water is not adequate as a sole strategy for controlling OPPPs, although it may have promise in conjunction with other approaches. PMID:26066310

  10. Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences▿ †

    PubMed Central

    Valster, Rinske M.; Wullings, Bart A.; Bakker, Geo; Smidt, Hauke; van der Kooij, Dick

    2009-01-01

    Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa. PMID:19465529

  11. Relationship between Organic Carbon and Opportunistic Pathogens in Simulated Glass Water Heaters.

    PubMed

    Williams, Krista; Pruden, Amy; Falkinham, Joseph O; Edwards, Marc; Williams, Krista; Pruden, Amy; Iii, Joseph O Falkinham; Edwards, Marc

    2015-01-01

    Controlling organic carbon levels in municipal water has been hypothesized to limit downstream growth of bacteria and opportunistic pathogens in premise plumbing (OPPPs). Here, the relationships between influent organic carbon (0-15,000 µg ozonated fulvic acid /L) and the number of total bacteria [16S rRNA genes and heterotrophic plate counts (HPCs)] and a wide range of OPPPs (gene copy numbers of Acanthamoeba polyphaga, Vermamoeba vermiformis, Legionella pneumophila, and Mycobacterium avium) were examined in the bulk water of 120-mL simulated glass water heaters (SGWHs). The SGWHs were operated at 32-37 °C, which is representative of conditions encountered at the bottom of electric water heaters, with water changes of 80% three times per week to simulate low use. This design presented advantages of controlled and replicated (triplicate) conditions and avoided other potential limitations to OPPP growth in order to isolate the variable of organic carbon. Over seventeen months, strong correlations were observed between total organic carbon (TOC) and both 16S rRNA gene copy numbers and HPC counts (avg. R2 > 0.89). Although M. avium gene copies were occasionally correlated with TOC (avg. R2 = 0.82 to 0.97, for 2 out of 4 time points) and over a limited TOC range (0-1000 µg/L), no other correlations were identified between other OPPPs and added TOC. These results suggest that reducing organic carbon in distributed water is not adequate as a sole strategy for controlling OPPPs, although it may have promise in conjunction with other approaches. PMID:26066310

  12. Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes.

    PubMed

    Santini, Sebastien; Jeudy, Sandra; Bartoli, Julia; Poirot, Olivier; Lescot, Magali; Abergel, Chantal; Barbe, Valérie; Wommack, K Eric; Noordeloos, Anna A M; Brussaard, Corina P D; Claverie, Jean-Michel

    2013-06-25

    Large dsDNA viruses are involved in the population control of many globally distributed species of eukaryotic phytoplankton and have a prominent role in bloom termination. The genus Phaeocystis (Haptophyta, Prymnesiophyceae) includes several high-biomass-forming phytoplankton species, such as Phaeocystis globosa, the blooms of which occur mostly in the coastal zone of the North Atlantic and the North Sea. Here, we report the 459,984-bp-long genome sequence of P. globosa virus strain PgV-16T, encoding 434 proteins and eight tRNAs and, thus, the largest fully sequenced genome to date among viruses infecting algae. Surprisingly, PgV-16T exhibits no phylogenetic affinity with other viruses infecting microalgae (e.g., phycodnaviruses), including those infecting Emiliania huxleyi, another ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to an emerging clade (the Megaviridae) clustering the viruses endowed with the largest known genomes, including Megavirus, Mimivirus (both infecting acanthamoeba), and a virus infecting the marine microflagellate grazer Cafeteria roenbergensis. Seventy-five percent of the best matches of PgV-16T-predicted proteins correspond to two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from a hypersaline lake in Antarctica (Organic Lake), the hosts of which are unknown. As for OLPVs and other Megaviridae, the PgV-16T sequence data revealed the presence of a virophage-like genome. However, no virophage particle was detected in infected P. globosa cultures. The presence of many genes found only in Megaviridae in its genome and the presence of an associated virophage strongly suggest that PgV-16T shares a common ancestry with the largest known dsDNA viruses, the host range of which already encompasses the earliest diverging branches of domain Eukarya. PMID:23754393

  13. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

    PubMed

    Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

    2014-03-18

    The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health. PMID:24591590

  14. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology

    PubMed Central

    Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

    2014-01-01

    The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine–thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine–cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health. PMID:24591590

  15. A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa.

    PubMed

    Al-Khodor, Souhaila; Price, Christopher T; Habyarimana, Fabien; Kalia, Awdhesh; Abu Kwaik, Yousef

    2008-11-01

    The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression

  16. A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa

    PubMed Central

    Al-khodor, Souhaila; Price, Christopher T.; Habyarimana, Fabien; Kalia, Awdhesh; Kwaik, Yousef Abu

    2011-01-01

    Summary The Dot/Icm type IV secretion system of L. pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbors 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages, and Acanthamoeba polyphaga. Single cell analyses of co-infections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in-cis within communal phagosomes harboring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in-trans within cells harboring the wild type strain in a different phagosome, indicating that AnkB is a transacting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner, and that the last 3 C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodeled by the RER. We show that at the post exponential phase of growth, the LetA/S and PmrA/B two component systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE, and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by

  17. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    PubMed Central

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  18. Fatal Granulomatous Acanthamoeba Encephalitis Mimicking a Stroke, Diagnosed by Correlation of Results of Sequential Magnetic Resonance Imaging, Biopsy, In Vitro Culture, Immunofluorescence Analysis, and Molecular Analysis▿

    PubMed Central

    McKellar, Mehri S.; Mehta, Lahar R.; Greenlee, John E.; Hale, Devon C.; Booton, Gregory C.; Kelly, Daryl J.; Fuerst, Paul A.; Sriram, Rama; Visvesvara, Govinda S.

    2006-01-01

    Amebic infections involving the central nervous system are rare and difficult to diagnose. Magnetic resonance imaging (MRI) at timed intervals may be helpful, where scans reveal enhancing lesions and increased signal. We report a unique case of granulomatous amebic encephalitis that was proven pathologically with progressive radiological findings on MRI. PMID:16988022

  19. Amoebal Endosymbiont Parachlamydia acanthamoebae Bn9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution

    PubMed Central

    Nakamura, Shinji; Matsuo, Junji; Ishida, Kasumi; Yamazaki, Sumire; Oguri, Satoshi; Shouji, Natsumi; Hayashi, Yasuhiro; Yoshida, Mitsutaka; Yimin; Yamaguchi, Hiroyuki

    2015-01-01

    Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7–1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37°C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30°C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30°C compared to at 37°C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells. PMID:25643359

  20. Giant viruses of amoebae as potential human pathogens.

    PubMed

    Colson, Philippe; La Scola, Bernard; Raoult, Didier

    2013-01-01

    Giant viruses infecting phagocytic protists are composed of mimiviruses, the record holders of particle and genome size amongst viruses, and marseilleviruses. Since the discovery in 2003 at our laboratory of the first of these giant viruses, the Mimivirus, a growing body of data has revealed that they are common inhabitants of our biosphere. Moreover, from the outset, the story of Mimivirus has been linked to that of patients exhibiting pneumonia and it was shown that patients developed antibodies to this amoebal pathogen. Since then, there have been several proven cases of human infection or colonization with giant viruses of amoebae, which are known to host several bacteria that are human pathogens. Mimiviruses and marseilleviruses represent a major challenge in human pathology, as virological procedures implemented to date have not used appropriate media to allow their culture, and molecular techniques have used filtration steps that likely prevented their detection. Nevertheless, there is an increasing body of evidence that mimiviruses might cause pneumonia and that humans carry marseilleviruses, and re-analyses of metagenomic databases have provided evidence that these giant viruses can be common in human samples. The proportion of human infections related to these giant mimiviruses and marseilleviruses and the precise short- and long-term consequences of these infections have been scarcely investigated so far and should be the subject of future works. PMID:24157884

  1. Corneal ulcers and infections

    MedlinePlus

    Bacterial keratitis; Fungal keratitis; Acanthamoeba keratitis; Herpes simplex keratitis ... occur in people with a suppressed immune system. Herpes simplex keratitis is a serious viral infection. It ...

  2. Infection cycles of large DNA viruses: Emerging themes and underlying questions

    SciTech Connect

    Mutsafi, Yael Fridmann-Sirkis, Yael; Milrot, Elad; Hevroni, Liron; Minsky, Abraham

    2014-10-15

    The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway.

  3. Use of Phage Antibodies to Distinguish Closely Related Species of Protozoan Parasites

    PubMed Central

    Paget, Timothy; Khan, Naveed; Temple, Graham; Hough, Victoria; Greenman, John

    2000-01-01

    Acanthamoeba are typically identified in the laboratory using culture and microscopic observation. In this paper we describe the isolation and specificity of antibody fragments that can be used for the identification of Acanthamoeba. A phage library expressing a large repertoire (approx. 5×109) of antibody fragments was used to generate two libraries one enriched for bacteriophage that exhibit genus specific binding and the other containing bacteriophage that bind specifically to pathogenic Acanthamoeba. Individual clones were isolated on the basis of binding by ELISA, and then flowcytometry and immunofluorescence were used for further characterisation. Four monoclonal antibodies were isolated, specific for Acanthamoeba at the generic level with clone HPPG6 exhibiting the highest level of binding. Furthermore clone HPPG55 was specific for pathogenic species of Acanthamoeba. PMID:11360833

  4. Chapter 42. Waterborne and Foodborne Parasites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter identifies the most prominent parasites in North America that are acquired through contaminated food and water including protozoa (Acanthamoeba, Naegleria, Entamoeba, Giardia, Cryptosporidium, Cystoisospora, Cyclospora, Toxoplasma, and Balantidium), nematodes (Trichinella, Angiostrongyl...

  5. Alphabetical Index of Parasitic Diseases

    MedlinePlus

    ... Infection ) Back To Top K Kala-azar (Leishmaniasis, Leishmania Infection) Keratitis ( Acanthamoeba Infection) Back To Top L Leishmaniasis (Kala-azar, Leishmania Infection) Lice Infestation (Body, Head, or Pubic Lice, ...

  6. Role of Contact Lens Wear, Bacterial Flora, and Mannose-Induced Pathogenic Protease in the Pathogenesis of Amoebic Keratitis

    PubMed Central

    Alizadeh, Hassan; Neelam, Sudha; Hurt, Michael; Niederkorn, Jerry Y.

    2005-01-01

    The ocular surface is continuously exposed to potential pathogens, including free-living amoebae. Acanthamoeba species are among the most ubiquitous amoebae, yet Acanthamoeba keratitis is remarkably rare. The pathogenesis of Acanthamoeba keratitis is a complex, sequential process. Here we show that Acanthamoeba keratitis is profoundly affected by mannosylated proteins on the ocular surface, which stimulate the amoebae to elaborate a 133-kDa pathogenic protease. The mannose-induced protease (MIP133) mediates apoptosis of the corneal epithelium, facilitates corneal invasion, and degrades the corneal stroma. We show that contact lens wear upregulates mannosylated proteins on the corneal epithelium, stimulates MIP133 secretion, and exacerbates corneal disease. Corynebacterium xerosis, a constituent of the ocular flora, contains large amounts of mannose and is associated with Acanthamoeba keratitis. The present results show that amoebae exposed to C. xerosis produce increased amounts of MIP133 and more severe corneal disease. Oral immunization with MIP133 mitigates Acanthamoeba keratitis and demonstrates the feasibility of antidisease vaccines for pathogens that resist immune elimination. PMID:15664950

  7. Structural studies of the Sputnik virophage.

    PubMed

    Sun, Siyang; La Scola, Bernard; Bowman, Valorie D; Ryan, Christopher M; Whitelegge, Julian P; Raoult, Didier; Rossmann, Michael G

    2010-01-01

    The virophage Sputnik is a satellite virus of the giant mimivirus and is the only satellite virus reported to date whose propagation adversely affects its host virus' production. Genome sequence analysis showed that Sputnik has genes related to viruses infecting all three domains of life. Here, we report structural studies of Sputnik, which show that it is about 740 A in diameter, has a T=27 icosahedral capsid, and has a lipid membrane inside the protein shell. Structural analyses suggest that the major capsid protein of Sputnik is likely to have a double jelly-roll fold, although sequence alignments do not show any detectable similarity with other viral double jelly-roll capsid proteins. Hence, the origin of Sputnik's capsid might have been derived from other viruses prior to its association with mimivirus. PMID:19889775

  8. The role of giant viruses of amoebas in humans.

    PubMed

    Colson, Philippe; Aherfi, Sarah; La Scola, Bernard; Raoult, Didier

    2016-06-01

    Since 2003, dozens of giant viruses that infect amoebas (GVA), including mimiviruses and marseilleviruses, have been discovered. These giants appear to be common in our biosphere. From the onset, their presence and possible pathogenic role in humans have been serendipitously observed or investigated using a broad range of technological approaches, including culture, electron microscopy, serology and various techniques based on molecular biology. The link between amoebal mimiviruses and pneumonia has been the most documented, with findings that fulfill several of the criteria considered as proof of viral disease causation. Regarding marseilleviruses, they have been mostly described in asymptomatic persons, and in a lymph node adenitis. The presence and impact of GVA in humans undoubtedly deserve further investigation in medicine. PMID:27131020

  9. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life

    PubMed Central

    Yutin, Natalya; Wolf, Yuri I.; Koonin, Eugene V.

    2015-01-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the “Megavirales”, there are three groups of giant viruses with genomes exceeding 500 kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5 Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the “Megavirales”. The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the “Megavirales” indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts. PMID:25042053

  10. Infections with free-living amebae.

    PubMed

    Visvesvara, Govinda S

    2013-01-01

    Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri are mitochondria-bearing, free-living eukaryotic amebae that have been known to cause infections of the central nervous system (CNS) of humans and other animals. Several species of Acanthamoeba belonging to several different genotypes cause an insidious and chronic disease, granulomatous amebic encephalitis (GAE), principally in immunocompromised hosts including persons infected with HIV/AIDS. Acanthamoeba spp., belonging to mostly group 2, also cause infection of the human cornea, Acanthamoeba keratitis. Balamuthia mandrillaris causes GAE in both immunocompromised and immunocompetent hosts mostly in the very young or very old individuals. Both Acanthamoeba spp. and B. mandrillaris also cause a disseminated disease including the lungs, skin, kidneys, and uterus. Naegleria fowleri, on the other hand, causes an acute and fulminating, necrotizing infection of the CNS called primary amebic meningoencephalitis (PAM) in children and young adults with a history of recent exposure to warm fresh water. Additionally, another free-living ameba Sappinia pedata, previously described as S. diploidea, also has caused a single case of amebic meningoencephalitis. In this review the biology of these amebae, clinical manifestations, molecular and immunological diagnosis, and epidemiological features associated with GAE and PAM are discussed. PMID:23829906

  11. Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba▿

    PubMed Central

    Lorenzo-Morales, Jacob; Martín-Navarro, Carmen M.; López-Arencibia, Atteneri; Santana-Morales, María A.; Afonso-Lehmann, Raquel N.; Maciver, Sutherland K.; Valladares, Basilio; Martínez-Carretero, Enrique

    2010-01-01

    Pathogenic strains of the genus Acanthamoeba are causative agents of severe infections, such as fatal encephalitis and a sight-threatening amoebic keratitis. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic, due to the existence of a highly resistant cyst stage in these amoebae. In previous studies, small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase from Acanthamoeba were designed and evaluated for future therapeutic use. The silencing of proteases resulted in Acanthamoeba failing to degrade human corneal cells, and silencing of glycogen phosphorylase caused amoebae to be unable to form mature cysts. After the siRNA design and concentration were optimized in order to avoid toxicity problems, cultures of Acanthamoeba were treated with a combination of both siRNAs, and cells were evaluated under an inverted microscope. This siRNA-based treatment dramatically affected the growth rate and cellular survival of the amoebae. These results were observed less than 48 h after the initiation of the treatment. In order to check possible toxic effects of the siRNA combination, three eukaryotic cell lines (HeLa, murine macrophages, and osteosarcoma cells) were treated with the same molecules, and cytotoxicity was examined by measuring lactate dehydrogenase release. The future use of the combination of these siRNAs is proposed as a potential therapeutic approach against pathogenic strains of Acanthamoeba. PMID:20855732

  12. A year long study of the presence of free living amoeba in Spain.

    PubMed

    Magnet, A; Fenoy, S; Galván, A L; Izquierdo, F; Rueda, C; Fernandez Vadillo, C; Del Aguila, C

    2013-12-01

    Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this

  13. Vannellid Species Isolated from Freshwater Source in a Park in Jamaica, West Indies

    PubMed Central

    Todd, Cheridah D.; Reyes-Batlle, María; Valladares, Basilio; Lindo, John F.; Lorenzo-Morales, Jacob

    2015-01-01

    Free-living amoebae (FLA) occupy a wide range of freshwater, marine, and soil habitats, and are opportunistic pathogens in human beings. While Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are well-known opportunistic organisms, Vannella epipetala is nonpathogenic. Sediments were collected from a freshwater source from a park in Jamaica to investigate the presence of FLA. Acanthamoeba and Naegleria spp. were not recovered; however, a Vannellid species identified by microscopy and PCR analysis as V. epipetala was isolated. These nonpathogens pose a threat to human beings as they may act as Trojan horses for microsporidian parasites and other pathogens, thereby facilitating their transmission to human beings. PMID:26512204

  14. Vannellid Species Isolated from Freshwater Source in a Park in Jamaica, West Indies.

    PubMed

    Todd, Cheridah D; Reyes-Batlle, María; Valladares, Basilio; Lindo, John F; Lorenzo-Morales, Jacob

    2015-01-01

    Free-living amoebae (FLA) occupy a wide range of freshwater, marine, and soil habitats, and are opportunistic pathogens in human beings. While Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are well-known opportunistic organisms, Vannella epipetala is nonpathogenic. Sediments were collected from a freshwater source from a park in Jamaica to investigate the presence of FLA. Acanthamoeba and Naegleria spp. were not recovered; however, a Vannellid species identified by microscopy and PCR analysis as V. epipetala was isolated. These nonpathogens pose a threat to human beings as they may act as Trojan horses for microsporidian parasites and other pathogens, thereby facilitating their transmission to human beings. PMID:26512204

  15. Family-Level Sampling of Mitochondrial Genomes in Coleoptera: Compositional Heterogeneity and Phylogenetics.

    PubMed

    Timmermans, Martijn J T N; Barton, Christopher; Haran, Julien; Ahrens, Dirk; Culverwell, C Lorna; Ollikainen, Alison; Dodsworth, Steven; Foster, Peter G; Bocak, Ladislav; Vogler, Alfried P

    2016-01-01

    Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML ("nonhomogeneous") algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees. PMID:26645679

  16. Family-Level Sampling of Mitochondrial Genomes in Coleoptera: Compositional Heterogeneity and Phylogenetics

    PubMed Central

    Timmermans, Martijn J. T. N.; Barton, Christopher; Haran, Julien; Ahrens, Dirk; Culverwell, C. Lorna; Ollikainen, Alison; Dodsworth, Steven; Foster, Peter G.; Bocak, Ladislav; Vogler, Alfried P.

    2016-01-01

    Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML (“nonhomogeneous”) algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees. PMID:26645679

  17. Structural data on hypertrehalosaemic neuropeptides from Cryptocercus punctulatus and Therea petiveriana: how do they fit into the phylogeny of cockroaches?

    PubMed Central

    Gäde, G.; Grandcolas, P.; Kellner, R.

    1997-01-01

    Hypertrehalosaemic neuropeptides from the corpora cardiaca of the cockroaches Cryptocercus punctulatus and Therea petiveriana were structurally analysed to gather phylogenetic information independent from that provided by morphoanatomical data. Isolation of the peptides by liquid chromatography and structural elucidation by Edman degradation and mass spectrometry revealed an identical octapeptide for both species: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2. This peptide, denoted Tem-HrTH, was previously found in tenebrionid beetles and in the cockroach Polyphaga aegyptiaca. Using this information for phylogenetic analysis yielded a peptide tree that supports the previous morphoanatomical data and thus places the woodroach Cryptocercus inside the cockroach subfamily Polyphaginae.

  18. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.

    PubMed

    Iyer, Lakshminarayan M; Balaji, S; Koonin, Eugene V; Aravind, L

    2006-04-01

    A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria

  19. Viruses in close associations with free-living amoebae.

    PubMed

    Scheid, Patrick

    2015-11-01

    As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses. PMID

  20. Investigational drug available directly from CDC for the treatment of infections with free-living amebae.

    PubMed

    2013-08-23

    Infections caused by free-living amebae (FLA) are severe and life-threatening. These infections include primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri and granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba species. Although several drugs have in vitro activity against FLA, mortality from these infections remains>90% despite treatment with combinations of drugs. PMID:23965830

  1. The 474-Kilobase-Pair Complete Genome Sequence of CeV-01B, a Virus Infecting Haptolina (Chrysochromulina) ericina (Prymnesiophyceae)

    PubMed Central

    Gallot-Lavallée, Lucie; Pagarete, António; Legendre, Matthieu; Santini, Sebastien; Sandaa, Ruth-Anne; Himmelbauer, Heinz; Ogata, Hiroyuki; Bratbak, Gunnar

    2015-01-01

    We report the complete genome sequence of CeV-01B, a large double-stranded DNA virus infecting the unicellular marine phytoplankton Haptolina (formerly Chrysochromulina) ericina. CeV-01B and its closest relative Phaeocystis globosa virus define an emerging subclade of the Megaviridae family with smaller genomes and particles than the originally described giant Mimiviridae infecting Acanthamoeba. PMID:26634761

  2. Protozoa: a novel Campylobacter reservoir?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous in vitro studies we found that Campylobacter jejuni remained viable for longer periods of time when they were cultivated in the presence of Tetrahymena pyriformis (ciliate) and Acanthamoeba castellanii (amoeba) than when they were in an independent planktonic state. Increased survival t...

  3. Presence of free-living amoebae in portable and stationary eye wash stations

    SciTech Connect

    Tyndall, R.L.; Lyle, M.M.; Ironside, K.S.

    1987-11-01

    Portable and stationary eye wash stations were tested for the presence of free-living amoebae. Such amoebae may be found in potable waters, and at least one genera, the Acanthamoebae, can cause severe infections when introduced into traumatized eyes. Concentrates or filtrates of water from eye wash stations were placed on nonnutrient agar plates seeded with Escherichia coli. Resultant outgrowths of free-living amoebae, which were morphologically identified as mixtures of Hartmannella and Acanthamoebae, were inoculated intranasally into weanling mice. Subsequently, brain and lung tissues from inject mice were tested for amoebae as an indication of persistent infection. Acanthamoebae and Hartmannella were detected in some eye wash stations at each of four test sites. Both portable and stationary stations harbored Acanthamoebae. Some of the isolates caused persistent pulmonary infection but were not isolated from brain tissue. Flushing stationary eye wash stations temporarily reduced the number of stations positive for amoebae. Treatment of portable stations with 25 ppm of free chlorine also reduced the number of stations harboring amoebae but caused corrosion in some of the stations.

  4. Draft Genome Sequence of Tokyovirus, a Member of the Family Marseilleviridae Isolated from the Arakawa River of Tokyo, Japan

    PubMed Central

    2016-01-01

    Members of the Marseilleviridae family are large DNA viruses with icosahedral particles that infect Acanthamoeba cells. This report presents a new Marseilleviridae family member discovered in a water/soil sample from a river in Tokyo, named Tokyovirus, with genome size of 370 to 380 kb. PMID:27284144

  5. Use of cell cultures as an indicator of pathogenicity of free-living amoebae.

    PubMed Central

    Cursons, R T; Brown, T J

    1978-01-01

    Results comparing the time needed for the development of cytopathic effects in cell cultures with that needed to cause death in mice using inocula of Naegleria and Acanthamoeba are presented. The significance of the source and concentration of the inocula is demonstrated. The use of cell cultures as an indicator of the pathogenicity of free-living amoebae is discussed. Images PMID:342543

  6. Complete Genome Sequence of a New Member of the Marseilleviridae Recovered from the Brackish Submarine Spring in the Cassis Port-Miou Calanque, France

    PubMed Central

    Doutre, Gabriel; Arfib, Bruno; Rochette, Pierre; Bonin, Patricia

    2015-01-01

    Marseilleviridae is a rapidly expanding family of Acanthamoeba-infecting large DNA viruses distributed worldwide. We report here the complete 349-kbp genome sequence of Port-Miou virus, which is surprisingly close to that of Lausannevirus (isolated from the Seine River upstream from Paris, France), despite the strong dissimilarities of their sampling locations. PMID:26607881

  7. Cutaneous acanthamoebiasis in AIDS.

    PubMed

    Torno, M S; Babapour, R; Gurevitch, A; Witt, M D

    2000-02-01

    Acanthamoeba is a recognized pathogen in the immunocompromised patient, commonly presenting as chronic or subacute encephalitis. However, cutaneous disease in the absence of CNS involvement is increasingly recognized, especially in the setting of chronic, nonhealing skin lesions in the patient with AIDS. We describe a patient with AIDS and cutaneous acanthamoebiasis and review our experience with treatment and diagnosis. PMID:10640931

  8. Absence of Chlamydia-like organisms in pigs.

    PubMed

    Van Gils, M; Aeby, S; Vanrompay, D; Greub, G

    2015-09-01

    Porcine reproductive failure, especially abortion, causes significant economic loss in the pig industry. Waddlia chondrophila and Parachlamydia acanthamoebae are potential abortigenic agents for pigs. Therefore, we investigated the presence of these two Chlamydia-like organisms in abortion-related samples originating from Belgian pig farms. All investigated samples remained negative. PMID:26137311

  9. Absence of Chlamydia-like organisms in pigs

    PubMed Central

    Van Gils, M.; Aeby, S.; Vanrompay, D.; Greub, G.

    2015-01-01

    Porcine reproductive failure, especially abortion, causes significant economic loss in the pig industry. Waddlia chondrophila and Parachlamydia acanthamoebae are potential abortigenic agents for pigs. Therefore, we investigated the presence of these two Chlamydia-like organisms in abortion-related samples originating from Belgian pig farms. All investigated samples remained negative. PMID:26137311

  10. Microbial diversities (16S and 18S rDNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

    EPA Science Inventory

    Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, e.g. Legionella pneumophila, via parasitization of free-living amoebae such as Acanthamoebae. Yet knowledge about the microbial composition of DW biofilms developed on common in-premise pl...

  11. Occurrence of free-living amoebae in streams of the Mexico Basin.

    PubMed

    Bonilla-Lemus, Patricia; Caballero Villegas, Adán S; Carmona Jiménez, Javier; Lugo Vázquez, Alfonso

    2014-11-01

    Free-living amoebae (FLA) are protozoa that are widely distributed in nature. They are important in the cycling of nutrients in aquatic food chains, but their distribution in natural aquatic environments is not well known. We conducted a survey to determine the presence and distribution of FLA and their relation to some physicochemical parameters in streams of the Mexico Basin in Central Mexico. Thirty-two sites from 18 streams were sampled. Samples were centrifuged and cultured onto NNA-media to isolate amoebae. Identifications were based on morphology. The pathogenicity of Acanthamoeba isolates was tested. Oxygen saturation, temperature, pH, specific conductance, water flow, dissolved reactive phosphorus, nitrites, nitrates, ammonia, dissolved inorganic nitrogen and coliforms were determined. One hundred-and-twenty FLA representing 18 genera were identified. The most frequent genera were Vannella, Rosculus and Acanthamoeba. The frequency of potentially pathogenic FLA was low and only 3 Acanthamoeba isolates were invasive in mice. The highest species richness of FLA was found in streams located into agriculture activity areas and those close to small villages that discharge wastewater into them. Water temperatures were always below 17°C. Oxygen saturation and pH were within the limits for the growth of most FLA. The presence of Acanthamoeba and Naegleria was low; nevertheless, they include potentially pathogenic species and can act as vectors and reservoirs for microbial pathogens and can produce human infections. PMID:25014111

  12. Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba.

    PubMed Central

    Alizadeh, H; Pidherney, M S; McCulley, J P; Niederkorn, J Y

    1994-01-01

    Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis. Images PMID:8132336

  13. Complete Genome Sequence of a New Member of the Marseilleviridae Recovered from the Brackish Submarine Spring in the Cassis Port-Miou Calanque, France.

    PubMed

    Doutre, Gabriel; Arfib, Bruno; Rochette, Pierre; Claverie, Jean-Michel; Bonin, Patricia; Abergel, Chantal

    2015-01-01

    Marseilleviridae is a rapidly expanding family of Acanthamoeba-infecting large DNA viruses distributed worldwide. We report here the complete 349-kbp genome sequence of Port-Miou virus, which is surprisingly close to that of Lausannevirus (isolated from the Seine River upstream from Paris, France), despite the strong dissimilarities of their sampling locations. PMID:26607881

  14. Draft Genome Sequence of Tokyovirus, a Member of the Family Marseilleviridae Isolated from the Arakawa River of Tokyo, Japan.

    PubMed

    Takemura, Masaharu

    2016-01-01

    Members of the Marseilleviridae family are large DNA viruses with icosahedral particles that infect Acanthamoeba cells. This report presents a new Marseilleviridae family member discovered in a water/soil sample from a river in Tokyo, named Tokyovirus, with genome size of 370 to 380 kb. PMID:27284144

  15. Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus

    PubMed Central

    Lartigue, Audrey; Jeudy, Sandra; Bertaux, Lionel; Abergel, Chantal

    2013-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF. PMID:23295487

  16. Sputnik, a virophage infecting the viral domain of life.

    PubMed

    Desnues, Christelle; Boyer, Mickaël; Raoult, Didier

    2012-01-01

    This chapter discusses the astonishing discovery of the Sputnik virophage, a new virus infecting giant viruses of the genera Mimivirus and Mamavirus. While other virophages have also since been described, this chapter focuses mainly on Sputnik, which is the best described. We detail the general properties of the virophage life cycle, as well as its hosts, genomic characteristics, ecology, and origin. In addition to genetic, phylogenetic, and structural evidence, the existence of virophages has deeply altered our view of the tripartite division of life to include the addition of a fourth domain constituted of the nucleocytoplasmic large DNA viruses, an important point that is discussed. PMID:22420851

  17. The origins of giant viruses, virophages and their relatives in host genomes

    PubMed Central

    2014-01-01

    Giant viruses have revealed a number of surprises that challenge conventions on what constitutes a virus. The Samba virus newly isolated in Brazil expands the known distribution of giant mimiviruses to a near-global scale. These viruses, together with the transposon-related virophages that infect them, pose a number of questions about their evolutionary origins that need to be considered in the light of the complex entanglement between host, virus and virophage genomes. See research article: http://www.virologyj.com/content/11/1/95. PMID:25184667

  18. Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus.

    PubMed

    Lartigue, Audrey; Jeudy, Sandra; Bertaux, Lionel; Abergel, Chantal

    2013-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF. PMID:23295487

  19. Structures of giant icosahedral eukaryotic dsDNA viruses

    PubMed Central

    Xiao, Chuan; Rossmann, Michael G.

    2011-01-01

    In the last twenty years, numerous giant, dsDNA, icosahedral viruses have been discovered and assigned to the nucleocytoplasmic large dsDNA virus (NCLDV) clade. The major capsid proteins of these viruses consist of two consecutive jelly-roll domains, assembled into trimers, with pseudo 6-fold symmetry. The capsomers are assembled into arrays that have either p6 (as in Paramecium bursaria Chlorella virus-1) or p3 symmetry (as in Mimivirus). Most of the NCLDV viruses have a membrane that separates the nucleocapsid from the external capsid. PMID:21909343

  20. The fossil record and macroevolutionary history of the beetles

    PubMed Central

    Smith, Dena M.; Marcot, Jonathan D.

    2015-01-01

    Coleoptera (beetles) is the most species-rich metazoan order, with approximately 380 000 species. To understand how they came to be such a diverse group, we compile a database of global fossil beetle occurrences to study their macroevolutionary history. Our database includes 5553 beetle occurrences from 221 fossil localities. Amber and lacustrine deposits preserve most of the beetle diversity and abundance. All four extant suborders are found in the fossil record, with 69% of all beetle families and 63% of extant beetle families preserved. Considerable focus has been placed on beetle diversification overall, however, for much of their evolutionary history it is the clade Polyphaga that is most responsible for their taxonomic richness. Polyphaga had an increase in diversification rate in the Early Cretaceous, but instead of being due to the radiation of the angiosperms, this was probably due to the first occurrences of beetle-bearing amber deposits in the record. Perhaps, most significant is that polyphagan beetles had a family-level extinction rate of zero for most of their evolutionary history, including across the Cretaceous–Palaeogene boundary. Therefore, focusing on the factors that have inhibited beetle extinction, as opposed to solely studying mechanisms that may promote speciation, should be examined as important determinants of their great diversity today. PMID:25788597

  1. A Comparative Analysis of Mitochondrial Genomes in Coleoptera (Arthropoda: Insecta) and Genome Descriptions of Six New Beetles

    PubMed Central

    Song, H.; Cameron, S. L.; Whiting, M. F.

    2008-01-01

    Coleoptera is the most diverse group of insects with over 360,000 described species divided into four suborders: Adephaga, Archostemata, Myxophaga, and Polyphaga. In this study, we present six new complete mitochondrial genome (mtgenome) descriptions, including a representative of each suborder, and analyze the evolution of mtgenomes from a comparative framework using all available coleopteran mtgenomes. We propose a modification of atypical cox1 start codons based on sequence alignment to better reflect the conservation observed across species as well as findings of TTG start codons in other genes. We also analyze tRNA-Ser(AGN) anticodons, usually GCU in arthropods, and report a conserved UCU anticodon as a possible synapomorphy across Polyphaga. We further analyze the secondary structure of tRNA-Ser(AGN) and present a consensus structure and an updated covariance model that allows tRNAscan-SE (via the COVE software package) to locate and fold these atypical tRNAs with much greater consistency. We also report secondary structure predictions for both rRNA genes based on conserved stems. All six species of beetle have the same gene order as the ancestral insect. We report noncoding DNA regions, including a small gap region of about 20 bp between tRNA-Ser(UCN) and nad1 that is present in all six genomes, and present results of a base composition analysis. PMID:18779259

  2. The fossil record and macroevolutionary history of the beetles.

    PubMed

    Smith, Dena M; Marcot, Jonathan D

    2015-04-22

    Coleoptera (beetles) is the most species-rich metazoan order, with approximately 380 000 species. To understand how they came to be such a diverse group, we compile a database of global fossil beetle occurrences to study their macroevolutionary history. Our database includes 5553 beetle occurrences from 221 fossil localities. Amber and lacustrine deposits preserve most of the beetle diversity and abundance. All four extant suborders are found in the fossil record, with 69% of all beetle families and 63% of extant beetle families preserved. Considerable focus has been placed on beetle diversification overall, however, for much of their evolutionary history it is the clade Polyphaga that is most responsible for their taxonomic richness. Polyphaga had an increase in diversification rate in the Early Cretaceous, but instead of being due to the radiation of the angiosperms, this was probably due to the first occurrences of beetle-bearing amber deposits in the record. Perhaps, most significant is that polyphagan beetles had a family-level extinction rate of zero for most of their evolutionary history, including across the Cretaceous-Palaeogene boundary. Therefore, focusing on the factors that have inhibited beetle extinction, as opposed to solely studying mechanisms that may promote speciation, should be examined as important determinants of their great diversity today. PMID:25788597

  3. High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences.

    PubMed

    Yuan, Ming-Long; Zhang, Qi-Lin; Zhang, Li; Guo, Zhong-Long; Liu, Yong-Jian; Shen, Yu-Ying; Shao, Renfu

    2016-11-01

    The Coleoptera (beetles) exhibits tremendous morphological, ecological, and behavioral diversity. To better understand the phylogenetics and evolution of beetles, we sequenced three complete mitogenomes from two families (Cleridae and Meloidae), which share conserved mitogenomic features with other completely sequenced beetles. We assessed the influence of six datasets and three inference methods on topology and nodal support within the Coleoptera. We found that both Bayesian inference and maximum likelihood with homogeneous-site models were greatly affected by nucleotide compositional heterogeneity, while the heterogeneous-site mixture model in PhyloBayes could provide better phylogenetic signals for the Coleoptera. The amino acid dataset generated more reliable tree topology at the higher taxonomic levels (i.e. suborders and series), where the inclusion of rRNA genes and the third positions of protein-coding genes improved phylogenetic inference at the superfamily level, especially under a heterogeneous-site model. We recovered the suborder relationships as (Archostemata+Adephaga)+(Myxophaga+Polyphaga). The series relationships within Polyphaga were recovered as (Scirtiformia+(Elateriformia+((Bostrichiformia+Scarabaeiformia+Staphyliniformia)+Cucujiformia))). All superfamilies within Cucujiformia were recovered as monophyletic. We obtained a cucujiform phylogeny of (Cleroidea+(Coccinelloidea+((Lymexyloidea+Tenebrionoidea)+(Cucujoidea+(Chrysomeloidea+Curculionoidea))))). This study showed that although tree topologies were sensitive to data types and inference methods, mitogenomic data could provide useful information for resolving the Coleoptera phylogeny at various taxonomic levels by using suitable datasets and heterogeneous-site models. PMID:27497607

  4. Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

    PubMed

    Milrot, Elad; Mutsafi, Yael; Fridmann-Sirkis, Yael; Shimoni, Eyal; Rechav, Katya; Gurnon, James R; Van Etten, James L; Minsky, Abraham

    2016-01-01

    The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway. PMID:26248343

  5. Open membranes are the precursors for assembly of large DNA viruses.

    PubMed

    Suárez, Cristina; Welsch, Sonja; Chlanda, Petr; Hagen, Wim; Hoppe, Simone; Kolovou, Androniki; Pagnier, Isabelle; Raoult, Didier; Krijnse Locker, Jacomine

    2013-11-01

    Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an 'open-eight' conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis. PMID:23751082

  6. A decade of improvements in Mimiviridae and Marseilleviridae isolation from amoeba.

    PubMed

    Pagnier, Isabelle; Reteno, Dorine-Gaelle Ikanga; Saadi, Hanene; Boughalmi, Mondher; Gaia, Morgan; Slimani, Meriem; Ngounga, Tatsiana; Bekliz, Meriem; Colson, Philippe; Raoult, Didier; La Scola, Bernard

    2013-01-01

    Since the isolation of the first giant virus, the Mimivirus, by T.J. Rowbotham in a cooling tower in Bradford, UK, and after its characterisation by our group in 2003, we have continued to develop novel strategies to isolate additional strains. By first focusing on cooling towers using our original time-consuming procedure, we were able to isolate a new lineage of giant virus called Marseillevirus and a new Mimivirus strain called Mamavirus. In the following years, we have accumulated the world's largest unique collection of giant viruses by improving the use of antibiotic combinations to avoid bacterial contamination of amoeba, developing strategies of preliminary screening of samples by molecular methods, and using a high-throughput isolation method developed by our group. Based on the inoculation of nearly 7,000 samples, our collection currently contains 43 strains of Mimiviridae (14 in lineage A, 6 in lineage B, and 23 in lineage C) and 17 strains of Marseilleviridae isolated from various environments, including 3 of human origin. This study details the procedures used to build this collection and paves the way for the high-throughput isolation of new isolates to improve the record of giant virus distribution in the environment and the determination of their pangenome. PMID:24157882

  7. Open membranes are the precursors for assembly of large DNA viruses

    PubMed Central

    Suárez, Cristina; Welsch, Sonja; Chlanda, Petr; Hagen, Wim; Hoppe, Simone; Kolovou, Androniki; Pagnier, Isabelle; Raoult, Didier; Locker, Jacomine Krijnse

    2014-01-01

    Summary Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convex side of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open-eight’ conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis. PMID:23751082

  8. Detection and identification of free-living amoeba from aquatic environment in different seasons in Taiwan

    NASA Astrophysics Data System (ADS)

    Tzeng, K.; Hsu, B.; Tsai, H.; Huang, P.; Tsai, J.; Kao, P.; Huang, K.; Chen, J.

    2013-12-01

    Free-living amoeba includes Acanthamoeba and Naegleria, which are widely distributed in water and soil. Human infection with free-living amoeba leads to serious illness, even lethal. For example, central nervous system infection will cause amoebic meningoencephalitis, and infections will cause amoebic keratitis. The presence of free-living amoeba in environment water can be used as a water quality indicator in ecosystem assessment. In Taiwan, reservoirs are indispensable because of the water source are limited by the steep terrain and the short river flow. Therefore, we need to pay more attention in the quality control of reservoirs water. The aims of this study are to investigate the presence of free-living amoeba in Taiwan reservoirs, and to compare the differences among seasons. At last, the identification and genotyping of Acanthamoeba and Naegleria are investigated. In this study, we use polymerase chain reaction with specific primers to analyze the presence of free-living amoeba in aquatic environment. We collected total 60 samples from reservoirs in Taiwan. The water samples are divided into two parts for both direct concentration method and culture method. The results show the different detection rates among seasons. For Acanthamoeba, the detection rates were 28.3% (17 of 60 water samples), 21.7% (13 of 60 water samples) and 8.3% (5 of 60 water samples) in autumn, winter and spring, respectively. For Naegleria, the detection rates were 6.7% (4 of 60 water samples), 0% (0 of 60 water samples) and 0% (0 of 60 water samples) were detected positive in autumn, winter and spring, respectively. Sequence analysis showed that the major genotypes in Acanthamoeba were T3, T4, T10 and T11 in autumn, T2, T4 and T10 in winter, T4 in spring. Due to the presences of Acanthamoeba and Naegleria in reservoirs, we should pay more attention in water quality monitoring to prevent the potential risks of diseases. Keywords: free-living amoeba, Acanthamoeba, Naegleria, polymerase

  9. In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures.

    PubMed

    Yera, Hélène; Dupouy-Camet, Jean; Jackson, Jonathan W; Sriram, Rama; Sweat, Stacey; Goldstein, Jason M; Visvesvara, Govinda S

    2015-09-01

    Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison. PMID:25980370

  10. Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods.

    PubMed

    Armand, B; Motazedian, M H; Asgari, Q

    2016-01-01

    Free-living amoebae (FLA) are the most abundant and widely distributed protozoa in the environment. An investigation was conducted to determine the presence of free-living amoebae (FLA), Acanthamoeba and Vermamoeba in waterfronts of parks and squares and tap water of Shiraz City, Iran. FLA are considered pathogenic for human. These ubiquitous organisms have been isolated from different environments such as water, soil, and air. Eighty-two water samples were collected from different places of Shiraz City during the summer of 2013. All samples were processed in Dept. of Parasitology and Mycology, Shiraz University of Medical Sciences, Fars, Iran. Samples were screened for FLA and identified by morphological characters in the cultures, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Overall, 48 samples were positive for Acanthamoeba and Vermamoeba in non-nutrient agar culture based on morphological characteristics. The PCR examination was done successfully. Sequencing results were revealed T4 (62.96 %) genotypes as the most common genotype of Acanthamoeba in the Shiraz water sources. In addition, T5 (33.33 %) and T15 (3.71 %) were isolated from water supplies. Vermamoeba vermiformis was known the dominant species from this genus. The high frequency of Acanthamoeba spp. and Vermamoeba in different environmental water sources of Shiraz is an alert for the public health related to water sources. The result highlights a need for taking more attention to water supplies in order to prevent illnesses related to free-living amoebae. PMID:26412057

  11. A proposed cascade of vascular events leading to granulomatous amoebic encephalitis.

    PubMed

    Baig, Abdul Mannan; Khan, Naveed Ahmed

    2015-11-01

    Granulomatous amoebic encephalitis due to Acanthamoeba is a chronic disease that almost always results in death. Hematogenous spread is a pre-requisite followed by amoebae invasion of the blood-brain barrier to enter the central nervous system. Given the systemic nature of this infection, a significant latent period of several months before the appearance of clinical manifestations is puzzling. Based on reported cases, here we propose pathogenetic mechanisms that explain the above described latency of the disease. PMID:26276705

  12. Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

    PubMed

    Trabelsi, Houaida; Dendana, F; Neji, S; Sellami, H; Cheikhrouhou, F; Makni, F; Ayadi, A

    2016-01-01

    Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial. PMID:26468150

  13. Temperature tolerance of pathogenic and nonpathogenic free-living amoebas.

    PubMed

    Griffin, J L

    1972-11-24

    Within tested strains of the genera Naegleria and Acanthamoeba the ability to grow at high temperatures seems directly related to virulence, with nonvirulent strains unable to grow at normal or elevated body temperatures. Outside these genera, nonvirulent Hartmannella and Tetramitus do grow at elevated temperatures, which suggests a barrier to pathogenicity other than temperature sensitivity. The high optimal temperature of pathogenic Naegleria apparently explains previous difficulty in obtaining isolates from the aquatic environment. PMID:5085984

  14. Balamuthia mandrillaris: in vitro interactions with selected protozoa and algae.

    PubMed

    Tapia, José L; Torres, Benjamin Nogueda; Visvesvara, Govinda S

    2013-01-01

    Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one-on-one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp. PMID:23790262

  15. Detection and Molecular Characterization of Potentially Pathogenic Free-living Amoebae from Water Sources in Kish Island, Southern Iran

    PubMed Central

    Niyyati, Maryam; Lasgerdi, Zohreh; Lorenzo-Morales, Jacob

    2015-01-01

    Amoebic keratitis, a sight-threatening corneal infection, mainly occurs in contact lens wearers who wash their eyes with tap water. The present research was conducted to identify the occurrence of potentially pathogenic free-living amoebae (FLA) in tap water sources on Kish Island, a tourist region in Iran. Amoebae were detected using a culture-enriched method and by polymerase chain reaction (PCR)/sequencing of the diagnostic fragment 3 region of the 18S rRNA gene of Acanthamoeba. In the case of other free-living amoebae species, PCR/sequencing analysis of the 18S rDNA was conducted. Results of this study showed the presence of Acanthamoeba belonging to T3, T4, T5, and T11 genotypes in tap water sources. Additionally, Vermamoebae vermiformis was detected in three water samples. This is the first report of the Acanthamoeba genotypes T3, T4, T5, and T11 and V. vermiformis species in tap water sources in a tourist region in Iran. PMID:25922581

  16. Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau.

    PubMed

    Baquero, Rocío A; Reyes-Batlle, María; Nicola, Graciela G; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Guillermo Esteban, J; Valladares, Basilio; Martínez-Carretero, Enrique; Piñero, José E; Lorenzo-Morales, Jacob

    2014-06-01

    Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa. PMID:24934796

  17. Detection of Balamuthia mandrillaris DNA in the storage case of contact lenses in Germany.

    PubMed

    Balczun, Carsten; Scheid, Patrick L

    2016-05-01

    Acanthamoeba spp. are frequently the etiological agents of a severe form of sight-threatening keratitis, called Acanthamoeba keratitis. The contact lens storage solution of a patient with keratitis of unknown genesis was screened using our diagnostic tools to detect potentially pathogenic free-living amoebae (FLA). Culture methods and a triplex quantitative real-time polymerase chain reaction (qPCR) targeting Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris were used in context of this routine screening. While no amoebae were detected by culture, qPCR specifically detected DNA of B. mandrillaris. This FLA is known as the etiological agent of a fatal form of encephalitis in humans and other mammals, Balamuthia amoebic encephalitis (BAE). A fragment of the 18S rDNA gene was amplified from the sample and showed 99 % sequence identity to B. mandrillaris sequences from GenBank. To the best of our knowledge, this is the first report of B. mandrillaris found in association with contact lenses. Although no viable amoeba was obtained by culturing efforts, the verification of B. mandrillaris DNA in the contact lens storage solution demonstrates how easily this pathogen might come into close contact with humans. PMID:26965426

  18. A Rickettsiales symbiont of amoebae with ancient features.

    PubMed

    Schulz, Frederik; Martijn, Joran; Wascher, Florian; Lagkouvardos, Ilias; Kostanjšek, Rok; Ettema, Thijs J G; Horn, Matthias

    2016-09-01

    The Rickettsiae comprise intracellular bacterial symbionts and pathogens infecting diverse eukaryotes. Here, we provide a detailed characterization of 'Candidatus Jidaibacter acanthamoeba', a rickettsial symbiont of Acanthamoeba. The bacterium establishes the infection in its amoeba host within 2 h where it replicates within vacuoles. Higher bacterial loads and accelerated spread of infection at elevated temperatures were observed. The infection had a negative impact on host growth rate, although no increased levels of host cell lysis were seen. Phylogenomic analysis identified this bacterium as member of the Midichloriaceae. Its 2.4 Mb genome represents the largest among Rickettsiales and is characterized by a moderate degree of pseudogenization and a high coding density. We found an unusually large number of genes encoding proteins with eukaryotic-like domains such as ankyrins, leucine-rich repeats and tetratricopeptide repeats, which likely function in host interaction. There are a total of three divergent, independently acquired type IV secretion systems, and 35 flagellar genes representing the most complete set found in an obligate intracellular Alphaproteobacterium. The deeply branching phylogenetic position of 'Candidatus Jidaibacter acanthamoeba' together with its ancient features place it closely to the rickettsial ancestor and helps to better understand the transition from a free-living to an intracellular lifestyle. PMID:25908022

  19. Survey of infectious etiologies of bovine abortion during mid- to late gestation in dairy herds.

    PubMed

    Barkallah, Mohamed; Gharbi, Yaakoub; Hassena, Amal Ben; Slima, Ahlem Ben; Mallek, Zouhir; Gautier, Michel; Greub, Gilbert; Gdoura, Radhouane; Fendri, Imen

    2014-01-01

    Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle. PMID:24662769

  20. Application of flow cytometry to studies of pathogenic free-living amoebae.

    PubMed Central

    Muldrow, L L; Tyndall, R L; Fliermans, C B

    1982-01-01

    Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading. PMID:6186196

  1. Free-living amoebae (FLA) co-occurring with legionellae in industrial waters.

    PubMed

    Scheikl, Ute; Sommer, Regina; Kirschner, Alexander; Rameder, Alexandra; Schrammel, Barbara; Zweimüller, Irene; Wesner, Wolfgang; Hinker, Manfred; Walochnik, Julia

    2014-08-01

    Legionella pneumophila is known as the causative agent of Legionnaires' disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000 CFU/100 ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time. PMID:25062389

  2. Effect of Common Drinking Water Disinfectants, Chlorine and Heat, on Free Legionella and Amoebae-Associated Legionella

    PubMed Central

    Cervero-Aragó, Sílvia; Rodríguez-Martínez, Sarah; Puertas-Bennasar, Antoni; Araujo, Rosa M.

    2015-01-01

    Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed. PMID:26241039

  3. Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau

    PubMed Central

    Baquero, Rocío A; Reyes-Batlle, María; Nicola, Graciela G; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Guillermo Esteban, J; Valladares, Basilio; Martínez-Carretero, Enrique; Piñero, José E; Lorenzo-Morales, Jacob

    2014-01-01

    Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa. PMID:24934796

  4. Chromosome comparison of 17 species / sub-species of African Goliathini (Coleoptera, Scarabaeidae, Cetoniinae)

    PubMed Central

    Dutrillaux, Anne-Marie; Dutrillaux, Bernard

    2016-01-01

    Abstract The mitotic karyotypes of 17 species of African Goliathini (Cetoniinae) are described using various chromosome banding techniques. All but one are composed of 20 chromosomes, mostly metacentric, forming a karyotype assumed to be close to that of the Polyphaga ancestor. The most derived karyotypes are those of Goliathus goliatus Drury, 1770, with eight pairs of acrocentrics and Chlorocana africana Drury, 1773, with only14 chromosomes. In species of the genera Cyprolais Burmeister, 1842, Megalorhina Westwood, 1847, Stephanocrates Kolbe, 1894 and Stephanorrhina Burmeister, 1842, large additions of variable heterochromatin are observed on both some particular autosomes and the X chromosome. Species of the genera Eudicella White, 1839 and Dicronorrhina Burmeister, 1842 share the same sub-metacentric X. Although each species possesses its own karyotype, it remains impossible to propose robust phylogenetic relationships on the basis of chromosome data only. PMID:27551348

  5. Reclassification of giant viruses composing a fourth domain of life in the new order Megavirales.

    PubMed

    Colson, Philippe; de Lamballerie, Xavier; Fournous, Ghislain; Raoult, Didier

    2012-01-01

    Interest in giant viruses has risen sharply since 2003, following the discovery of the Mimivirus and four other protist-infecting giant viruses that are linked to the nucleocytoplasmic large DNA viruses (NCLDVs). Despite considerable heterogeneity in hosts and genome sizes, the NCLDVs have been shown to be monophyletic based on analyses of their sequences and gene repertoires and recent studies have proposed that these viruses share a common ancient ancestor and compose a fourth domain of life. In addition, several characteristics of these giant viruses contradict or do not match the criteria used for the canonical definition of viruses, and the NCLDV denomination is not completely appropriate. We propose here to define a new viral order named Megavirales. PMID:22508375

  6. Giants among larges: how gigantism impacts giant virus entry into amoebae.

    PubMed

    Rodrigues, Rodrigo Araújo Lima; Abrahão, Jônatas Santos; Drumond, Betânia Paiva; Kroon, Erna Geessien

    2016-06-01

    The proposed order Megavirales comprises the nucleocytoplasmic large DNA viruses (NCLDV), infecting a wide range of hosts. Over time, they co-evolved with different host cells, developing various strategies to penetrate them. Mimiviruses and other giant viruses enter cells through phagocytosis, while Marseillevirus and other large viruses explore endocytosis and macropinocytosis. These differing strategies might reflect the evolution of those viruses. Various scenarios have been proposed for the origin and evolution of these viruses, presenting one of the most enigmatic issues to surround these microorganisms. In this context, we believe that giant viruses evolved independently by massive gene/size gain, exploring the phagocytic pathway of entry into amoebas. In response to gigantism, hosts developed mechanisms to evade these parasites. PMID:27039270

  7. Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic

    PubMed Central

    Allen, Lisa Zeigler; Allen, Eric E; Badger, Jonathan H; McCrow, John P; Paulsen, Ian T; Elbourne, Liam DH; Thiagarajan, Mathangi; Rusch, Doug B; Nealson, Kenneth H; Williamson, Shannon J; Venter, J Craig; Allen, Andrew E

    2012-01-01

    Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ∼5.8 million predicted proteins across seven sites, from three different size classes: 0.1–0.8, 0.8–3.0 and 3.0–200.0 μm. Taxonomic and metabolic analyses suggest that sequences from the 0.1–0.8 μm size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8–200 μm) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially. PMID:22278668

  8. Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan.

    PubMed

    Tung, Min-Che; Hsu, Bing-Mu; Tao, Chi-Wei; Lin, Wei-Chen; Tsai, Hsiu-Feng; Ji, Dar-Der; Shen, Shu-Min; Chen, Jung-Sheng; Shih, Feng-Cheng; Huang, Yu-Li

    2013-08-01

    Naegleria fowleri can cause primary amoebic meningoencephalitis, a rapidly developing and highly lethal infectious disease. The first confirmed case of primary amoebic meningoencephalitis in Taiwan was reported in November 2011, in which the patient visited a thermal spring recreational area 1 week prior to hospitalisation. Water sampling was performed to verify the presence of Naegleria at the facility. According to our results, 32% and 20% of recreational water samples were contaminated with Naegleria spp. and Acanthamoeba spp., respectively. The genotypes of Naegleria identified at the hot spring included N. fowleri, Naegleria australiensis and Naegleria lovaniensis. Using PCR, it was determined that the strain of N. fowleri in one sample possessed the same genotype 2 as the clinical isolate. Thus, the thermal spring was suggested to be the likely source of infection. This is the first known instance of simultaneously isolating N. fowleri from both a patient as well as from a hot spring in Taiwan. Following this initial study, the pools at the thermal spring recreational area were drained, scrubbed and disinfected, and a follow-up study was performed 1 month later. Naegleria fowleri was not detected in follow-up testing; however, other Naegleria spp. were identified. We postulate that the biofilm in the waterlines may have provided a reservoir for free-living amoebae. The presence/absence of Acanthamoeba and Naegleria spp. did not differ significantly with any measured parameters related to water quality; however, a high percentage of the thermal water pool samples were contaminated with Naegleria or Acanthamoeba. Thus, amoebic contamination may present a serious threat to the health of humans who engage in leisure activities at thermal springs. PMID:23665128

  9. Pathogenesis of amoebic encephalitis: Are the amoebae being credited to an 'inside job' done by the host immune response?

    PubMed

    Baig, Abdul Mannan

    2015-08-01

    Pathogenic free living amoeba like Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal "amoebic meningoencephalitis" by acquiring different route of entries to the brain. The host immune response to these protist pathogens differs from each another, as evidenced by the postmortem gross and microscopic findings from the brains of the affected patients. Cited with the expression of 'brain eating amoeba' when the infection is caused by N. fowleri, this expression is making its way into parasitology journals and books. The impression that it imparts is, as if the brain damage is substantially due to the enzymes and toxins produced by this amoeba. A detailed review of the literature, analysis of archived specimens and with our experimental assays, here we establish that with N. fowleri, Acanthamoeba and Balamuthia spp., the infections result in an extensive brain damage that in fact is substantially caused by the host immune response rather than the amoeba. Due to the comparatively larger sizes of these pathogens and the prior exposure of the amoebal antigen to the human body, the host immune system launches an amplified response that not only breaches the blood brain barrier (BBB), but also becomes the major cause of brain damage in Amoebic meningoencephalitis. It is our understanding that for N. fowleri the host immune response is dominated by acute inflammatory cytokines and that, in cases of Acanthamoeba and Balamuthia spp., it is the type IV hypersensitivity reaction that fundamentally not only contributes to disruption and leakiness of the blood brain barrier (BBB) but also causes the neuronal damage. The further intensification of brain damage is done by toxins and enzymes secreted by the amoeba, which causes the irreversible brain damage. PMID:25930186

  10. Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia

    PubMed Central

    Richard, Reena Leeba; Ithoi, Init; Abd Majid, Mohamad Azlan; Wan Sulaiman, Wan Yusoff; Tan, Tian Chye; Nissapatorn, Veeranoot; Lim, Yvonne Ai Lian

    2016-01-01

    The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites. PMID:27367710

  11. Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

    PubMed

    Richard, Reena Leeba; Ithoi, Init; Abd Majid, Mohamad Azlan; Wan Sulaiman, Wan Yusoff; Tan, Tian Chye; Nissapatorn, Veeranoot; Lim, Yvonne Ai Lian

    2016-01-01

    The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites. PMID:27367710

  12. Development of a new contact lens multipurpose solution: Comparative analysis of microbiological, biological and clinical performance

    PubMed Central

    Kilvington, Simon; Huang, Ling; Kao, Eugenia; Powell, Charles H.

    2010-01-01

    Purpose Antimicrobial, cytotoxicity and clinical performance of a new soft contact lens multi-purpose solution (COMPLETE® RevitaLens) based on polyquaternium-1 and alexidine dihydrochloride (NuMPS) was evaluated. Methods Antimicrobial efficacy was assessed according to ISO 14729 for both biocidal and regimen performance against bacteria and fungi. Acanthamoeba efficacy was tested along with ability to retain antimicrobial activity on partial evaporation. In vitro cytotoxicity of NuMPS and OPTI-FREE® RepleniSH® MPS (MPS-3) was assessed based on ISO 10993-5 and United States Pharmacopeia (USP) methods. In addition, a 3 month, double-masked, parallel group clinical trial comparing safety and acceptability with respect to MPS-3 was conducted with 4 silicone hydrogel (SiHy) and FDA Group IV lens types. Results NuMPS showed broad antimicrobial efficacy, including Acanthamoeba, giving a 3–4 log10 reduction in viability after 6 hours contact time. NuMPS also passed ISO 14729 regimens with SiHy and etafilcon lenses for bacteria, fungi and also Acanthamoeba. The cytotoxicity of NuMPS was equivalent or better compared to MPS-3. In the clinical trial, there was no statistically significant between-group difference in corneal staining (p > 0.05). Patients using MPS-3 had more adverse events than patients using NuMPS: 11.8 % (11/93) versus 2.8 % (5/177), respectively, (p < 0.05). There were no differences noted in cleanliness or wearing comfort (p > 0.05). Conclusion Taken together, the results of these studies indicate that the NuMPS is a novel and effective soft contact lens care solution.

  13. Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species.

    PubMed

    De La Cruz, E M; Pollard, T D

    1996-11-12

    We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C. Filaments of S. cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S. cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments [Kron et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4466-4470]. The temperature dependence of the rate constants was interpreted according to transition state theory. There is a small enthalpic difference (delta H++) between the ground states and the transition state. Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++). At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0). The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length. Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin. The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e. "breathe") to expose the phalloidin binding site [De La Cruz, E. M., & Pollard, T. D. (1994) Biochemistry 33, 14387-14392]. Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more

  14. Occurrence and Control of Legionella in Recycled Water Systems

    PubMed Central

    Jjemba, Patrick K.; Johnson, William; Bukhari, Zia; LeChevallier, Mark W.

    2015-01-01

    Legionella pneumophila is on the United States Environmental Protection Agency (USEPA) Candidate Contaminant list (CCL) as an important pathogen. It is commonly encountered in recycled water and is typically associated with amoeba, notably Naegleria fowleri (also on the CCL) and Acanthamoeba sp. No legionellosis outbreak has been linked to recycled water and it is important for the industry to proactively keep things that way. A review was conducted examine the occurrence of Legionella and its protozoa symbionts in recycled water with the aim of developing a risk management strategy. The review considered the intricate ecological relationships between Legionella and protozoa, methods for detecting both symbionts, and the efficacy of various disinfectants. PMID:26140674

  15. Occurrence and Control of Legionella in Recycled Water Systems.

    PubMed

    Jjemba, Patrick K; Johnson, William; Bukhari, Zia; LeChevallier, Mark W

    2015-01-01

    Legionella pneumophila is on the United States Environmental Protection Agency (USEPA) Candidate Contaminant list (CCL) as an important pathogen. It is commonly encountered in recycled water and is typically associated with amoeba, notably Naegleria fowleri (also on the CCL) and Acanthamoeba sp. No legionellosis outbreak has been linked to recycled water and it is important for the industry to proactively keep things that way. A review was conducted examine the occurrence of Legionella and its protozoa symbionts in recycled water with the aim of developing a risk management strategy. The review considered the intricate ecological relationships between Legionella and protozoa, methods for detecting both symbionts, and the efficacy of various disinfectants. PMID:26140674

  16. Neuroparasitic Infections: Cestodes, Trematodes, and Protozoans

    PubMed Central

    Walker, M.D.; Zunt, J.R.

    2009-01-01

    Parasitic infection of the nervous system can produce a variety of symptoms and signs. Because symptoms of infection are often mild or nonspecific, diagnosis can be difficult. Familiarity with basic epidemiological characteristics and distinguishing radiographic findings can increase the likelihood of detection and proper treatment of parasitic infection of the nervous system. This article discusses the clinical presentation, diagnosis, and treatment for some of the more common infections of the nervous system caused by cestodes, trematodes and protozoans: Echinococcus spp., Spirometra spp. (sparganosis), Paragonimus spp., Schistosoma spp., Trypanosoma spp., Naegleria fowlerii, Acanthamoeba histolytica, and Balamuthia mandrillaris. PMID:16170739

  17. Recurrent corneal ulceration in presence of synthetic microfibrils

    PubMed Central

    Barsam, A; Patel, N; Laganowski, HC; Perry, HD

    2011-01-01

    Recurrence of microbial keratitis in the presence of protozoal infection is very rare and infrequently reported unless predisposing factors are present. The association of recurrent microbial keratitis and synthetic microfibrils has never previously been reported to our knowledge. This single interventional case study describes the clinical course and treatment of a contact lens wearer who was treated for Acanthamoeba keratitis with superinfection from bacterial organisms in the presence of synthetic microfibrils. The presence of synthetic fibrils on a corneal ulcer base may act as a nidus for pathological organisms and interfere with normal corneal healing. This may result in infection recurrence and the growth of resistant opportunistic organisms. PMID:21750618

  18. [IMPACT OF CASPIAN SEA LEVEL FLUCTUATIONS ON THE EPIZOOTIC ACTIVITY OF THE CASPIAN SANDY NATURAL PLAGUE FOCUS].

    PubMed

    Popov, N V; Udovikov, A I; Eroshenko, G A; Karavaeva, T B; Yakovlev, S A; Porshakov, A M; Zenkevich, E S; Kutyrev, V V

    2016-01-01

    There is evidence that in 1923-2014 the sharp aggravations of the epizootic situation of plague in the area of its Caspian sandy natural focus after long interepizootic periods are in time with the ups of the Caspian Sea in the extrema of 11-year solar cycles. There were cases of multiple manifestations of plague in the same areas in the epizootic cycles of 1946-1954, 1979-1996, 2001, and 2013-2014. The paper considers the possible role of amebae of the genus Acanthamoeba and nematodes, the representatives of the orders Rhabditida and Tylenchida in the microfocal pattern of plague manifestations. PMID:27029140

  19. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  20. Response of the protistan community of a rice field soil to different oxygen tensions.

    PubMed

    Takenouchi, Yuriko; Iwasaki, Kazufumi; Murase, Jun

    2016-07-01

    Heterotrophic protists in soil are grazers that control the biomass and community structure of bacteria, thereby enhancing nutrient recycling. Oxygen regulates the microeukaryotic community, but little is known about its response to microoxic conditions. Here we studied the impact of oxygen tension on culturable heterotrophic protists in a rice field soil. The number of protists, dominated by amoeba and flagellates, under oxygen tensions ranging from atmospheric level (21%) to below the Pasteur point (0.08%) were similar (10(4) cells g(-1) dry soil); no protists were detected under anoxic conditions. DGGE fingerprinting of microeukaryotes demonstrated a shift in the community structure depending on the oxygen tension during growth. Both common and specific amoeba and flagellates were identified at different oxygen tensions. Amoeba isolates (Acanthamoeba sp. and Hartmannella sp.) grew to the same extent under the oxygen tensions tested; the Acanthamoeba sp. isolate migrated more slowly under the lowest tension (0.08%). Our results demonstrated that amoeba and flagellates in soil adapt to a wide range of oxygen tensions with a shift in community structure. This suggests an ability to search for food in soil environments such as the oxic-anoxic interface of flooded soil or inside soil aggregates that are inaccessible to ciliates. PMID:27183973

  1. Microbiological quality in Finnish public swimming pools and whirlpools with special reference to free living amoebae: a risk factor for contact lens wearers?

    PubMed Central

    Vesaluoma, M; Kalso, S; Jokipii, L; Warhurst, D; Pönkä, A; Tervo, T

    1995-01-01

    To assess the possible risk of microbial keratitis associated with swimming or bathing in public pools, the microbiological quality as well as the presence of free living amoebae in 16 halogenated swimming pools and whirlpools, located in Helsinki, Finland, was determined. Five additional whirlpools situated in the ferries cruising from Finland to Sweden were included in the study. Other parameters investigated were the total bacterial count, identification of Pseudomonas aeruginosa and Staphylococcus aureus, measurement of free residual and combined chlorine, potassium permanganate index, urine, pH, and turbidity. Amoebae were detected in 41% of the pool water samples studied. Seven of 11 whirlpools and four of 10 swimming pools were shown to contain amoebae. An Acanthamoeba species was isolated from only one outdoor swimming pool; the other amoebae belonged to the genera Vexillifera, Flabellula, Hartmannella, and Rugipes. Although not a single verified case of Acanthamoeba keratitis has been found in Finland, the findings show that there is a theoretical risk of amoebic and bacterial keratitis associated with swimming or bathing in properly cleaned public pools. Consequently, we do not recommend swimming or bathing with contact lenses. PMID:7696241

  2. Acanthamoebicidal activity of Fusarium sp. Tlau3, an endophytic fungus from Thunbergia laurifolia Lindl.

    PubMed

    Boonman, Narumon; Wiyakrutta, Suthep; Sriubolmas, Nongluksna; Dharmkrong-at Chusattayanond, Araya

    2008-10-01

    A fungal endophyte identified as Fusarium sp. Tlau3 was isolated from fresh twig of Thunbergia laurifolia Lindl., a Thai medicinal plant collected from the forest of Chiang Mai Province, Northern Thailand. The fungus was grown on a medium containing yeast extracts and sucrose. The fungal metabolites were isolated from the culture broth by dichloromethane extraction, isooctane/methanol then n-butanol/water partitions, and fractionation with Sephadex LH 20 column chromatography. Acanthamoebicidal fractions were found to induce the formation of large contractile vacuole (LCV) in trophozoites of an Acanthamoeba clinical isolate, leading to cell lysis under isotonic and hypotonic conditions within 1 h. In hypertonic condition, LCV formation was also induced but without cell lysis. Acridine orange staining of the treated cells revealed increased intracellular acidity, implying an increased proton pumping or a vacuolar proton-ATPase (V-ATPase) stimulation. Scanning electron microscopy showed cell membrane damage with intact cytoplasmic organelles. Our finding has indicated that contractile vacuoles of Acanthamoeba trophozoites are the primary target of the amoebicidal substance(s) from this endophytic fungus. PMID:18633646

  3. Detection of novel Chlamydiae and Legionellales from human nasal samples of healthy volunteers.

    PubMed

    Corsaro, Daniele; Venditti, Danielle

    2015-07-01

    Chlamydiae are intracellular bacterial parasites of eukaryotes, ranging from amoebae to humans. They comprise many novel members and are investigated as emerging pathogens. Environmental studies highlighted similarities between the ecologies of chlamydiae and legionellae, both groups being important agents of respiratory infections. Herein, we analyzed nasal samples from healthy persons, searching for the presence of amoebae, chlamydiae and legionellae. From a total of 25 samples, we recovered by PCR eight samples positive to chlamydiae and six samples positive to legionellae. Among these samples, four were positive to both organisms. The sequencing of 16S rDNAs allowed to identify (i) among Chlamydiae: Parachlamydia acanthamoebae, Chlamydophila psittaci, Chlamydophila felis, and members of Rhabdochlamydiaceae, Simkaniaceae and E6 lineage and (ii) among Legionellaceae: Legionella longbeachae, Legionella bozemanii and Legionella impletisoli. Unexpectedly, we also recovered Diplorickettsia sp. Amoebae collected from nasal mucosae, Acanthamoeba and Vermamoeba, were endosymbiont-free, and chlamydiae revealed refractory to amoeba coculture. This study shows common exposure to chlamydiae and legionellae and suggests open air activities like gardening as a probable additional source of infection. PMID:25697709

  4. New insight into non-healing corneal ulcers: iatrogenic crystals

    PubMed Central

    Livingstone, I; Stefanowicz, F; Moggach, S; Connolly, J; Ramamurthi, S; Mantry, S; Ramaesh, K

    2013-01-01

    Aims To characterise and correlate crystalline precipitations implicated in non-healing corneal ulceration in two patients with a previous history of acanthamoeba keratitis. Materials and methods Persistence of acanthamoeba and secondary bacterial infection was excluded with negative corneal scrapes. Confocal microscopy identified crystal-like deposits within the corneal stroma. To investigate possible precipitating combinations, all concurrent treatments at the time of presentation were mixed in wells, with observation of precipitate formation. Precipitates were observed with phase-contrast microscopy, and subsequently characterised via crystallography techniques and electrospray ionisation mass spectrometry. Results Combinations of dexamethasone 0.1% minims and chlorhexidine gluconate 0.2% formed an amorphous material characterised by electrospray ionisation mass spectrometry as an insoluble chlorhexidine salt. Combinations of chloramphenicol drops and timolol 0.5% formed a crystal identified via X-ray crystallography as santite (K(B5O6(OH)4).(H2O)2). This is a borate mineral identified in nature, arising from thermal springs, but never reported in biological tissues. Clinical improvement was observed following the cessation of the implicated precipitating combinations. Conclusion Our observations suggest iatrogenic precipitate formation, with a potential deleterious effect upon healing. The substrates for these precipitates include several frequently prescribed topical ophthalmic treatments. These findings shed new light on the aetiopathogenesis of non-healing corneal ulceration, and have broad implications on topical prescribing for this challenging condition. PMID:23558213

  5. Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa.

    PubMed

    Chavatte, N; Lambrecht, E; Van Damme, I; Sabbe, K; Houf, K

    2016-07-30

    Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT. PMID:27369581

  6. Plant extracts as natural amoebicidal agents.

    PubMed

    Derda, Monika; Hadaś, Edward; Thiem, Barbara

    2009-02-01

    Strains of Acanthamoeba sp. constitute a factor contributing to the occurrence of chronic granulomatous amoebic encephalitis, keratitis, pneumonia, as well as inflammations of other organs. Treatment of these diseases is very difficult and not always effective. A majority of these infections have been fatal. The aim of our study was to examine the amoebicidal or amoebistatic activity of plant extracts from Rubus chamaemorus, Pueraria lobata, Solidago virgaurea and Solidago graminifolia. For the purpose of isolation of pharmacologically active substances, we used the aboveground parts of plants, together with flowers, roots and leaves. It was established that extracts from S. virgauera, P. lobata and R. chamaemorus displayed chemotherapeutic properties in vitro in concentrations of approximately 0.01-0.05 mg extract/mL, i.e., in concentrations of 0.350 microg/mL expressed in ellagic acid for R. chamaemorus and 0.053 microg/mL expressed in puerarin for P. lobata. Therapeutic index values is 3.5-20. As a result of in vivo experiments, it was found out that, following therapy using the extracts, animals infected with Acanthamoeba sp. survived for an extended period (2.5-3 times longer). It was determined that plant extracts may be used both externally and internally in the case of a combined therapy for acanthamoebiasis. The tested extracts are not toxic for animals. PMID:19050923

  7. Some secrets are revealed: parasitic keratitis amoebae as vectors of the scarcely described pandoraviruses to humans.

    PubMed

    Scheid, Patrick; Balczun, Carsten; Schaub, Günter A

    2014-10-01

    In this article, the results of a long effort to derive valuable phylogenetic data about an extraordinary spore-like infectious particle (endocytobiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis are presented. The development of these endocytobionts has already been demonstrated with electron microscopic photo sequences, leading to a relevant model of its development presented here. The molecular biological investigation following the discovery of two other Pandoravirus species within aquatic sediments in 2013 led to the taxonomic affiliation of our endocytobiont with the genus Pandoravirus. A range of endocytobionts (intracellular biofilms) have been found in recent years, among which are several viruses which obligatorily proliferate within free-living amoebae. In human medicine, foreign objects which are placed in or on humans cause problems with microorganisms in biofilms. Contact lenses are especially important, because they are known as a source of a rapid formation of biofilm. These were the first Pandoraviruses described, and because this is additionally the first documented association with humans, we have clearly demonstrated how easily such (viral) endocytobionts can be transferred to humans. This case counts as an example of parasites acting as vectors of phylogenetically different microorganisms especially when living sympatric within their biocoenosis of biofilms. As the third part of the "Pandoravirus trilogy", it finally reveals the phylogenetic nature of these "extraordinary endocytobionts" within Acanthamoebae. PMID:25033816

  8. Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance

    PubMed Central

    Rohr, Ute; Weber, Susanne; Michel, Rolf; Selenka, Fidelis; Wilhelm, Michael

    1998-01-01

    Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53°C. PMID:9572957

  9. Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila.

    PubMed

    Dey, Rafik; Bodennec, Jacques; Mameri, Mouh Oulhadj; Pernin, Pierre

    2009-01-01

    Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis, known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella (L. pneumophila, Paris), but not of others belonging to the same serogroup (L. pneumophila, Philadelphia and L. pneumophila, Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3-4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila. Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium. PMID:19016880

  10. Free-living amebic meningoencephalitides: comparative study.

    PubMed

    Martínez, A J

    1977-01-01

    Primary amebic meningoencephalitis (PAM) is an infectious disease essentially confined to the central nervous system (CNS) and caused by free-living ameboflagellates of the genus Naegleria (N) and Acanthamoeba (A). N produces an acute fulminant, usually fatal hemorrhagic-necrotizing meningoencephalitis, associated with an inflammatory reaction composed of neutrophils, eosinophils, macrophages and a few lymphocytes. The incubation period is between 3-7 days. Typically, PAM occurs in healthy, young individuals, who frequently have a history of swimming or washing their face in infested waters. The portal of entry into the CNS is through the olfactory neuroepithelium, at the level of the cribriform plate and invasion of the amyelinic submucosal nervous plexus. Trophozoites are the only amebic forms found in the lesions. By contrast, A produces a sub-acute or chronic granulamotous meningoencephalitis (AM) with multinucleated foreign body giant cells, lymphocytes and monocytes. Cysts and trophozoites may be present in the lesions. AM have been reported in chronically ill debilitated individuals, some of them under immunosuppressive therapy, without history of recent swimming. The portal of entry into the CNS appears to be by hematogenous route. The incubation period is unknown, but perhaps more than 10 days. This comparison indicates that infection due to Naegleria produces a clearly defined clinco-pathological entity, which differs significantly from that due to Acanthamoeba, and both species of amebus should be considered in the differential diagnosis of amebic meningoencephalitis. PMID:616542

  11. Estimating Tempo and Mode of Y Chromosome Turnover: Explaining Y Chromosome Loss With the Fragile Y Hypothesis

    PubMed Central

    Blackmon, Heath; Demuth, Jeffery P.

    2014-01-01

    Chromosomal sex determination is phylogenetically widespread, having arisen independently in many lineages. Decades of theoretical work provide predictions about sex chromosome differentiation that are well supported by observations in both XY and ZW systems. However, the phylogenetic scope of previous work gives us a limited understanding of the pace of sex chromosome gain and loss and why Y or W chromosomes are more often lost in some lineages than others, creating XO or ZO systems. To gain phylogenetic breadth we therefore assembled a database of 4724 beetle species’ karyotypes and found substantial variation in sex chromosome systems. We used the data to estimate rates of Y chromosome gain and loss across a phylogeny of 1126 taxa estimated from seven genes. Contrary to our initial expectations, we find that highly degenerated Y chromosomes of many members of the suborder Polyphaga are rarely lost, and that cases of Y chromosome loss are strongly associated with chiasmatic segregation during male meiosis. We propose the “fragile Y” hypothesis, that recurrent selection to reduce recombination between the X and Y chromosome leads to the evolution of a small pseudoautosomal region (PAR), which, in taxa that require XY chiasmata for proper segregation during meiosis, increases the probability of aneuploid gamete production, with Y chromosome loss. This hypothesis predicts that taxa that evolve achiasmatic segregation during male meiosis will rarely lose the Y chromosome. We discuss data from mammals, which are consistent with our prediction. PMID:24939995

  12. Estimating tempo and mode of Y chromosome turnover: explaining Y chromosome loss with the fragile Y hypothesis.

    PubMed

    Blackmon, Heath; Demuth, Jeffery P

    2014-06-01

    Chromosomal sex determination is phylogenetically widespread, having arisen independently in many lineages. Decades of theoretical work provide predictions about sex chromosome differentiation that are well supported by observations in both XY and ZW systems. However, the phylogenetic scope of previous work gives us a limited understanding of the pace of sex chromosome gain and loss and why Y or W chromosomes are more often lost in some lineages than others, creating XO or ZO systems. To gain phylogenetic breadth we therefore assembled a database of 4724 beetle species' karyotypes and found substantial variation in sex chromosome systems. We used the data to estimate rates of Y chromosome gain and loss across a phylogeny of 1126 taxa estimated from seven genes. Contrary to our initial expectations, we find that highly degenerated Y chromosomes of many members of the suborder Polyphaga are rarely lost, and that cases of Y chromosome loss are strongly associated with chiasmatic segregation during male meiosis. We propose the "fragile Y" hypothesis, that recurrent selection to reduce recombination between the X and Y chromosome leads to the evolution of a small pseudoautosomal region (PAR), which, in taxa that require XY chiasmata for proper segregation during meiosis, increases the probability of aneuploid gamete production, with Y chromosome loss. This hypothesis predicts that taxa that evolve achiasmatic segregation during male meiosis will rarely lose the Y chromosome. We discuss data from mammals, which are consistent with our prediction. PMID:24939995

  13. Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA*

    PubMed Central

    Imamura, Kayo; Averill, April; Wallace, Susan S.; Doublié, Sylvie

    2012-01-01

    Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174–26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site. PMID:22170059

  14. Diversity and dynamics of algal Megaviridae members during a harmful brown tide caused by the pelagophyte, Aureococcus anophagefferens.

    PubMed

    Moniruzzaman, Mohammad; Gann, Eric R; LeCleir, Gary R; Kang, Yoonja; Gobler, Christopher J; Wilhelm, Steven W

    2016-05-01

    Many giant dsDNA algal viruses share a common ancestor with Mimivirus--one of the largest viruses, in terms of genetic content. Together, these viruses form the proposed 'Megaviridae' clade of nucleocytoplasmic large DNA viruses. To gauge Megaviridae diversity, we designed degenerate primers targeting the major capsid protein genes of algae-infecting viruses within this group and probed the clade's diversity during the course of a brown tide bloom caused by the harmful pelagophyte,Aureococcus anophagefferens We amplified target sequences in water samples from two distinct locations (Weesuck Creek and Quantuck Bay, NY) covering 12 weeks concurrent with the proliferation and demise of a bloom. In total, 475 amplicons clustered into 145 operational taxonomic units (OTUs) at 97% identity. One OTU contained 19 sequences with ≥97% identity to AaV, a member of the Megaviridae clade that infects A. anophagefferens, suggesting AaV was present during the bloom. Unifrac analysis showed clear temporal patterns in algal Megaviridae dynamics, with a shift in the virus community structure that corresponded to the Aureococcus bloom decline in both locations. Our data provide insights regarding the environmental relevance of algal Megaviridae members and raise important questions regarding their phylodynamics across different environmental gradients. PMID:26985013

  15. Challenging the state-of-the-art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

    PubMed Central

    Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J. Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V.; Craig, Timothy K.; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C.; van Raaij, Mark J.; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

    2014-01-01

    For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, over 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this paper, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict trans-membrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin IL-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fibre protein gp17 from bacteriophage T7; the Bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. PMID:24318984

  16. Updates and achievements in virology.

    PubMed

    Buonaguro, Franco M; Campadelli-Fiume, Gabriella; De Giuli Morghen, Carlo; Palù, Giorgio

    2010-07-01

    The 4th European Congress of Virology, hosted by the Italian Society for Virology, attracted approximately 1300 scientists from 46 countries worldwide. It also represented the first conference of the European Society for Virology, which was established in Campidoglio, Rome, Italy in 2009. The main goal of the meeting was to share research activities and results achieved in European virology units/institutes and to strengthen collaboration with colleagues from both western and developing countries. The worldwide representation of participants is a testament to the strength and attraction of European virology. The 5-day conference brought together the best of current virology; topics covered all three living domains (bacteria, archaea and eucarya), with special sessions on plant and veterinary virology as well as human virology, including two oral presentations on mimiviruses. The conference included five plenary sessions, 31 workshops, one hepatitis C virus roundtable, ten special workshops and three poster sessions, as well as 45 keynote lectures, 191 oral presentations and 845 abstracts. Furthermore, the Gesellschaft fur Virologie Loeffler-Frosch medal award was given to Peter Vogt for his long-standing career and achievements; the Gardner Lecture of the European Society for Clinical Virology was presented by Yoshihiro Kawaoka, and the Pioneer in Virology Lecture of the Italian Society for Virology was presented by Ulrich Koszinowski. PMID:20624042

  17. Structure of Sputnik, a virophage, at 3.5-Å resolution.

    PubMed

    Zhang, Xinzheng; Sun, Siyang; Xiang, Ye; Wong, Jimson; Klose, Thomas; Raoult, Didier; Rossmann, Michael G

    2012-11-01

    "Sputnik" is a dsDNA virus, referred to as a virophage, that is coassembled with Mimivirus in the host amoeba. We have used cryo-EM to produce an electron density map of the icosahedral Sputnik virus at 3.5-Å resolution, sufficient to verify the identity of most amino acids in the capsid proteins and to establish the identity of the pentameric protein forming the fivefold vertices. It was also shown that the virus lacks an internal membrane. The capsid is organized into a T = 27 lattice in which there are 260 trimeric capsomers and 12 pentameric capsomers. The trimeric capsomers consist of three double "jelly-roll" major capsid proteins creating pseudohexameric capsomer symmetry. The pentameric capsomers consist of five single jelly-roll proteins. The release of the genome by displacing one or more of the pentameric capsomers may be the result of a low-pH environment. These results suggest a mechanism of Sputnik DNA ejection that probably also occurs in other big icosahedral double jelly-roll viruses such as Adenovirus. In this study, the near-atomic resolution structure of a virus has been established where crystallization for X-ray crystallography was not feasible. PMID:23091035

  18. Unifying view of stem-loop hairpin RNA as origin of current and ancient parasitic and non-parasitic RNAs, including in giant viruses.

    PubMed

    Seligmann, Hervé; Raoult, Didier

    2016-06-01

    Putatively, stem-loop RNA hairpins explain networks of selfish elements and RNA world remnants. Their genomic density increases with intracellular lifestyle, especially when comparing giant viruses and their virophages. RNA protogenomes presumably templated for mRNAs and self-replicating stem-loops, ancestors of modern genes and parasitic sequences, including tRNAs and rRNAs. Primary and secondary structure analyses suggest common ancestry for t/rRNAs and parasitic RNAs, parsimoniously link diverse RNA metabolites (replication origins, tRNAs, ribozymes, riboswitches, miRNAs and rRNAs) to parasitic RNAs (ribosomal viroids, Rickettsia repeated palindromic elements (RPE), stem-loop hairpins in giant viruses, their virophages, and transposable retrovirus-derived elements). Results indicate ongoing genesis of small RNA metabolites, and common ancestry or similar genesis for rRNA and retroviral sequences. Assuming functional integration of modular duplicated RNA hairpins evolutionarily unifies diverse molecules, postulating stem-loop hairpin RNAs as origins of genetic innovation, ancestors of rRNAs, retro- and Mimivirus sequences, and cells. PMID:26716728

  19. Giant Viruses of Amoebas: An Update.

    PubMed

    Aherfi, Sarah; Colson, Philippe; La Scola, Bernard; Raoult, Didier

    2016-01-01

    During the 12 past years, five new or putative virus families encompassing several members, namely Mimiviridae, Marseilleviridae, pandoraviruses, faustoviruses, and virophages were described. In addition, Pithovirus sibericum and Mollivirus sibericum represent type strains of putative new giant virus families. All these viruses were isolated using amoebal coculture methods. These giant viruses were linked by phylogenomic analyses to other large DNA viruses. They were then proposed to be classified in a new viral order, the Megavirales, on the basis of their common origin, as shown by a set of ancestral genes encoding key viral functions, a common virion architecture, and shared major biological features including replication inside cytoplasmic factories. Megavirales is increasingly demonstrated to stand in the tree of life aside Bacteria, Archaea, and Eukarya, and the megavirus ancestor is suspected to be as ancient as cellular ancestors. In addition, giant amoebal viruses are visible under a light microscope and display many phenotypic and genomic features not found in other viruses, while they share other characteristics with parasitic microbes. Moreover, these organisms appear to be common inhabitants of our biosphere, and mimiviruses and marseilleviruses were isolated from human samples and associated to diseases. In the present review, we describe the main features and recent findings on these giant amoebal viruses and virophages. PMID:27047465

  20. Plant genomes enclose footprints of past infections by giant virus relatives

    PubMed Central

    Maumus, Florian; Epert, Aline; Nogué, Fabien; Blanc, Guillaume

    2014-01-01

    Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb–2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants. PMID:24969138

  1. Taxonomic distribution of large DNA viruses in the sea

    PubMed Central

    Monier, Adam; Claverie, Jean-Michel; Ogata, Hiroyuki

    2008-01-01

    Background Viruses are ubiquitous and the most abundant biological entities in marine environments. Metagenomics studies are increasingly revealing the huge genetic diversity of marine viruses. In this study, we used a new approach - 'phylogenetic mapping' - to obtain a comprehensive picture of the taxonomic distribution of large DNA viruses represented in the Sorcerer II Global Ocean Sampling Expedition metagenomic data set. Results Using DNA polymerase genes as a taxonomic marker, we identified 811 homologous sequences of likely viral origin. As expected, most of these sequences corresponded to phages. Interestingly, the second largest viral group corresponded to that containing mimivirus and three related algal viruses. We also identified several DNA polymerase homologs closely related to Asfarviridae, a viral family poorly represented among isolated viruses and, until now, limited to terrestrial animal hosts. Finally, our approach allowed the identification of a new combination of genes in 'viral-like' sequences. Conclusion Albeit only recently discovered, giant viruses of the Mimiviridae family appear to constitute a diverse, quantitatively important and ubiquitous component of the population of large eukaryotic DNA viruses in the sea. PMID:18598358

  2. DNA Viruses: The Really Big Ones (Giruses)

    PubMed Central

    Van Etten, James L.; Lane, Leslie C.; Dunigan, David D.

    2010-01-01

    Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses. PMID:20690825

  3. Genomic comparison of closely related Giant Viruses supports an accordion-like model of evolution.

    PubMed

    Filée, Jonathan

    2015-01-01

    Genome gigantism occurs so far in Phycodnaviridae and Mimiviridae (order Megavirales). Origin and evolution of these Giant Viruses (GVs) remain open questions. Interestingly, availability of a collection of closely related GV genomes enabling genomic comparisons offer the opportunity to better understand the different evolutionary forces acting on these genomes. Whole genome alignment for five groups of viruses belonging to the Mimiviridae and Phycodnaviridae families show that there is no trend of genome expansion or general tendency of genome contraction. Instead, GV genomes accumulated genomic mutations over the time with gene gains compensating the different losses. In addition, each lineage displays specific patterns of genome evolution. Mimiviridae (megaviruses and mimiviruses) and Chlorella Phycodnaviruses evolved mainly by duplications and losses of genes belonging to large paralogous families (including movements of diverse mobiles genetic elements), whereas Micromonas and Ostreococcus Phycodnaviruses derive most of their genetic novelties thought lateral gene transfers. Taken together, these data support an accordion-like model of evolution in which GV genomes have undergone successive steps of gene gain and gene loss, accrediting the hypothesis that genome gigantism appears early, before the diversification of the different GV lineages. PMID:26136734

  4. Genomic comparison of closely related Giant Viruses supports an accordion-like model of evolution

    PubMed Central

    Filée, Jonathan

    2015-01-01

    Genome gigantism occurs so far in Phycodnaviridae and Mimiviridae (order Megavirales). Origin and evolution of these Giant Viruses (GVs) remain open questions. Interestingly, availability of a collection of closely related GV genomes enabling genomic comparisons offer the opportunity to better understand the different evolutionary forces acting on these genomes. Whole genome alignment for five groups of viruses belonging to the Mimiviridae and Phycodnaviridae families show that there is no trend of genome expansion or general tendency of genome contraction. Instead, GV genomes accumulated genomic mutations over the time with gene gains compensating the different losses. In addition, each lineage displays specific patterns of genome evolution. Mimiviridae (megaviruses and mimiviruses) and Chlorella Phycodnaviruses evolved mainly by duplications and losses of genes belonging to large paralogous families (including movements of diverse mobiles genetic elements), whereas Micromonas and Ostreococcus Phycodnaviruses derive most of their genetic novelties thought lateral gene transfers. Taken together, these data support an accordion-like model of evolution in which GV genomes have undergone successive steps of gene gain and gene loss, accrediting the hypothesis that genome gigantism appears early, before the diversification of the different GV lineages. PMID:26136734

  5. Giant Viruses of Amoebas: An Update

    PubMed Central

    Aherfi, Sarah; Colson, Philippe; La Scola, Bernard; Raoult, Didier

    2016-01-01

    During the 12 past years, five new or putative virus families encompassing several members, namely Mimiviridae, Marseilleviridae, pandoraviruses, faustoviruses, and virophages were described. In addition, Pithovirus sibericum and Mollivirus sibericum represent type strains of putative new giant virus families. All these viruses were isolated using amoebal coculture methods. These giant viruses were linked by phylogenomic analyses to other large DNA viruses. They were then proposed to be classified in a new viral order, the Megavirales, on the basis of their common origin, as shown by a set of ancestral genes encoding key viral functions, a common virion architecture, and shared major biological features including replication inside cytoplasmic factories. Megavirales is increasingly demonstrated to stand in the tree of life aside Bacteria, Archaea, and Eukarya, and the megavirus ancestor is suspected to be as ancient as cellular ancestors. In addition, giant amoebal viruses are visible under a light microscope and display many phenotypic and genomic features not found in other viruses, while they share other characteristics with parasitic microbes. Moreover, these organisms appear to be common inhabitants of our biosphere, and mimiviruses and marseilleviruses were isolated from human samples and associated to diseases. In the present review, we describe the main features and recent findings on these giant amoebal viruses and virophages. PMID:27047465

  6. [Investigation of potentially pathogenic free-living amoebae and their in vivo pathogenicity in water supplies of Turkey].

    PubMed

    Yazar, Süleyman; Gürbüz, Esra; Sönmez, Mehmet Fatih; Çetinkaya, Ülfet; Kuk, Salih

    2016-07-01

    Free-living amoebae (FLA) are found widely in soil and water in the nature. Among them in which potentially pathogenic for humans and animals are known as "potential pathogenic free-living amoebae (PPFLA)". PPFLA are characterized as the causes of clinical manifestations leading to death especially in immunosuppressed people. Four genus of PPFLA (Acanthamoeba, Naegleria, Balamuthia and Sappinia) are known to be pathogenic to humans. The aims of this study were to investigate the presence of PPFLA in the water supplies in Turkey and to determine their in vivo pathogenicity. A total of 664 water samples were collected from the ponds, rivers, streams and wells found in provinces located at different regions (central, western, eastern and southeastern regions) of Turkey. These samples were initially inoculated in the monoxenic culture media and evaluated by both microscopy and polymerase chain reaction (PCR) in terms of the presence of FLA. The samples identified as positive were then cultured in axenic media, the growth of amoebae that were confirmed microscopically, were than studied with PCR for molecular characterization. The isolates that were found positive by PCR from axenic cultures were inoculated intranasally to immunocompetent and immunodeficient (athymic) [BALB/c Rag2(-/-) gamma(c)(-/-)] BALB/c mice followed by the evaluation on the 21st day by histopathological and molecular methods to investigate their in vivo pathogenicity. In our study, 143 water samples were detected as positive in monoxenic cultures and 41 of them were detected as positive in axenic cultures. Twenty of 41 samples detected as positive in axenic culture could be continued in culture for three months. As a result of PCR using primers common to SYA, only nine have been identified from 20 samples as positive. According to the result of the PCR with specific primers, all (n= 9) were positive for Acanthamoeba sp., eight for Sappini sp. and five for Balamuthia mandrillaris, while none was

  7. Management of granulomatous amebic encephalitis: Laboratory diagnosis and treatment

    PubMed Central

    Parija, Subhash Chandra; Dinoop, KP; Venugopal, Hrudya

    2015-01-01

    Granulomatous amebic encephalitis is a life-threatening central nervous system (CNS) infection caused by the free-living amoebae Acanthamoeba spp., Balamuthia mandrillaris and Sappinia pedata. The disease has a subacute to chronic onset affecting commonly the immunocompromised population with high mortality rate. The diagnosis of this disease entity requires high suspicion with appropriate sample collection and testing by the laboratory experts. Radiological investigations are nonspecific and commonly confused with CNS tuberculosis, neurocysticercosis, disseminated encephalomyelitis, viral encephalitis etc., delaying the accurate diagnosis of these cases. Early diagnosis plays a crucial role in the survival of these cases since appropriate management can be initiated. No single drug is effective; hence multiple antibiotics targeting various proteins or receptors are required for successful treatment. A combination of surgical and medical interventions involving multiple specialty experts is required to prevent death and morbidity in survivors. PMID:25709949

  8. Characterization of a Drinking Water Distribution Pipeline Terminally Colonized by Naegleria fowleri.

    PubMed

    Morgan, Matthew J; Halstrom, Samuel; Wylie, Jason T; Walsh, Tom; Kaksonen, Anna H; Sutton, David; Braun, Kalan; Puzon, Geoffrey J

    2016-03-15

    Free-living amoebae, such as Naegleria fowleri, Acanthamoeba spp., and Vermamoeba spp., have been identified as organisms of concern due to their role as hosts for pathogenic bacteria and as agents of human disease. In particular, N. fowleri is known to cause the disease primary amoebic meningoencephalitis (PAM) and can be found in drinking water systems in many countries. Understanding the temporal dynamics in relation to environmental and biological factors is vital for developing management tools for mitigating the risks of PAM. Characterizing drinking water systems in Western Australia with a combination of physical, chemical and biological measurements over the course of a year showed a close association of N. fowleri with free chlorine and distance from treatment over the course of a year. This information can be used to help design optimal management strategies for the control of N. fowleri in drinking-water-distribution systems. PMID:26853055

  9. Presence of pathogenic microorganisms in power-plant cooling waters. Report for October 1, 1979-September 30, 1981

    SciTech Connect

    Tyndall, R.L.

    1982-10-01

    Cooling waters from eleven geographically disparate power plants were tested for the presence of Naegleria fowleri and Legionella pneumophila (LDB). Control source waters for each plant were also tested for these pathogens. Water from two of the eleven plants contained pathogenic Naegleria, and infectious Legionella were found in seven of the test sites. Pathogenic Naegleria were not found in control waters, but infectious Legionella were found in five of the eleven control source water sites. Concentrations of nitrite, sulfate, and total organic carbon correlated with the concentrations of LDB. A new species of Legionella was isolated from one of the test sites. In laboratory tests, both Acanthamoeba and Naegleria were capable of supporting the growth of Legionella pneumophila.

  10. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    PubMed

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  11. Cocultivation of Legionella pneumophila and free-living amoebae

    SciTech Connect

    Tyndall, R.L.; Domingue, E.L.

    1982-10-01

    Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophia as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. roryba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth. 20 references, 1 figure, 4 tables.

  12. Testate amoeba Rhogostoma minus Belar, 1921, associated with nodular gill disease of rainbow trout, Oncorhynchus mykiss (Walbaum).

    PubMed

    Dyková, I; Tyml, T

    2016-05-01

    The case study targeted to determine the aetiology of nodular gill disease (NGD) of farmed rainbow trout. The methods included microscopical examination of gill material in fresh, culturing of isolated organisms, histology, transmission electron microscopy and molecular biology identification. The results revealed an intravital colonization of fish gills by the testate amoeba Rhogostoma minus Belar, 1921. Rhogostoma infection was found in all fish examined microscopically (15/15); in contrast, naked amoebae related to fully developed NGD lesions were found in minority of these fish (5/15). They belonged to four genera, Acanthamoeba, Vermamoeba, Naegleria and Vannella. Results presented in this study contribute to the mosaic of findings that contrary to amoebic gill disease of marine fish turn attention to the possibility of the heterogeneous, multi-amoeba-species and multifactorial aetiology of NGD. PMID:25952929

  13. Events during eucaryotic rRNA transcription initiation and elongation: Conversion from the closed to the open promoter complex requires nucleotide substrates

    SciTech Connect

    Bateman, E.; Paule, M.R.

    1988-05-01

    Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyro-carbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

  14. Survival of Campylobacter jejuni in Waterborne Protozoa

    PubMed Central

    Snelling, W. J.; McKenna, J. P.; Lecky, D. M.; Dooley, J. S. G.

    2005-01-01

    The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers. PMID:16151149

  15. Emerging chlamydial infections.

    PubMed

    Corsaro, Daniele; Venditti, Danielle

    2004-01-01

    Chlamydiae are important intracellular bacterial pathogens of vertebrates. In the last years, novel members of this group have been discovered: Parachlamydia acanthamoebae and Simkania negevensis seems to be emerging respiratory human pathogens, while Waddlia chondrophila might be a new agent of bovine abortion. Various species have been showed to infect also the herpetofauna and fishes, and some novel chlamydiae are endosymbionts of arthropods. In addition, molecular studies evidenced a huge diversity of chlamydiae from both environmental and clinical samples, most of such a diversity could be formed by novel lineages of chlamydiae. Experimental studies showed that free-living amoebae may support multiplication of various chlamydiae, then could play an important role as reservoir/vector of chlamydial infections. Here we reviewed literature data concerning chlamydial infections, with a particular emphasis on the novely described chlamydial organisms. PMID:15239381

  16. Compositions and methods for pathogen transport

    DOEpatents

    El-Etr, Sahar; Farquar, George R.

    2016-01-26

    This disclosure provides a method for transporting a pathogen under ambient conditions, by culturing the pathogen with an amoeba under conditions that favor the incorporation of the pathogen into a trophozoite, starving the amoeba until it encysts, then culturing under conditions that favor conversion of the amoeba back to a trophozoite. In one aspect, the conditions that favor incorporation of the pathogen into the cyst of the amoeba comprises contacting the pathogen with the amoeba in an iron rich environment. Virus and/or bacteria are pathogens that can be transported by the disclosed method. Amoeba that are useful in the disclosed methods include, without limitation Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria gruberi. The disclosed methods have utility in: transporting pathogens from military field hospitals and clinics to the laboratory; transporting pathogens from global satellite laboratories to clinical laboratories; long term storage of pathogens; enriching contaminated patient samples for pathogens of interest; biosurveillance and detection efforts.

  17. Free-living pathogenic and nonpathogenic amoebae in Maryland soils.

    PubMed Central

    Sawyer, T K

    1989-01-01

    Tests for potentially pathogenic amoebae were carried out on soil samples from the following sites: (i) farmlands fertilized with municipal sewage wastes, (ii) a stream receiving sewage effluent from a sludge lagoon, (iii) a ravine receiving storm runoff from a cattle farm, (iv) farmlands not fertilized with sewage wastes, and (v) a vegetated shoreline of a waterfront estate not used for farming or livestock production. Study sites were located on the eastern shore of Maryland, bordered to the north by Delaware and to the south by Virginia. Twenty-four species of soil amoebae, including five potentially pathogenic Acanthamoeba species (members of the family Acanthamoebidae), were identified. All of the sites yielded two or more of the potential pathogens. PMID:2757373

  18. Coprology of Panthera tigris altaica and Felis bengalensis euptilurus from the Russian Far East.

    PubMed

    González, P; Carbonell, E; Urios, V; Rozhnov, V V

    2007-08-01

    Fecal samples from the Siberian tiger (Panthera tigris altaica) and the Amur cat (Felis bengalensis euptilurus) from Far Eastern Russia, were examined for parasites. A natural sedimentation methodology was used and a complete examination of all the sediment was performed. This fecal investigation allowed us to isolate and identify several developmental stages of gastrointestinal, hepatic, and respiratory parasites. Five parasites were found from P. t. altaica: 11 trematodes (Platynosomum fastosum) and 4 nematodes (Strongyloides sp., Ancylostomatidae, Toxascaris leonina, and Toxocara cati). Five parasites were found from F. b. euptilurus: 1 cestode (Diplopylidium sp.) and 4 nematodes (Trichuris sp., Ancylostomatidae, Toxascaris leonina, and Aelurostrongylus abstrusus). In addition, trophozoites of the amoeba Acanthamoeba sp. were detected in tiger feces. PMID:17918383

  19. The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin.

    PubMed Central

    Ampe, C; Vandekerckhove, J

    1987-01-01

    We have carried out a primary structure analysis of the F-actin capping proteins of Physarum polycephalum. Cap42(b) was completely sequenced and was found to be identical with Physarum actin. Approximately 88% of the sequence of cap42(a) was determined. Cap42(a) and fragmin were found to be identical by amino acid composition, isoelectric point, mol. wt, elution time on reversed-phase chromatography and amino acid sequence of their tryptic peptides. The available sequence of cap42(a) is greater than 36% homologous with the NH2-terminal 42-kd domain of human gelsolin. A highly homologous region of 16 amino acids is also shared between cap42(a), gelsolin and the Acanthamoeba profilins. Cap42(a) binds two actin molecules in a similar way to gelsolin suggesting a mechanism of F-actin modulation that has been conserved during evolution. Images Fig. 1. Fig. 3. Fig. 4. PMID:2832154

  20. UV inactivation of pathogenic and indicator microorganisms

    SciTech Connect

    Chang, J.C.; Ossoff, S.F.; Lobe, D.C.; Dorfman, M.H.; Dumais, C.M.; Qualls, R.G.; Johnson, J.D.

    1985-06-01

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.

  1. An investigation into the antimicrobial mechanisms of action of two contact lens biocides using electron microscopy.

    PubMed

    Codling, Caroline E; Hann, Anthony C; Maillard, Jean-Yves; Russell, A Denver

    2005-12-01

    Polyquaternium-1 (PQ-1) and myristamidopropyl dimethylamine (MAPD) are biocides used commercially in a contact lens disinfecting solutions. Electron microscopy was used to provide further evidence on the mechanism(s) of action of these agents against a wide range of ocular pathogens including bacteria, fungi and protozoa. Both PQ-1 and MAPD caused multiple forms of damage to the organisms tested, evidenced by structural alterations, blebbing, leakage and cell destruction. The extent of damage and the selectivity against specific type of microorganisms was consistent with the antimicrobial activity of these agents. Although electron microscopy is a powerful tool, it has its limitations when used to examine the mode of action of biocides. Indeed, there was no evidence of gross structural alteration to Acanthamoeba castellani or Aspergillus fumigatus following treatment. PMID:16332500

  2. A strange endocytobiont revealed as largest virus.

    PubMed

    Scheid, P

    2016-06-01

    A lot of endocytobionts (or endosymbionts) have been discovered within free-living amoebae in recent years. In this article the results of a long lasting effort to derive valuable data about an extraordinary spore-like infectious microorganism (endocytobiont, endosymbiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens case of a patient with keratitis, are presented. It took some time until this endocytobiont could be attributed to the genus Pandoravirus following a publication of two other pandoraviruses isolated from aquatic environments. Consequently the molecular biological investigation led to the taxonomic affiliation of the endocytobiont with the genus Pandoravirus and to the description of a new Pandoravirus species, Pandoravirus inopinatum after whole-genome sequencing in 2015. The fact that it was isolated from a contact lens container of a keratitis patient gives another dimension to these findings showing paradigmatically, how readily these 'new' giant viruses get to humans. PMID:27016694

  3. srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda).

    PubMed

    Baverstock, P R; Illana, S; Christy, P E; Robinson, B S; Johnson, A M

    1989-05-01

    A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent. PMID:2622334

  4. Evaluation of military field-water quality: Volume 6, Infectious organisms of military concern associated with nonconsumptive exposure: Assessment of health risks and recommendations for establishing related standards

    SciTech Connect

    Cooper, R.C.; Olivieri, A.W.; Danielson, R.E.; Badger, P.G.

    1986-02-01

    This study is an assessment of the risk of illness due to exposure to water-related (i.e., water-based, water-washed) infectious organisms. The organisms under consideration are Aeromonas spp., Leptospira spp., Pseudomonas spp., Staphylococcus spp., non-cholerae Vibrio spp., Acanthamoeba spp., Balantidium coli, Naegleria spp., Ascaris lumbricoides, Dracunculus medinesis, Schistosoma spp., and the agents responsible for cercarial dermatitis (i.e., Trichobilharzia, Gigantobilharzia, and Austrobilharzia). Evaluation of the risk to disease associated with the above pathogens requires information in specific areas such as dose response, concentration of agents in the environment, and environmental persistence. The existing body of knowledge concerning these agents ranges from speculation to established fact. Unfortunately, areas of information critical to risk assessment are frequently unavailable. Because of this lack of data, the risk assessment presented is semiquantitative and limited to the presentation of an environmental classification scheme. 14 refs., 2 figs., 57 tabs.

  5. Species specificity of a monoclonal antibody produced to Naegleria fowleri and partial characterization of its antigenic determinant.

    PubMed

    Réveiller, F L; Marciano-Cabral, F; Pernin, P; Cabanes, P A; Legastelois, S

    2000-08-01

    Monoclonal antibody (Mab) 5D12 against Naegleria fowleri was analyzed for species specificity. Mab 5D12 reacted with a ubiquitous epitope present on the membrane of N. fowleri but not with soluble antigens. The Mab did not react with N. lovaniensis, N. gruberi, N. australiensis, or Acanthamoeba castellanii. The decreased reactivity of Mab 5D12 with N. fowleri observed after periodate oxidation, after digestion of carbohydrate moieties by three glycosidases, or after treatment of amebas with tunicamycin strongly suggests that the antigenic determinant has a polysaccharide component. Inhibition of the reactivity of Mab 5D12 by soluble saccharides supports the idea that N-acetyl or amino groups may play an important role in the recognition of the carbohydrate component of the epitope by the Mab. The specificity of Mab 5D12 makes this an ideal reagent for the identification of N. fowleri in environmental samples or in clinical specimens. PMID:10952262

  6. Temperature stress: reacting and adapting: lessons from poikilotherms.

    PubMed

    Harwood, John L

    2007-10-01

    Acanthamoeba castellanii (A. castellanii) is a common soil- or water-borne protozoon that feeds on bacteria by phagocytosis. A. castellanii can grow between 4 and 32 degrees C and has to adapt quickly to chilling in order to survive. We have identified a Delta12-fatty acid desaturase as key to low temperature adaptation. The activity of this enzyme is mainly increased through gene expression and new protein synthesis. Interestingly, the activity can also be altered independently by dissolved oxygen levels. In addition, we have identified a gene for the Delta12-desaturase, which, when expressed in yeast, catalyses Delta15-desaturation also. Moreover, it is also capable of producing very unusual n-1 polyunsaturated products. PMID:17584990

  7. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  8. Granulomatous amebic encephalitis following hematopoietic stem cell transplantation

    PubMed Central

    Doan, Ninh; Rozansky, Gregory; Nguyen, Ha Son; Gelsomino, Michael; Shabani, Saman; Mueller, Wade; Johnson, Vijay

    2015-01-01

    Background: Granulomatous amebic encephalitis (GAE) is rare, but often fatal. The infection has been documented predominantly among the immunocompromised population or among those with chronic disease. To date, however, there have only been eight cases regarding the infection following hematopoietic stem cell transplantation (HSCT). Case Description: A 62-year-old female with a history of relapsed diffuse large B-cell lymphoma, recently underwent peripheral blood autologous stem cell transplant after BEAM conditioning (day 0). On day +15, she began to exhibit worsening fatigue, generalized weakness, and fever. Symptoms progressed to nausea, emesis, somnolence, confusion, and frontal headaches over the next few days. Imaging demonstrated multifocal ill-defined vasogenic edema with patchy enhancement. The patient was started on broad antibiotics, antifungals, and seizure prophylaxis. Evaluation for bacterial, fungal, mycobacterial, and viral etiologies was fruitless. Her mental status progressively deteriorated. On day +22, she exhibited severe lethargy and went into pulseless electrical activity arrest, requiring chest compressions. The episode lasted <2 min and her pulse was restored. She was taken to the operating room for a brain biopsy. Postoperatively, her right pupil began to dilate compared to the left; she demonstrated extensor posturing in her upper extremities and withdrawal in her lower extremities. Repeat computed tomography demonstrated progressive edema. Given poor prognosis and poor neurological examination, the family opted for withdrawal of care. Final pathology was consistent with Acanthamoeba GAE. Conclusion: The authors report the third case of GAE after autologous stem cell transplant, and the ninth case overall after HSCT. This case is unusual due to its rapid clinical presentation after HSCT compared to prior literature. The case highlights the need for high suspicion of Acanthamoeba infection in this patient population. PMID:26539322

  9. Isolation and Molecular Identification of Vermamoeba vermiformis Strains from Soil Sources in El Hierro Island, Canary Islands, Spain.

    PubMed

    Reyes-Batlle, María; Wagner, Carolina; Zamora-Herrera, Jonadab; Vargas-Mesa, Alejandro; Sifaoui, Ines; González, Ana C; López-Arencibia, Atteneri; Valladares, Basilio; Martínez-Carretero, Enrique; Piñero, José E; Lorenzo-Morales, Jacob

    2016-07-01

    Free-living amoebae (FLA) are widely distributed protozoa in the environment and have been isolated from many sources such as dust, soil and water. Furthermore, some genera/species of FLA such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. are also able to cause opportunistic infections in humans and other animals. More recently, FLA have been reported to be environmental carriers of pathogenic bacteria, fungi and viruses, and thus have gained further importance from the public health point of view. Among them, Acanthamoeba spp. and Vermamoeba vermiformis have been described in many occasions as the most common carriers of pathogens of high medical relevance such as Legionella pneumophila and Mycobacterium spp. In this study, 24 soil samples were collected from the island of El Hierro, Canary Islands, Spain, in order to check for the presence of V. vermiformis strains in these samples. Soil samples were cultured on 2 % non-nutrient agar plates covered with a thin layer of heat-killed E. coli and checked daily for the presence of Vermamoeba. After a week, V. vermiformis amoebae were observed in 5 of the 24 processed samples (20.8 %) incubated at room temperature and 37 °C. Molecular characterization was carried out by amplifying the 18S rDNA gene and DNA sequencing, confirming that the isolated strains belonged to Vermamoeba vermiformis species. The high percentage of V. vermiformis in the studied soil sources should raise awareness in the region since these amoebae are potential environmental carriers of pathogens of high medical relevance. PMID:27056561

  10. Distribution of free-living amoebae in a treatment system of textile industrial wastewater.

    PubMed

    Ramirez, Elizabeth; Robles, Esperanza; Martinez, Blanca; Ayala, Reynaldo; Sainz, Guadalupe; Martinez, Maria Elena; Gonzalez, Maria Elena

    2014-11-01

    Free-living amoebae have been found in soil, air and a variety of aquatic environments, but few studies have been conducted on industrial wastewater and none on wastewater from the textile industry. The aim of this study was to determine the presence and distribution of free-living amoebae in a biological treatment system that treats textile industrial wastewater. Samples were taken from input, aeration tank, sedimentation tank and output. Samples were centrifuged at 1200g for 15min, the sediment was seeded on non-nutritive agar with Enterobacter aerogenes (NNE) and the plates were incubated at 30 and 37°C. Free-living amoebae were present in all stages of the treatment system. The highest number of amoebic isolates was found in the aeration tank and no seasonal distribution was observed during the year. A total of 14 amoeba genera were isolated: Acanthamoeba, Echinamoeba, Korotnevella, Mayorella, Naegleria, Platyamoeba, Saccamoeba, Stachyamoeba, Thecamoeba, Vahlkampfia, Vannella, Vermamoeba, Vexillifera and Willaertia. The most frequently found amoebae were Acanthamoeba and Vermamoeba which were found in all treatment system stages. The constant presence and diversity of free-living amoebae in the treatment system were important findings due to the characteristics of the wastewater from the textile plant in terms of the residue content from colorants, fixers, carriers, surfactants, etc., used in fabric dyeing and finishing processes. The factors that determined the presence and distribution of amoebae in the activated sludge system were their capacity to form cysts, which allowed them to resist adverse conditions; food availability; an average temperature of 27-33°C; dissolved oxygen in average concentrations above 2mg/L, and pH in a range of 5.9-7.1. PMID:25072828

  11. The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology.

    PubMed

    Whiting, M F; Carpenter, J C; Wheeler, Q D; Wheeler, W C

    1997-03-01

    Phylogenetic relationships among the holometabolous insect orders were inferred from cladistic analysis of nucleotide sequences of 18S ribosomal DNA (rDNA) (85 exemplars) and 28S rDNA (52 exemplars) and morphological characters. Exemplar outgroup taxa were Collembola (1 sequence), Archaeognatha (1), Ephemerida (1), Odonata (2), Plecoptera (2), Blattodea (1), Mantodea (1), Dermaptera (1), Orthoptera (1), Phasmatodea (1), Embioptera (1), Psocoptera (1), Phthiraptera (1), Hemiptera (4), and Thysanoptera (1). Exemplar ingroup taxa were Coleoptera: Archostemata (1), Adephaga (2), and Polyphaga (7); Megaloptera (1); Raphidioptera (1); Neuroptera (sensu stricto = Planipennia): Mantispoidea (2), Hemerobioidea (2), and Myrmeleontoidea (2); Hymenoptera: Symphyta (4) and Apocrita (19); Trichoptera: Hydropsychoidea (1) and Limnephiloidea (2); Lepidoptera: Ditrysia (3); Siphonaptera: Pulicoidea (1) and Ceratophylloidea (2); Mecoptera: Meropeidae (1), Boreidae (1), Panorpidae (1), and Bittacidae (2); Diptera: Nematocera (1), Brachycera (2), and Cyclorrhapha (1); and Strepsiptera: Corioxenidae (1), Myrmecolacidae (1), Elenchidae (1), and Stylopidae (3). We analyzed approximately 1 kilobase of 18S rDNA, starting 398 nucleotides downstream of the 5' end, and approximately 400 bp of 28S rDNA in expansion segment D3. Multiple alignment of the 18S and 28S sequences resulted in 1,116 nucleotide positions with 24 insert regions and 398 positions with 14 insert regions, respectively. All Strepsiptera and Neuroptera have large insert regions in 18S and 28S. The secondary structure of 18S insert 23 is composed of long stems that are GC rich in the basal Strepsiptera and AT rich in the more derived Strepsiptera. A matrix of 176 morphological characters was analyzed for holometabolous orders. Incongruence length difference tests indicate that the 28S + morphological data sets are incongruent but that 28S + 18S, 18S + morphology, and 28S + 18S + morphology fail to reject the hypothesis of

  12. Fauna Europaea: Coleoptera 2 (excl. series Elateriformia, Scarabaeiformia, Staphyliniformia and superfamily Curculionoidea).

    PubMed

    Audisio, Paolo; Alonso Zarazaga, Miguel-Angel; Slipinski, Adam; Nilsson, Anders; Jelínek, Josef; Taglianti, Augusto Vigna; Turco, Federica; Otero, Carlos; Canepari, Claudio; Kral, David; Liberti, Gianfranco; Sama, Gianfranco; Nardi, Gianluca; Löbl, Ivan; Horak, Jan; Kolibac, Jiri; Háva, Jirí; Sapiejewski, Maciej; Jäch, Manfred; Bologna, Marco Alberto; Biondi, Maurizio; Nikitsky, Nikolai B; Mazzoldi, Paolo; Zahradnik, Petr; Wegrzynowicz, Piotr; Constantin, Robert; Gerstmeier, Roland; Zhantiev, Rustem; Fattorini, Simone; Tomaszewska, Wioletta; Rücker, Wolfgang H; Vazquez-Albalate, Xavier; Cassola, Fabio; Angelini, Fernando; Johnson, Colin; Schawaller, Wolfgang; Regalin, Renato; Baviera, Cosimo; Rocchi, Saverio; Cianferoni, Fabio; Beenen, Ron; Schmitt, Michael; Sassi, David; Kippenberg, Horst; Zampetti, Marcello Franco; Trizzino, Marco; Chiari, Stefano; Carpaneto, Giuseppe Maria; Sabatelli, Simone; de Jong, Yde

    2015-01-01

    Fauna Europaea provides a public web-service with an index of scientific names (including synonyms) of all living European land and freshwater animals, their geographical distribution at country level (up to the Urals, excluding the Caucasus region), and some additional information. The Fauna Europaea project covers about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. This represents a huge effort by more than 400 contributing specialists throughout Europe and is a unique (standard) reference suitable for many users in science, government, industry, nature conservation and education. Coleoptera represent a huge assemblage of holometabolous insects, including as a whole more than 200 recognized families and some 400,000 described species worldwide. Basic information is summarized on their biology, ecology, economic relevance, and estimated number of undescribed species worldwide. Little less than 30,000 species are listed from Europe. The Coleoptera 2 section of the Fauna Europaea database (Archostemata, Myxophaga, Adephaga and Polyphaga excl. the series Elateriformia, Scarabaeiformia, Staphyliniformia and the superfamily Curculionoidea) encompasses 80 families (according to the previously accepted family-level systematic framework) and approximately 13,000 species. Tabulations included a complete list of the families dealt with, the number of species in each, the names of all involved specialists, and, when possible, an estimate of the gaps in terms of total number of species at an European level. A list of some recent useful references is appended. Most families included in the Coleoptera 2 Section have been updated in the most recent release of the Fauna Europaea index, or are ready to be updated as soon as the FaEu data management environment completes its migration from Zoological Museum Amsterdam to Berlin Museum für Naturkunde. PMID

  13. Fauna Europaea: Coleoptera 2 (excl. series Elateriformia, Scarabaeiformia, Staphyliniformia and superfamily Curculionoidea)

    PubMed Central

    Alonso Zarazaga, Miguel-Angel; Slipinski, Adam; Nilsson, Anders; Jelínek, Josef; Taglianti, Augusto Vigna; Turco, Federica; Otero, Carlos; Canepari, Claudio; Kral, David; Liberti, Gianfranco; Sama, Gianfranco; Nardi, Gianluca; Löbl, Ivan; Horak, Jan; Kolibac, Jiri; Háva, Jirí; Sapiejewski, Maciej; Jäch, Manfred; Bologna, Marco Alberto; Biondi, Maurizio; Nikitsky, Nikolai B.; Mazzoldi, Paolo; Zahradnik, Petr; Wegrzynowicz, Piotr; Constantin, Robert; Gerstmeier, Roland; Zhantiev, Rustem; Fattorini, Simone; Tomaszewska, Wioletta; Rücker, Wolfgang H.; Vazquez-Albalate, Xavier; Cassola, Fabio; Angelini, Fernando; Johnson, Colin; Schawaller, Wolfgang; Regalin, Renato; Baviera, Cosimo; Rocchi, Saverio; Cianferoni, Fabio; Beenen, Ron; Schmitt, Michael; Sassi, David; Kippenberg, Horst; Zampetti, Marcello Franco; Trizzino, Marco; Chiari, Stefano; Carpaneto, Giuseppe Maria; Sabatelli, Simone

    2015-01-01

    Abstract Fauna Europaea provides a public web-service with an index of scientific names (including synonyms) of all living European land and freshwater animals, their geographical distribution at country level (up to the Urals, excluding the Caucasus region), and some additional information. The Fauna Europaea project covers about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. This represents a huge effort by more than 400 contributing specialists throughout Europe and is a unique (standard) reference suitable for many users in science, government, industry, nature conservation and education. Coleoptera represent a huge assemblage of holometabolous insects, including as a whole more than 200 recognized families and some 400,000 described species worldwide. Basic information is summarized on their biology, ecology, economic relevance, and estimated number of undescribed species worldwide. Little less than 30,000 species are listed from Europe. The Coleoptera 2 section of the Fauna Europaea database (Archostemata, Myxophaga, Adephaga and Polyphaga excl. the series Elateriformia, Scarabaeiformia, Staphyliniformia and the superfamily Curculionoidea) encompasses 80 families (according to the previously accepted family-level systematic framework) and approximately 13,000 species. Tabulations included a complete list of the families dealt with, the number of species in each, the names of all involved specialists, and, when possible, an estimate of the gaps in terms of total number of species at an European level. A list of some recent useful references is appended. Most families included in the Coleoptera 2 Section have been updated in the most recent release of the Fauna Europaea index, or are ready to be updated as soon as the FaEu data management environment completes its migration from Zoological Museum Amsterdam to Berlin Museum für Naturkunde

  14. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

    SciTech Connect

    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  15. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

    PubMed Central

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-01-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

  16. Revisiting the genome packaging in viruses with lessons from the "Giants".

    PubMed

    Chelikani, Venkata; Ranjan, Tushar; Kondabagil, Kiran

    2014-10-01

    Genome encapsidation is an essential step in the life cycle of viruses. Viruses either use some of the most powerful ATP-dependent motors to compel the genetic material into the preformed capsid or make use of the positively charged proteins to bind and condense the negatively charged genome in an energy-independent manner. While the former is a hallmark of large DNA viruses, the latter is commonly seen in small DNA and RNA viruses. Discoveries of many complex giant viruses such as mimivirus, megavirus, pandoravirus, etc., belonging to the nucleo-cytoplasmic large DNA virus (NCLDV) superfamily have changed the perception of genome packaging in viruses. From what little we have understood so far, it seems that the genome packaging mechanism in NCLDVs has nothing in common with other well-characterized viral packaging systems such as the portal-terminase system or the energy-independent system. Recent findings suggest that in giant viruses, the genome segregation and packaging processes are more intricately coupled than those of other viral systems. Interestingly, giant viral packaging systems also seem to possess features that are analogous to bacterial and archaeal chromosome segregation. Although there is a lot of diversity in terms of host range, type of genome, and genome size among viruses, they all seem to use three major types of independent innovations to accomplish genome encapsidation. Here, we have made an attempt to comprehensively review all the known viral genome packaging systems, including the one that is operative in giant viruses, by proposing a simple and expanded classification system that divides the viral packaging systems into three large groups (types I-III) on the basis of the mechanism employed and the relatedness of the major packaging proteins. Known variants within each group have been further classified into subgroups to reflect their unique adaptations. PMID:24998349

  17. MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes.

    PubMed

    Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2016-01-01

    The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a 'dark matter.' We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about the

  18. Planktonic Microbes in the Gulf of Maine Area

    PubMed Central

    Li, William K. W.; Andersen, Robert A.; Gifford, Dian J.; Incze, Lewis S.; Martin, Jennifer L.; Pilskaln, Cynthia H.; Rooney-Varga, Juliette N.; Sieracki, Michael E.; Wilson, William H.; Wolff, Nicholas H.

    2011-01-01

    In the Gulf of Maine area (GoMA), as elsewhere in the ocean, the organisms of greatest numerical abundance are microbes. Viruses in GoMA are largely cyanophages and bacteriophages, including podoviruses which lack tails. There is also evidence of Mimivirus and Chlorovirus in the metagenome. Bacteria in GoMA comprise the dominant SAR11 phylotype cluster, and other abundant phylotypes such as SAR86-like cluster, SAR116-like cluster, Roseobacter, Rhodospirillaceae, Acidomicrobidae, Flavobacteriales, Cytophaga, and unclassified Alphaproteobacteria and Gammaproteobacteria clusters. Bacterial epibionts of the dinoflagellate Alexandrium fundyense include Rhodobacteraceae, Flavobacteriaceae, Cytophaga spp., Sulfitobacter spp., Sphingomonas spp., and unclassified Bacteroidetes. Phototrophic prokaryotes in GoMA include cyanobacteria that contain chlorophyll (mainly Synechococcus), aerobic anoxygenic phototrophs that contain bacteriochlorophyll, and bacteria that contain proteorhodopsin. Eukaryotic microalgae in GoMA include Bacillariophyceae, Dinophyceae, Prymnesiophyceae, Prasinophyceae, Trebouxiophyceae, Cryptophyceae, Dictyochophyceae, Chrysophyceae, Eustigmatophyceae, Pelagophyceae, Synurophyceae, and Xanthophyceae. There are no records of Bolidophyceae, Aurearenophyceae, Raphidophyceae, and Synchromophyceae in GoMA. In total, there are records for 665 names and 229 genera of microalgae. Heterotrophic eukaryotic protists in GoMA include Dinophyceae, Alveolata, Apicomplexa, amoeboid organisms, Labrynthulida, and heterotrophic marine stramenopiles (MAST). Ciliates include Strombidium, Lohmaniella, Tontonia, Strobilidium, Strombidinopsis and the mixotrophs Laboea strobila and Myrionecta rubrum (ex Mesodinium rubra). An inventory of selected microbial groups in each of 14 physiographic regions in GoMA is made by combining information on the depth-dependent variation of cell density and the depth-dependent variation of water volume. Across the entire GoMA, an estimate for the

  19. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

    PubMed Central

    Smith, Paul; Ho, C. Kiong; Takagi, Yuko; Djaballah, Hakim

    2016-01-01

    ABSTRACT Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. PMID:26908574

  20. Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases

    PubMed Central

    Prakash, Aishwarya; Eckenroth, Brian E.; Averill, April M.; Imamura, Kayo; Wallace, Susan S.; Doublié, Sylvie

    2013-01-01

    Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process. PMID:24120312

  1. Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA-Packaging Motors with Revolving Mechanism.

    PubMed

    Pi, Fengmei; Zhao, Zhengyi; Chelikani, Venkata; Yoder, Kristine; Kvaratskhelia, Mamuka; Guo, Peixuan

    2016-09-15

    The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Recently discovered was a third type of biomotor, including the viral DNA-packaging motor, beside the bacterial DNA translocases, that uses a revolving mechanism without rotation. By analogy, rotation resembles the Earth's rotation on its own axis, while revolving resembles the Earth's revolving around the Sun (see animations at http://rnanano.osu.edu/movie.html). Herein, we review the structures of viral dsDNA-packaging motors, the stoichiometries of motor components, and the motion mechanisms of the motors. All viral dsDNA-packaging motors, including those of dsDNA/dsRNA bacteriophages, adenoviruses, poxviruses, herpesviruses, mimiviruses, megaviruses, pandoraviruses, and pithoviruses, contain a high-stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometries of target complexes that work sequentially. PMID:27356896

  2. MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes

    PubMed Central

    Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2016-01-01

    The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter.’ We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about

  3. Waterborne parasites: a current status from the Philippines

    PubMed Central

    2014-01-01

    Background Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. Methods A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. Results Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p < 0.01) and Giardia and nitrate (r = 0.502, p < 0.01). Conclusion This study was aimed to create greater awareness of parasitic contamination in the water environment in the Philippines and also to

  4. Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

    PubMed Central

    Al-Quadan, Tasneem; Santic, Marina; Habyarimana, Fabien; Kalia, Awdhesh; Kwaik, Yousef Abu

    2009-01-01

    The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the 9L10P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-9L10P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-9L10P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination

  5. Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa.

    PubMed

    Price, Christopher T; Al-Khodor, Souhaila; Al-Quadan, Tasneem; Santic, Marina; Habyarimana, Fabien; Kalia, Awdhesh; Kwaik, Yousef Abu

    2009-12-01

    The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved

  6. Charles Darwin, beetles and phylogenetics.

    PubMed

    Beutel, Rolf G; Friedrich, Frank; Leschen, Richard A B

    2009-11-01

    changed dramatically. With very large data sets and high throughput sampling, phylogenetic questions can be addressed without prior knowledge of morphological characters. Nevertheless, molecular studies have not lead to the great breakthrough in beetle systematics--yet. Especially the phylogeny of the extremely species rich suborder Polyphaga remains incompletely resolved. Coordinated efforts of molecular workers and of morphologists using innovative techniques may lead to more profound insights in the near future. The final aim is to develop a well-founded phylogeny, which truly reflects the evolution of this immensely species rich group of organisms. PMID:19760277

  7. Charles Darwin, beetles and phylogenetics

    NASA Astrophysics Data System (ADS)

    Beutel, Rolf G.; Friedrich, Frank; Leschen, Richard A. B.

    2009-11-01

    . This has changed dramatically. With very large data sets and high throughput sampling, phylogenetic questions can be addressed without prior knowledge of morphological characters. Nevertheless, molecular studies have not lead to the great breakthrough in beetle systematics—yet. Especially the phylogeny of the extremely species rich suborder Polyphaga remains incompletely resolved. Coordinated efforts of molecular workers and of morphologists using innovative techniques may lead to more profound insights in the near future. The final aim is to develop a well-founded phylogeny, which truly reflects the evolution of this immensely species rich group of organisms.

  8. Detection of Free-Living Amoebae Using Amoebal Enrichment in a Wastewater Treatment Plant of Gauteng Province, South Africa

    PubMed Central

    Muchesa, P.; Mwamba, O.; Barnard, T. G.; Bartie, C.

    2014-01-01

    Free-living amoebae pose a potential health risk in water systems as they may be pathogenic and harbor potential pathogenic bacteria known as amoebae resistant bacteria. Free-living amoebae were observed in 150 (87.2%) of the environmental water samples. In particular, Acanthamoeba sp. was identified in 22 (12.8%) using amoebal enrichment and confirmed by molecular analysis. FLA were isolated in all 8 stages of the wastewater treatment plant using the amoebal enrichment technique. A total of 16 (9.3%) samples were positive for FLA from influent, 20 (11.6%) from bioreactor feed, 16 (9.3%) from anaerobic zone, 16 (9.3%) from anoxic zone, 32 (18.6%) from aerators, 16 (9.3%) from bioreactor effluent, 11 (6.4%) from bioreactor final effluent, and 45 (26.2%) from maturation pond. This study provides baseline information on the occurrence of amoebae in wastewater treatment plant. This has health implications on receiving water bodies as some FLA are pathogenic and are also involved in the transmission and dissemination of pathogenic bacteria. PMID:25530964

  9. Isolation and Molecular Characterization of Free-Living Amoebae from Different Water Sources in Italy

    PubMed Central

    Montalbano Di Filippo, Margherita; Santoro, Maristella; Lovreglio, Piero; Monno, Rosa; Capolongo, Carmen; Calia, Carla; Fumarola, Luciana; D’Alfonso, Rossella; Berrilli, Federica; Di Cave, David

    2015-01-01

    Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans. PMID:25811766

  10. A divergent Cardinium found in daddy long-legs (Arachnida: Opiliones).

    PubMed

    Chang, Jin; Masters, Amber; Avery, Amanda; Werren, John H

    2010-11-01

    Recent studies indicate that a newly described bacterial endosymbiont, Cardinium, is widespread in arthropods and induces different reproductive manipulations in hosts. In this study, we used a portion of the 16S rRNA gene of the Cardinium to screen 16 Opilionid species from the suborder Palptores. We found the incidence of Cardinium in these Opiliones was significantly higher than in other pooled arthropods (31.2% versus 7.2%, P=0.007). Phylogenetic analyses using maximum parsimony (MP) and Bayesian analysis revealed two distinct clades in Opiliones. One is a divergent monophyletic clade with strong support that has so far not been found in other arthropods, and a second one contains Cardinium both from Opiliones and other arthropods. There is not complete concordance of the Cardinium strains with host phylogeny, suggesting some horizontal movement of the bacteria among Opiliones. Although the divergence in the sequenced 16S rRNA region between the Cardinium infecting Opiliones and Cardinium from other arthropods is greater than among Cardinium found in other arthropods, all are monophyletic with respect to the outgroup bacteria (endosymbionts of Acanthamoeba). Based on high pairwise genetic distances, deep branch, and a distinct phylogenetic grouping, we conclude that some Opiliones harbor a newly discovered Cardinium clade. PMID:20515696

  11. Novel Clade of Alphaproteobacterial Endosymbionts Associated with Stinkbugs and Other Arthropods

    PubMed Central

    Kikuchi, Yoshitomo; Meng, Xian Ying; Koga, Ryuichi; Fukatsu, Takema

    2012-01-01

    Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade. PMID:22504806

  12. Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment.

    PubMed

    Alleron, Laëtitia; Merlet, Nicole; Lacombe, Christian; Frère, Jacques

    2008-11-01

    Legionella pneumophila, a facultative intracellular human pathogen, can persist for long periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing systems is often difficult. We tested L. pneumophila survival after monochloramine treatment. Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal-yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed after >4 months after treatment. These results demonstrate for the first time that L. pneumophila could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L. pneumophila in water networks is generally done by enumeration on standard solid medium. This method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long periods and should be resuscitated by amoeba. These cells constitute potential sources of contamination and should be taken into account in monitoring water networks. PMID:18839249

  13. Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

    PubMed

    Schnare, M N; Gray, M W

    1982-03-25

    In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista. PMID:7079176

  14. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.

    PubMed

    Al-Bana, Badii H; Haddad, Moreen T; Garduño, Rafael A

    2014-02-01

    Legionella pneumophila is an intracellular bacterial parasite of freshwater protozoa and an accidental waterborne human pathogen. L. pneumophila is highly pleomorphic showing several forms that differentiate within its developmental cycle. In water, L. pneumophila produces viable but non-culturable cells (VBNCCs), which remain largely uncharacterized. We produced VBNCCs from two developmental forms of L. pneumophila [stationary phase forms (SPFs) and mature infectious forms (MIFs)] in two water microcosms [double-deionized (dd) and tap water] at 45°C. In contrast with SPFs, MIFs upheld a robust ultrastructure and high viability in the two water microcosms. In dd-water, MIFs and SPFs lost their culturability faster than in tap water and did not consume their poly-β-hydroxybutyrate inclusions. Resuscitation in Acanthamoeba castellani was only possible for VBNCCs produced from SPFs in tap water. Addition of salts to dd-water prolonged L. pneumophila culturability to tap water levels, suggesting that L. pneumophila requires ions to maintain its readiness to resume growth. VBNCCs resisted detergent lysis and digestion in the ciliate Tetrahymena, except for VBNCCs produced from SPFs in dd-water. L. pneumophila VBNCCs thus show distinct traits according to its originating developmental form and the surrounding water microcosm. PMID:23968544

  15. The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation, Autoaggregation, and Pathogen-Phagocyte Interactions

    PubMed Central

    Abdel-Nour, Mena; Duncan, Carla; Prashar, Akriti; Rao, Chitong; Ginevra, Christophe; Jarraud, Sophie; Low, Donald E.; Ensminger, Alexander W.; Terebiznik, Mauricio R.

    2014-01-01

    Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae. PMID:24334670

  16. 187-gene phylogeny of protozoan phylum Amoebozoa reveals a new class (Cutosea) of deep-branching, ultrastructurally unique, enveloped marine Lobosa and clarifies amoeba evolution.

    PubMed

    Cavalier-Smith, Thomas; Chao, Ema E; Lewis, Rhodri

    2016-06-01

    Monophyly of protozoan phylum Amoebozoa, and subdivision into subphyla Conosa and Lobosa each with different cytoskeletons, are well established. However early diversification of non-ciliate lobose amoebae (Lobosa) is poorly understood. To clarify it we used recently available transcriptomes to construct a 187-gene amoebozoan tree for 30 species, the most comprehensive yet. This robustly places new genus Atrichosa (formerly lumped with Trichosphaerium) within lobosan class Tubulinea, not Discosea as previously supposed. We identified an earliest diverging lobosan clade comprising marine amoebae armoured by porose scaliform cell-envelopes, here made a novel class Cutosea with two pseudopodially distinct new families. Cutosea comprise Sapocribrum, ATCC PRA-29 misidentified as 'Pessonella', plus from other evidence Squamamoeba. We confirm that Acanthamoeba and ATCC 50982 misidentified as Stereomyxa ramosa are closely related. Discosea have a strongly supported major subclade comprising Thecamoebida plus Glycostylida (suborders Dactylopodina, Stygamoebina; Vannellina) phylogenetically distinct from Centramoebida. Stygamoeba is sister to Dactylopodina. Himatismenida are either sister to Centramoebida or deeper branching. Discosea usually appear holophyletic (rarely paraphyletic). Paramoeba transcriptomes include prokinetoplastid Perkinsela-like endosymbiont sequences. Cunea, misidentified as Mayorella, is closer to Paramoeba than Vexillifera within holophyletic Dactylopodina. Taxon-rich site-heterogeneous rDNA trees confirm cutosan distinctiveness, allow improved conosan taxonomy, and reveal previous dictyostelid tree misrooting. PMID:27001604

  17. Corneal infection in Shandong peninsula of China: a 10-year retrospective study on 578 cases

    PubMed Central

    Pan, Xiao-Jing; Jiang, Tao; Zhu, Hai; Liu, Peng-Peng; Zhou, Zhan-Yu; Mao, Alexander J.

    2016-01-01

    AIM To determine the epidemiological characteristics, clinical signs, laboratory findings, and outcomes in patients with corneal infection in Shandong peninsula of China. METHODS The medical records of 578 inpatients (578 eyes) with corneal infection were reviewed retrospectively for demographic characteristics, risk factors, seasonal variation, clinical signs, laboratory findings, and treatment strategy. Patient history, ocular examination findings using slit-lamp biomicroscopy, laboratory findings resulted from microbiological cultures, and treatment. RESULTS Fungal keratitis constituted 58.48% of cases of infectious keratitis among the inpatients, followed by herpes simplex keratitis (20.76%), bacterial keratitis (19.03%) and acanthamoeba keratitis (1.73%). The most common risk factor was corneal trauma (71.80%). The direct microscopic examination (338 cases) using potassium hydroxide (KOH) wet mounts was positive in 296 cases (87.57%). Among the 298 fungal culture-positive cases, Fusarium species were the most common isolates (70.47%). A total of 517 cases (89.45%) received surgical intervention, including 255 (44.12%) cases of penetrating keratoplasty, 74 (12.80%) cases of lamellar keratoplasty which has become increasingly popular, and 77 cases (13.32%) of evisceration or enucleation. CONCLUSION At present, infectious keratitis is a primary corneal disease causing blindness in China. With Fusarium species being the most commonly identified pathogens, fungal keratitis is the leading cause of severe infectious corneal ulcers in Shandong peninsula of China. PMID:26949610

  18. Microbiological Spectrum of Brain Abscess at a Tertiary Care Hospital in South India: 24-Year Data and Review

    PubMed Central

    Lakshmi, V.; Umabala, P.; Anuradha, K.; Padmaja, K.; Padmasree, C.; Rajesh, A.; Purohit, A. K.

    2011-01-01

    Intracranial abscesses are life-threatening infections that pose a diagnostic challenge not only to the neurosurgeon but also to the microbiologists. Detailed studies documenting the spectrum of infecting agents involved in brain abscesses are limited from India. Materials and Methods. This is a retrospective analysis of 352 samples from 1987 to 2010 analyzed at a tertiary care hospital in South India from 1987 to 2010, to document the changing trends with time. Results. The age of the patients ranged from 2 to 80 years, a larger number of males being affected. Otogenic infections were the most common cause while cryptogenic abscesses were 20%. Gram stain and culture positivity were 78% each. Gram-positive and negative facultative aerobes and obligate anaerobes were also on the rise. Unusual organisms, like Burkholderia pseudomallei, Salmonella typhi, Nocardia species, Cladosporium bantiana, Fonsecaea pedrosoi, Entamoeba histolytica, and Acanthamoeba were also isolated and/or detected from the brain abscesses aspirate or resected tissue. Summary. New and emerging pathogens associated with brain abscess, especially in immunosuppressed individuals, have renewed the necessity of an early detection, and it will be of great value in appropriate management of patients with brain abscess. PMID:22191080

  19. The ADF/cofilin family: actin-remodeling proteins

    PubMed Central

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms. PMID:12049672

  20. A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication.

    PubMed

    Goodwin, Ian P; Kumova, Ogan K; Ninio, Shira

    2016-08-01

    The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts. PMID:27421957

  1. Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria

    PubMed Central

    Lambrecht, Ellen; Baré, Julie; Chavatte, Natascha; Bert, Wim; Sabbe, Koen

    2015-01-01

    The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed. PMID:26070667

  2. Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

    PubMed Central

    Wang, Hong; Edwards, Marc; Falkinham, Joseph O.

    2012-01-01

    The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems. PMID:22752174

  3. Fungal and Parasitic Infections of the Eye

    PubMed Central

    Klotz, Stephen A.; Penn, Christopher C.; Negvesky, Gerald J.; Butrus, Salim I.

    2000-01-01

    The unique structure of the human eye as well as exposure of the eye directly to the environment renders it vulnerable to a number of uncommon infectious diseases caused by fungi and parasites. Host defenses directed against these microorganisms, once anatomical barriers are breached, are often insufficient to prevent loss of vision. Therefore, the timely identification and treatment of the involved microorganisms are paramount. The anatomy of the eye and its surrounding structures is presented with an emphasis upon the association of the anatomy with specific infection of fungi and parasites. For example, filamentous fungal infections of the eye are usually due to penetrating trauma by objects contaminated by vegetable matter of the cornea or globe or, by extension, of infection from adjacent paranasal sinuses. Fungal endophthalmitis and chorioretinitis, on the other hand, are usually the result of antecedent fungemia seeding the ocular tissue. Candida spp. are the most common cause of endogenous endophthalmitis, although initial infection with the dimorphic fungi may lead to infection and scarring of the chorioretina. Contact lens wear is associated with keratitis caused by yeasts, filamentous fungi, and Acanthamoebae spp. Most parasitic infections of the eye, however, arise following bloodborne carriage of the microorganism to the eye or adjacent structures. PMID:11023963

  4. Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

    PubMed Central

    Cirillo, J D; Falkow, S; Tompkins, L S; Bermudez, L E

    1997-01-01

    Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium. PMID:9284149

  5. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    SciTech Connect

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-05-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with (..gamma..-/sup 32/P)ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo.

  6. Corneal cross linking and infectious keratitis: a systematic review with a meta-analysis of reported cases

    PubMed Central

    2013-01-01

    Background Collagen cross linking (CXL) of the cornea has been developed recently as a new treatment for multidrug-resistant infectious keratitis. The aim of this study is to summarize the previously published data and evaluate the effectiveness of this treatment. Results The search identified 12 articles. The number of eyes was 104. The infectious keratitis was associated with bacteria in 58 eyes (57f%): Gram-positive bacteria in 44 (43%; 4 of which were infected with Mycobacterium (3.6%)) and Gram-negative bacteria in 14 eyes (13%), fungus in 13 eyes (12%), and Acanthamoeba in 7 eyes (7%). In 26 eyes (25%), the microbiological culture was negative or not performed. The mean time of re-epithelization after CXL was 20.7±28.1 days (minimum of 3, maximum of 145). Sixteen eyes underwent deep or lamellar keratoplasty. The pooled analysis suggested that CXL has a favorable effect on the block of corneal melting in 85% (95%; CI 0.77, 0.91) of eyes. Conclusion Although randomized controlled trials are needed, the available evidence supports the use of CXL in the treatment of infectious keratitis. PMID:23718849

  7. The CpxRA two-component system contributes to Legionella pneumophila virulence.

    PubMed

    Tanner, Jennifer R; Li, Laam; Faucher, Sébastien P; Brassinga, Ann Karen C

    2016-06-01

    The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation. PMID:26934669

  8. Sensitivity of Vermamoeba (Hartmannella) vermiformis cysts to conventional disinfectants and protease.

    PubMed

    Fouque, Emilie; Héchard, Yann; Hartemann, Philippe; Humeau, Philippe; Trouilhé, Marie-Cécile

    2015-06-01

    Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts. PMID:26042964

  9. Emerging protozoal pathogens in India: How prepared are we to face the threat?

    PubMed Central

    Parija, Subhash Chandra; Giri, Sidhartha

    2012-01-01

    Emerging protozoal pathogens have become a major threat to human health. The number of protozoal pathogens causing human disease has been on the rise since the last two to three decades. Significant increase in the number of immunocompromised people, increase in international travel, deforestation, and widespread urban dwellings are some of the factors contributing to this changing epidemiology of protozoal diseases. Apart from Naegleria and Acanthamoeba, other free-living amoebae like Balamuthia and Sappinia are being reported to cause meningoencephalitis in humans. Plasmodium knowlesi, a zoonotic malarial parasite, has become a major cause of human malaria in Southeast Asia. Trypanosoma evansi and Trypanosoma lewisi, which normally infect horses and rodents respectively, have been reported to cause human trypanosomiasis in India. Balantidium coli is emerging as an important cause of dysentery especially in the immunocompromised population. In India, where a significant proportion of population lives in close proximity to cattle and pigs, B. coli can emerge as a significant pathogen in cases of dysentery, especially in the immunocompromised population. Babesia microti has become an important cause of transfusion transmitted babesiosis (TTB) in countries like the United States. As Babesia can be misdiagnosed as Plasmodium and blood transfusion is becoming common in India, it is necessary to develop diagnostic tests to rule out this pathogen in blood donors. Increased awareness among clinicians, pathologists, and microbiologists along with other factors like constant surveillance, improved diagnostic tests, and a high index of suspicion are important to detect and properly treat such emerging protozoal pathogens in humans. PMID:23508066

  10. Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

    PubMed Central

    Schulz, Frederik; Lagkouvardos, Ilias; Wascher, Florian; Aistleitner, Karin; Kostanjšek, Rok; Horn, Matthias

    2014-01-01

    Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations. PMID:24500618

  11. The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

    PubMed

    Schaap, Pauline; Barrantes, Israel; Minx, Pat; Sasaki, Narie; Anderson, Roger W; Bénard, Marianne; Biggar, Kyle K; Buchler, Nicolas E; Bundschuh, Ralf; Chen, Xiao; Fronick, Catrina; Fulton, Lucinda; Golderer, Georg; Jahn, Niels; Knoop, Volker; Landweber, Laura F; Maric, Chrystelle; Miller, Dennis; Noegel, Angelika A; Peace, Rob; Pierron, Gérard; Sasaki, Taeko; Schallenberg-Rüdinger, Mareike; Schleicher, Michael; Singh, Reema; Spaller, Thomas; Storey, Kenneth B; Suzuki, Takamasa; Tomlinson, Chad; Tyson, John J; Warren, Wesley C; Werner, Ernst R; Werner-Felmayer, Gabriele; Wilson, Richard K; Winckler, Thomas; Gott, Jonatha M; Glöckner, Gernot; Marwan, Wolfgang

    2016-01-01

    Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer. PMID:26615215

  12. The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis.

    PubMed

    Griffin, J L

    1983-05-01

    From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing. PMID:6631781

  13. Enhanced bacterial metabolism of a Pseudomonas strain in response to the addition of culture filtrate of a bacteriophagous amoeba.

    PubMed

    Levrat, P; Pussard, M; Alabouvette, C

    1992-02-21

    In a previous work, Levrat et al. [21] showed an enhancement of the production of pyoverdin (siderophore) by Pseudomonas putida in the presence of amoeba. To explain the mechanism of stimulation, the hypothesis of production of stimulatory factors by amoeba was proposed. Filtrates of both mixed culture of bacteria and amoeba (Pseudomonas putida + Acanthamoeba castellanii) and of axenic culture of amoeba were added to the culture medium of Pseudomonas. The production of pyoverdin was increased in the presence of the filtrates. The maximum stimulation was observed with a 6 to 8 day old mixed culture filtrate at 2% final concentration. A higher amount of filtrate did not enhance the stimulation. General metabolisms like ammonium production or respiration were also enhanced in the presence of filtrate of mixed cultures. Filtrates of axenic culture of amoeba were also able to stimulate the production of pyoverdin by Pseudomonas. This stimulation of the bacterial metabolism was not correlated with a higher growth of the bacterial population. Then, the enhancement of the bacterial metabolic activity was not due to a rapid recycling of the bacterial biomass but rather to a production of stimulatory factors by amoeba. PMID:23194985

  14. Metacommunity analysis of amoeboid protists in grassland soils

    PubMed Central

    Fiore-Donno, Anna Maria; Weinert, Jan; Wubet, Tesfaye; Bonkowski, Michael

    2016-01-01

    This study reveals the diversity and distribution of two major ubiquitous groups of soil amoebae, the genus Acanthamoeba and the Myxomycetes (plasmodial slime-moulds) that are rarely, if ever, recovered in environmental sampling studies. We analyzed 150 grassland soil samples from three Biodiversity Exploratories study regions in Germany. We developed specific primers targeting the V2 variable region in the first part of the small subunit of the ribosomal RNA gene for high-throughput pyrotag sequencing. From ca. 1 million reads, applying very stringent filtering and clustering parameters to avoid overestimation of the diversity, we obtained 273 acanthamoebal and 338 myxomycete operational taxonomic units (OTUs, 96% similarity threshold). This number is consistent with the genetic diversity known in the two investigated lineages, but unequalled to date by any environmental sampling study. Only very few OTUs were identical to already known sequences. Strikingly different OTUs assemblages were found between the three German regions (PerMANOVA p.value = 0.001) and even between sites of the same region (multiple-site Simpson-based similarity indices <0.4), showing steep biogeographical gradients. PMID:26750872

  15. Architecture and host interface of environmental chlamydiae revealed by electron cryotomography

    PubMed Central

    Pilhofer, Martin; Aistleitner, Karin; Ladinsky, Mark S.; König, Lena; Horn, Matthias; Jensen, Grant J.

    2016-01-01

    Summary Chlamydiae comprise important pathogenic and symbiotic bacteria that alternate between morphologically and physiologically different life stages during their developmental cycle. Using electron cryotomography, we characterize the ultrastructure of the developmental stages of three environmental chlamydiae: Parachlamydia acanthamoebae, Protochlamydia amoebophila and Simkania negevensis. We show that chemical fixation and dehydration alter the cell shape of Parachlamydia and that the crescent body is not a developmental stage, but an artefact of conventional electron microscopy. We further reveal type III secretion systems of environmental chlamydiae at macromolecular resolution and find support for a chlamydial needle-tip protein. Imaging bacteria inside their host cells by cryotomography for the first time, we observe marked differences in inclusion morphology and development as well as host organelle recruitment between the three chlamydial organisms, with Simkania inclusions being tightly enveloped by the host endoplasmic reticulum. The study demonstrates the power of electron cryotomography to reveal structural details of bacteria–host interactions that are not accessible using traditional methods. PMID:24118768

  16. Allovahlkampfia spelaea Causing Keratitis in Humans

    PubMed Central

    Tolba, Mohammed Essa Marghany; Huseein, Enas Abdelhameed Mahmoud; Farrag, Haiam Mohamed Mahmoud; Mohamed, Hanan El Deek; Kobayashi, Seiki; Suzuki, Jun; Ali, Tarek Ahmed Mohamed; Sugano, Sumio

    2016-01-01

    Background Free-living amoebae are present worldwide. They can survive in different environment causing human diseases in some instances. Acanthamoeba sp. is known for causing sight-threatening keratitis in humans. Free-living amoeba keratitis is more common in developing countries. Amoebae of family Vahlkampfiidae are rarely reported to cause such affections. A new genus, Allovahlkampfia spelaea was recently identified from caves with no data about pathogenicity in humans. We tried to identify the causative free-living amoeba in a case of keratitis in an Egyptian patient using morphological and molecular techniques. Methods Pathogenic amoebae were culture using monoxenic culture system. Identification through morphological features and 18S ribosomal RNA subunit DNA amplification and sequencing was done. Pathogenicity to laboratory rabbits and ability to produce keratitis were assessed experimentally. Results Allovahlkampfia spelaea was identified as a cause of human keratitis. Whole sequence of 18S ribosomal subunit DNA was sequenced and assembled. The Egyptian strain was closely related to SK1 strain isolated in Slovenia. The ability to induce keratitis was confirmed using animal model. Conclusions This the first time to report Allovahlkampfia spelaea as a human pathogen. Combining both molecular and morphological identification is critical to correctly diagnose amoebae causing keratitis in humans. Use of different pairs of primers and sequencing amplified DNA is needed to prevent misdiagnosis. PMID:27415799

  17. Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria.

    PubMed

    Lambrecht, Ellen; Baré, Julie; Chavatte, Natascha; Bert, Wim; Sabbe, Koen; Houf, Kurt

    2015-08-15

    The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed. PMID:26070667

  18. Free-living amoebae used to isolate consortia capable of degrading trichloroethylene

    SciTech Connect

    Tyndall, R.L.; Katz, D.S.; Little, C.D.; Kennedy, J.R.

    1991-12-31

    The interaction of protozoa with prokaryotes is well documented. These interactions can be either ecto- or endosymbiotic. An example of photosynthetic symbiosis is the well-defined interaction between paramecium and entrapped Chlorella. Paramecium can also form symbiotic relationships with gram-negative heterotrophic bacteria. Jeon has described an interaction between amoebae and engulfed bacteria, that eventuated into a dependency of the amoebae on the presence of the engulfed bacterium. Free-living amoebae and tetrahymena can engulf and subsequently provide the necessary niche for the replication of Legionella. Acanthamoebae trophozoites and cysts can harbor and support the replication of unidentified gram-negative bacteria. King has recently shown that bacteria associated with free-living amoebae are more resistant to toxic environments. Assuming that methylotrophic bacteria in situ are a part of a mixed community, and based on our observations that bacteria associated with protozoa may not be easily isolated by standard techniques, we attempted to use protozoa as a tool to isolate TCE-degrading bacteria.

  19. Identification of Legionella pneumophila Genes Important for Infection of Amoebas by Signature-Tagged Mutagenesis

    PubMed Central

    Polesky, Andrea H.; Ross, Julianna T. D.; Falkow, Stanley; Tompkins, Lucy S.

    2001-01-01

    Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes. PMID:11159993

  20. Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations.

    PubMed Central

    Paszko-Kolva, C; Yamamoto, H; Shahamat, M; Sawyer, T K; Morris, G; Colwell, R R

    1991-01-01

    Forty eyewash units were sampled for protozoa, bacteria, and fungi. Total heterotrophic bacterial counts on nutrient agar and R2A agar (Difco Laboratories, Detroit, Mich.) ranged from 0 to 10(5) CFU/ml, with Pseudomonas spp. being the most frequently isolated. Total counts of 10(4) and 10(8) cells per ml were obtained with the acridine orange staining procedure. All samples were examined for Legionella spp. by direct fluorescent-antibody staining and by culturing on buffered charcoal-yeast extract agar containing alpha-ketoglutarate and glycine and supplemented with cycloheximide, vancomycin, and polymyxin B. DNA-DNA hybridization was used to confirm identification of the Legionella isolates. Legionellae were detected in 35 of 40 (87.5%) samples by direct fluorescent-antibody staining, with 3 samples yielding both Legionella spp. and amoebae. Amoebae identified as Hartmannella, Vahlkampfia, Acanthamoeba, and Cochliopodium spp. were detected in 19 of 40 (47:5%) samples. Sabouraud dextrose agar was used to obtain a crude estimate of viable fungal populations, pH, hardness, and ammonia, alkalinity, chlorine, copper, and iron contents were recorded for all water samples collected from eyewash stations; 33% of the samples had greater than or equal to 10 mg of CO2 per liter. It is concluded that eyewash stations not regularly flushed and/or cleaned and used to flush traumatized eye tissue may be a source of infection and can contaminate laboratory environments via aerosol transmission. PMID:2036003

  1. Distribution System Water Quality Affects Responses of Opportunistic Pathogen Gene Markers in Household Water Heaters.

    PubMed

    Wang, Hong; Masters, Sheldon; Falkinham, Joseph O; Edwards, Marc A; Pruden, Amy

    2015-07-21

    Illustrative distribution system operation and management practices shaped the occurrence and persistence of Legionella spp., nontuberculous mycobacteria (NTM), Pseudomonas aeruginosa, and two amoebae host (Acanthamoeba spp., Vermamoeba vermiformis) gene markers in the effluent of standardized simulated household water heaters (SWHs). The interplay between disinfectant type (chlorine or chloramine), water age (2.3-5.7 days) and materials (polyvinyl chloride (PVC), cement or iron) in upstream simulated distribution systems (SDSs) profoundly influenced levels of pathogen gene markers in corresponding SWH bulk waters. For example, Legionella spp. were 3-4 log higher in SWHs receiving water from chloraminated vs chlorinated SDSs, because of disinfectant decay from nitrification. By contrast, SWHs fed with chlorinated PVC SDS water not only harbored the lowest levels of all pathogen markers, but effluent from the chlorinated SWHs were even lower than influent levels in several instances (e.g., 2 log less Legionella spp. and NTM for PVC and 3-5 log less P. aeruginosa for cement). However, pathogen gene marker influent levels correlated positively to effluent levels in the SWHs (P < 0.05). Likewise, microbial community structures were similar between SWHs and the corresponding SDS feed waters. This study highlights the importance and challenges of distribution system management/operation to help control opportunistic pathogens. PMID:26121595

  2. Black cobra (Naja naja karachiensis) lysates exhibit broad-spectrum antimicrobial activities

    PubMed Central

    Sagheer, Mehwish; Siddiqui, Ruqaiyyah; Iqbal, Junaid; Khan, Naveed Ahmed

    2014-01-01

    It is hypothesized that animals living in polluted environments possess antimicrobials to counter pathogenic microbes. The fact that snakes feed on germ-infested rodents suggests that they encounter pathogenic microbes and likely possess antimicrobials. The venom is used only to paralyze the rodent, but the ability of snakes to counter potential infections in the gut due to disease-ridden rodents requires robust action of the immune system against a broad range of pathogens. To test this hypothesis, crude lysates of different organs of Naja naja karachiensis (black cobra) were tested for antimicrobial properties. The antimicrobial activities of extracts were tested against selected bacterial pathogens (neuropathogenic Escherichia coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumonia), protist (Acanthamoeba castellanii), and filamentous fungus (Fusarium solani). The findings revealed that plasma and various organ extracts of N. n. karachiensis exhibited antimicrobial activity against E. coli K1, MRSA, P. aeruginosa, S. pneumoniae, A. castellanii, and F. solani in a concentration-dependent manner. The results of this study are promising for the development of new antimicrobials. PMID:24625321

  3. Ultraviolet radiation for the sterilization of contact lenses

    SciTech Connect

    Gritz, D.C.; Lee, T.Y.; McDonnell, P.J.; Shih, K.; Baron, N. )

    1990-10-01

    Two sources of ultraviolet (UV) radiation with peak wavelengths in the UV-C or UV-B ranges were compared for their ability to sterilize contact lenses infected with Pseudomonas aeruginosa, Streptococcus pneumoniae, Acanthamoeba castellani, Candida albicans, and Aspergillus niger. Also examined was the effect of prolonged UV light exposure on soft and rigid gas permeable (RGP) contact lenses. The UV-C lamp (253.7 nm, 250 mW/cm2 at 1 cm) was germicidal for all organisms within 20 minutes but caused destruction of the soft lens polymers within 6 hours of cumulative exposure. UV-C caused damage to RGP lenses in less than 100 hours. The UV-B lamp (290-310 nm, 500 mW/cm2 at 1 cm) was germicidal for all organisms tested (except Aspergillus) with a 180-minute exposure and caused less severe changes in the soft lens polymers than did the UV-C lamp, although cumulative exposure of 300 hours did substantially weaken the soft lens material. RGP materials were minimally affected by exposure to 300 hours of UV-B. Ultraviolet light is an effective germicidal agent but is injurious to soft lens polymers; its possible utility in the sterilization of RGP lenses and lens cases deserves further study.

  4. Abundance, diversity and community composition of free-living protozoa on vegetable sprouts.

    PubMed

    Chavatte, N; Lambrecht, E; Van Damme, I; Sabbe, K; Houf, K

    2016-05-01

    Interactions with free-living protozoa (FLP) have been implicated in the persistence of pathogenic bacteria on food products. In order to assess the potential involvement of FLP in this contamination, detailed knowledge on their occurrence, abundance and diversity on food products is required. In the present study, enrichment and cultivation methods were used to inventory and quantify FLP on eight types of commercial vegetable sprouts (alfalfa, beetroot, cress, green pea, leek, mung bean, red cabbage and rosabi). In parallel, total aerobic bacteria and Escherichia coli counts were performed. The vegetable sprouts harbored diverse communities of FLP, with Tetrahymena (ciliate), Bodo saltans and cercomonads (flagellates), and Acanthamoeba and Vannella (amoebae) as the dominant taxa. Protozoan community composition and abundance significantly differed between the sprout types. Beetroot harbored the most abundant and diverse FLP communities, with many unique species such as Korotnevella sp., Vannella sp., Chilodonella sp., Podophrya sp. and Sphaerophrya sp. In contrast, mung bean sprouts were species-poor and had low FLP numbers. Sampling month and company had no significant influence, suggesting that seasonal and local factors are of minor importance. Likewise, no significant relationship between protozoan community composition and bacterial load was observed. PMID:26742616

  5. Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants.

    PubMed

    Dupuy, Mathieu; Berne, Florence; Herbelin, Pascaline; Binet, Marie; Berthelot, Nelsie; Rodier, Marie-Hélène; Soreau, Sylvie; Héchard, Yann

    2014-03-01

    Free-living amoebae are naturally present in water. These protozoa could be pathogenic and could also shelter pathogenic bacteria. Thus, they are described as a potential hazard for health. Also, free-living amoebae have been described to be resistant to biocides, especially under their cyst resistant form. There are several studies on amoeba treatments but none of them compare sensitivity of trophozoites and cysts from different genus to various water disinfectants. In our study, we tested chlorine, monochloramine and chlorine dioxide on both cysts and trophozoites from three strains, belonging to the three main genera of free-living amoebae. The results show that, comparing cysts to trophozoites inactivation, only the Acanthamoeba cysts were highly more resistant to treatment than trophozoites. Comparison of the disinfectant efficiency led to conclude that chlorine dioxide was the most efficient treatment in our conditions and was particularly efficient against cysts. In conclusion, our results would help to adapt water treatments in order to target free-living amoebae in water networks. PMID:23932411

  6. Application of cattle slurry containing Mycobacterium avium subsp. paratuberculosis (MAP) to grassland soil and its effect on the relationship between MAP and free-living amoeba.

    PubMed

    Salgado, M; Alfaro, M; Salazar, F; Badilla, X; Troncoso, E; Zambrano, A; González, M; Mitchell, R M; Collins, M T

    2015-01-30

    Slurry from dairy farms is commonly used to fertilize crops and pastures. This mixture of manure, urine and water can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP) is a major concern. Persistence of MAP in soil and infection of soil Acanthamoeba was evaluated by culture, real-time IS900 PCR, and by staining of amoeba with acid-fast and vital stains comparing soils irrigated with MAP-spiked or control dairy farm slurry. MAP DNA was detected in soil for the 8 month study duration. MAP was detected by PCR from more soil samples for plots receiving MAP-spiked slurry (n=61/66) than from soils receiving control slurry (n=10/66 samples). Vital stains verified that intracellular MAP in amoeba was viable. More MAP was found in amoeba at the end of the study than immediately after slurry application. There was no relationship between MAP presence in soil and in amoeba over time. Infection of amoeba by MAP provides a protected niche for the persistence and even possibly the replication of MAP in soils. As others have suggested, MAP-infected amoeba may act like a "Trojan horse" providing a means for persistence in soils and potentially a source of infection for grazing animals. PMID:25448447

  7. Contact Lens Wearer Demographics and Risk Behaviors for Contact Lens-Related Eye Infections--United States, 2014.

    PubMed

    Cope, Jennifer R; Collier, Sarah A; Rao, Maya M; Chalmers, Robin; Mitchell, G Lynn; Richdale, Kathryn; Wagner, Heidi; Kinoshita, Beth T; Lam, Dawn Y; Sorbara, Luigina; Zimmerman, Aaron; Yoder, Jonathan S; Beach, Michael J

    2015-08-21

    Contact lenses provide safe and effective vision correction for many Americans. However, contact lens wearers risk infection if they fail to wear, clean, disinfect, and store their contact lenses as directed. Over the past decade, CDC has investigated several multistate outbreaks of serious eye infections among contact lens wearers, including Acanthamoeba keratitis. Each investigation identified frequent contact lens hygiene-related risk behaviors among patients. To guide prevention efforts, a population-based survey was used to estimate the number of contact lens wearers aged ≥18 years in the United States. A separate online survey of contact lens wearers assessed the prevalence of contact lens hygiene-related risk behaviors. Approximately 99% of wearers reported at least one contact lens hygiene risk behavior. Nearly one third of contact lens wearers reported having experienced a previous contact lens-related red or painful eye requiring a doctor's visit. An estimated 40.9 million U.S. adults wear contact lenses, and many could be at risk for serious eye infections because of poor contact lens wear and care behaviors. These findings have informed the creation of targeted prevention messages aimed at contact lens wearers such as keeping all water away from contact lenses, discarding used disinfecting solution from the case and cleaning with fresh solution each day, and replacing their contact lens case every 3 months. PMID:26292204

  8. Molecular phylogenetic position of hexactinellid sponges in relation to the Protista and Demospongiae.

    PubMed

    West, L; Powers, D

    1993-01-01

    Although it is generally accepted that the first multicellular organisms arose from unicellular ancestors, the phylogenetic relationships linking these groups remain unclear. Anatomical, physiological, and molecular studies of current multicellular organisms with relatively simple body organization suggest key characteristics of the earliest multicellular lineages. Glass sponges, the Hexactinellida, possess cellular characteristics that resemble some unicellular protistan organisms. These unique sponges were abundant in shallow seas of the early Cambrian, but they are currently restricted to polar habitats or very deep regions of the world oceans. Due in part to their relative inaccessibility, their potential significance to the early phylogeny of the eukaryotic kingdoms has been largely overlooked. We used sequences of the 18s ribosomal RNA gene of Farrea occa, a representative of the deep-water hexactinellid sponges, and Coelocarteria singaporense, a representative of the more common demosponges, and compared them with selected ribosomal RNA gene sequences available within the Protista. Using four computational methods for phylogenetic analysis of ribosomal DNA sequences, we found that the hexactinellid sponge-demosponge cluster is most closely related to Volvox and Acanthamoeba. PMID:8364691

  9. Intermediary Metabolism in Protists: a Sequence-based View of Facultative Anaerobic Metabolism in Evolutionarily Diverse Eukaryotes

    PubMed Central

    Ginger, Michael L.; Fritz-Laylin, Lillian K.; Fulton, Chandler; Cande, W. Zacheus; Dawson, Scott C.

    2011-01-01

    Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2–3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H2 in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. PMID:21036663

  10. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  11. Association between Legionella pneumophila and amoebae in water.

    PubMed

    Henke, M; Seidel, K M

    1986-09-01

    The presence of amoebae and Legionella pneumophila in ground-water, drinking water supplies and whirlpools was investigated. Volumes of 10 to 1,000 ml were concentrated by membrane filtration. L. pneumophila was detected on buffered charcoal yeast extract (BCYE) agar, and amoebae by inverting filters on nonnutrient agar plates seeded with Escherichia coli that were incubated at 37 C for up to 12 days. In 65% of the samples positive for L. pneumophila amoebae were also detected. L. pneumophila and amoebae were detected together in 38% of warm drinking water samples. The highest isolation temperature for amoebae was 57 C, but fewer amoebae were detected above than below 50 C. In cold drinking water, amoebae were found in 88% of samples. The presence of L. pneumophila and amoebae in whirlpool waters (42%) presents a risk for man. Fresh environmental isolates of an Acanthamoeba species and L. pneumophila serogroup 4 were used for laboratory experiments. The amoebae supported intracellular multiplication of L. pneumophila in Chang's medium and autoclaved tap water, as shown by colony-forming unit (CFU) counts, direct fluorescent antibody test and Gimenez staining. Results confirmed that interaction between L. pneumophila and amoebae could occur in nature, and that the latter could act as hosts for legionellae and support their growth. PMID:3793452

  12. Molecular diversity and high virulence of Legionella pneumophila strains isolated from biofilms developed within a warm spring of a thermal spa

    PubMed Central

    2013-01-01

    Background Several cases of legionellosis have been diagnosed in the same French thermal spa in 1986, 1994 and 1997. L. pneumophila serogroup 1 (Lp1) strains have been isolated from several patients, but the source of contamination was not identified despite the presence of different Lp1 in water samples of the three natural springs feeding the spa at this period. Results Our strategy was to investigate L. pneumophila (Lp) strains from natural biofilms developed in a sulphur-rich warm spring of this contaminated site. Biofilm analysis revealed the presence of three Lp serogroups (Lp1, Lp10 and Lp12). Surprisingly, Lp10 and Lp12 were not reported in the previous described studies from water samples. Besides, the new seven Lp1 we isolated exhibit a high molecular diversity and have been differentiated in five classes according to their DNA genome patterns obtained by PFGE and mip sequences. It must be noted that these DNA patterns are original and unknown in databases. Interestingly, the 27 Lp environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acanthamoeba castellanii than those of known Lp1 epidemic strains. Conclusion The characteristics of Legionella pneumophila Lp1 strains isolated from the warm spring are in agreement with their presence in biofilms and their probable long-term persistence in this ecosystem. PMID:23350929

  13. Genotyping of Environmental and Clinical Stenotrophomonas maltophilia Isolates and their Pathogenic Potential

    PubMed Central

    Adamek, Martina; Overhage, Jörg; Bathe, Stephan; Winter, Josef; Fischer, Reinhard; Schwartz, Thomas

    2011-01-01

    Stenotrophomonas maltophilia is a highly versatile species with useful biotechnological potential but also with pathogenic properties. In light of possible differences in virulence characteristics, knowledge about genomic subgroups is therefore desirable. Two different genotyping methods, rep-PCR fingerprinting and partial gyrB gene sequencing were used to elucidate S. maltophilia intraspecies diversity. Rep-PCR fingerprinting revealed the presence of 12 large subgroups, while gyrB gene sequencing distinguished 10 subgroups. For 8 of them, the same strain composition was shown with both typing methods. A subset of 59 isolates representative for the gyrB groups was further investigated with regards to their pathogenic properties in a virulence model using Dictyostelium discoideum and Acanthamoeba castellanii as host organisms. A clear tendency towards accumulation of virulent strains could be observed for one group with A. castellanii and for two groups with D. discoideum. Several virulent strains did not cluster in any of the genetic groups, while other groups displayed no virulence properties at all. The amoeba pathogenicity model proved suitable in showing differences in S. maltophilia virulence. However, the model is still not sufficient to completely elucidate virulence as critical for a human host, since several strains involved in human infections did not show any virulence against amoeba. PMID:22110692

  14. Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity.

    PubMed

    Loiseau, Clémence; Schlusselhuber, Margot; Bigot, Renaud; Bertaux, Joanne; Berjeaud, Jean-Marc; Verdon, Julien

    2015-06-01

    A contaminant bacterial strain was found to exhibit an antagonistic activity against Legionella pneumophila, the causative agent of Legionnaires' disease. The bacterial strain was identified as a Bacillus subtilis and named B. subtilis AM1. PCR analysis revealed the presence of the sfp gene, involved in the biosynthesis of surfactin, a lipopeptide with versatile bioactive properties. The bioactive substances were extracted from AM1 cell-free supernatant with ethyl acetate and purified using reversed phase HPLC (RP-HPLC). Subsequent ESI-MS analyses indicated the presence of two active substances with protonated molecular ions at m/z 1008 and 1036 Da, corresponding to surfactin isoforms. Structures of lipopeptides were further determined by tandem mass spectrometry and compared to the spectra of a commercially available surfactin mixture. Surfactin displays an antibacterial spectrum almost restricted to the Legionella genus (MICs range 1-4 μg/mL) and also exhibits a weak activity toward the amoeba Acanthamoeba castellanii, known to be the natural reservoir of L. pneumophila. Anti-biofilm assays demonstrated that 66 μg/mL of surfactin successfully eliminated 90 % of a 6-day-old biofilm. In conclusion, this study reveals for the first time the potent activity of surfactin against Legionella sp. and preformed biofilms thus providing new directions toward the use and the development of lipopeptides for the control of Legionella spread in the environment. PMID:25573468

  15. Free-living amoebae and their associated bacteria in Austrian cooling towers: a 1-year routine screening.

    PubMed

    Scheikl, Ute; Tsao, Han-Fei; Horn, Matthias; Indra, Alexander; Walochnik, Julia

    2016-09-01

    Free-living amoebae (FLA) are widely spread in the environment and known to cause rare but often serious infections. Besides this, FLA may serve as vehicles for bacterial pathogens. In particular, Legionella pneumophila is known to replicate within FLA thereby also gaining enhanced infectivity. Cooling towers have been the source of outbreaks of Legionnaires' disease in the past and are thus usually screened for legionellae on a routine basis, not considering, however, FLA and their vehicle function. The aim of this study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR-based screening system for various groups of FLA was established. Three cooling towers were screened every 2 weeks over the period of 1 year for FLA and Legionella spp., by culture and molecular methods in parallel. Altogether, 83.3 % of the cooling tower samples were positive for FLA, Acanthamoeba being the dominating genus. Interestingly, 69.7 % of the cooling tower samples were not suitable for the standard Legionella screening due to their high organic burden. In the remaining samples, positivity for Legionella spp. was 25 % by culture, but overall positivity was 50 % by molecular methods. Several amoebal isolates revealed intracellular bacteria. PMID:27177720

  16. Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

    PubMed

    Trigui, Hana; Paquet, Valérie E; Charette, Steve J; Faucher, Sébastien P

    2016-05-01

    Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence ofC. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized thatC. jejunicould be packaged under aerobic conditions by the amoebaAcanthamoeba castellaniior the ciliateTetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellaniidid not produce MLBs containingC. jejuni In contrast, when incubated withT. pyriformis,C. jejuniwas ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics ofC. jejuniculturability showed that packaging increased the survival ofC. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests thatT. pyriformismay increase the risk of persistence ofC. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging. PMID:26921427

  17. Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

    PubMed

    Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

    2016-03-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater a