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Sample records for mycobacterium dna elicits

  1. Inactivation of Mycobacterium tuberculosis for DNA Typing Analysis

    PubMed Central

    Bemer-Melchior, P.; Drugeon, H. B.

    1999-01-01

    DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months. PMID:10364613

  2. Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.

    PubMed

    Mo, Yongkai; Quanquin, Natalie M; Vecino, William H; Ranganathan, Uma Devi; Tesfa, Lydia; Bourn, William; Derbyshire, Keith M; Letvin, Norman L; Jacobs, William R; Fennelly, Glenn J

    2007-10-01

    Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant-albeit only 1.7-fold-increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120(h)(E)) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude

  3. Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization▿

    PubMed Central

    Mo, Yongkai; Quanquin, Natalie M.; Vecino, William H.; Ranganathan, Uma Devi; Tesfa, Lydia; Bourn, William; Derbyshire, Keith M.; Letvin, Norman L.; Jacobs, William R.; Fennelly, Glenn J.

    2007-01-01

    Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude

  4. The DNA-binding network of Mycobacterium tuberculosis

    PubMed Central

    Minch, Kyle J.; Rustad, Tige R.; Peterson, Eliza J. R.; Winkler, Jessica; Reiss, David J.; Ma, Shuyi; Hickey, Mark; Brabant, William; Morrison, Bob; Turkarslan, Serdar; Mawhinney, Chris; Galagan, James E.; Price, Nathan D.; Baliga, Nitin S.; Sherman, David R.

    2015-01-01

    Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression. PMID:25581030

  5. [Detection of Mycobacterium leprae DNA in nasal swab].

    PubMed

    Pontes, Ana Rosa Botelho; Almeida, Maria das Graças Carvalho; Xavier, Marília Brasil; Quaresma, Juarez Antonio Simões; Yassui, Edna Aoba

    2008-01-01

    Studies have demonstrated high sensibility of the polimerase chain reaction (PCR) technique in the identification of the Mycobacterium leprae DNA . This study aimed to evalue the PCR sensibility at the detection of the M. leprae DNA in nasal swab of leprosy patients and to compare the results with the bacilloscopy and multibacillary (MBs) and paucibacilares (PBs) forms. Nasal secretion samples of 24 leprosy patients were collected, and were preserved in one and two lise's solution. The PCR results were highly significant (p <0.0000) and they revealed grater sensibility than bacilloscopy, in several clinical forms. Nevertheless, still different studies are necessary, testing new markers and preservatives, with the purpose of lifting up the sensibility of this technique, in nasal secretion samples. PMID:19009116

  6. Structural and Thermodynamic Signatures of DNA Recognition by Mycobacterium tuberculosis DnaA

    SciTech Connect

    Tsodikov, Oleg V.; Biswas, Tapan

    2011-09-06

    An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 {angstrom} resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 {angstrom}). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.

  7. Mycobacterium avium serovars 2 and 8 infections elicit unique activation of the host macrophage immune responses.

    PubMed

    Cebula, B R; Rocco, J M; Maslow, J N; Irani, V R

    2012-12-01

    Mycobacterium avium is an opportunistic pathogen whose pathogenesis is attributed to its serovar-specific glycopeptidolipid (ssGPL), which varies among its 31 serovars. To determine if the presence and type of ssGPLs contribute to M. avium pathogenesis, we infected murine macrophages (mφs) with two M. avium wild type (wt) serovars (2 and 8) and their serovar-null strains. We examined the influence of ssGPL (presence and type) on cytokine production in non-activated (-IFN-γ) and activated (+IFN-γ) mφs, and the bacterial intra-mφ survival over a 6-day infection process. Serovar-2 infections activated TNF-α production that increased over the 6 day period and was capable of controlling the intra-mφ serovar-2 null strain. In contrast, the serovar-8 infection stimulated a strong pro-inflammatory response, but was incapable of removing the invading pathogen, maybe through IL-10 production. It was clear that the intracellular growth of serovar-null in contrast to the wt M. avium strains was easily controlled. Based on our findings and the undisputed fact that M. avium ssGPL is key to its pathogenesis, we conclude that it is not appropriate to dissect the pathogenesis of one M. avium serovar and apply those findings to other serovars. PMID:22991047

  8. The cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy

    PubMed Central

    MacDuff, Donna A.; Kimmey, Jacqueline M.; Diner, Elie J.; Olivas, Joanna; Vance, Russell E.; Stallings, Christina L.; Virgin, Herbert W.; Cox, Jeffery S.

    2015-01-01

    Summary Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolicDNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections. PMID:26048136

  9. The Cytosolic Sensor cGAS Detects Mycobacterium tuberculosis DNA to Induce Type I Interferons and Activate Autophagy.

    PubMed

    Watson, Robert O; Bell, Samantha L; MacDuff, Donna A; Kimmey, Jacqueline M; Diner, Elie J; Olivas, Joanna; Vance, Russell E; Stallings, Christina L; Virgin, Herbert W; Cox, Jeffery S

    2015-06-10

    Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolic DNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections. PMID:26048136

  10. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization.

    PubMed

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-10-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts. PMID:23851482

  11. Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization

    PubMed Central

    Zhang, Chunhua; Jin, Ke; Xiao, Yanling; Cheng, Ying; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2013-01-01

    Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts. PMID:23851482

  12. Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

    PubMed Central

    Balasingham, Seetha V.; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L.; Laerdahl, Jon K.; Bohr, Vilhelm A.; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB. PMID:22615856

  13. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  14. Detection of Mycobacterium ulcerans DNA in the Environment, Ivory Coast

    PubMed Central

    Tian, Roger Bi Diangoné; Niamké, Sébastian; Tissot-Dupont, Hervé; Drancourt, Michel

    2016-01-01

    Background Ivory Coast is a West African country with the highest reported cases of Buruli ulcer, a disabling subcutaneous infection due to Mycobacterium ulcerans. However, the prevalence of environmental M. ulcerans is poorly known in this country. Methods We collected 496 environmental specimens consisting of soil (n = 100), stagnant water (n = 200), plants (n = 100) and animal feces (n = 96) in Ivory Coast over five months in the dry and wet seasons in regions which are free of Buruli ulcer (control group A; 250 specimens) and in regions where the Buruli ulcer is endemic (group B; 246 specimens). After appropriate total DNA extraction incorporating an internal control, the M. ulcerans IS2404 and KR-B gene were amplified by real-time PCR in samples. In parallel, a calibration curve was done for M. ulcerans Agy99 IS2404 and KR-B gene. Results Of 460 samples free of PCR inhibition, a positive real-time PCR detection of insertion sequence IS2404 and KR-B gene was observed in 1/230 specimens in control group A versus 9/230 specimens in group B (P = 0.02; Fisher exact test). Positive specimens comprised seven stagnant water specimens, two feces specimens confirmed to be of Thryonomys swinderianus (agouti) origin by real-time PCR of the cytb gene; and one soil specimen. Extrapolation from the calibration curves indicated low inoculums ranging from 1 to 102 mycobacteria/mL. Conclusion This study confirms the presence of M. ulcerans in the watery environment surrounding patients with Buruli ulcer in Ivory Coast. It suggests that the agouti, which is in close contacts with populations, could play a role in the environmental cycle of M. ulcerans, as previously suggested for the closely related possums in Australia. PMID:26982581

  15. Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase

    SciTech Connect

    Biswas, Tapan; Tsodikov, Oleg V.

    2009-01-15

    Hexameric DnaB helicase unwinds the DNA double helix during replication of genetic material in bacteria. DnaB is an essential bacterial protein; therefore, it is an important potential target for antibacterial drug discovery. We report a crystal structure of the N-terminal region of DnaB from the pathogen Mycobacterium tuberculosis (MtDnaBn), determined at 2.0 {angstrom} resolution. This structure provides atomic resolution details of formation of the hexameric ring of DnaB by two distinct interfaces. An extensive hydrophobic interface stabilizes a dimer of MtDnaBn by forming a four-helix bundle. The other, less extensive, interface is formed between the dimers, connecting three of them into a hexameric ring. On the basis of crystal packing interactions between MtDnaBn rings, we suggest a model of a helicase-primase complex that explains previously observed effects of DnaB mutations on DNA priming.

  16. Eliciting antigen-specific egg-yolk IgY with naked DNA.

    PubMed

    Romito, M; Viljoen, G J; Du Plessis, D H

    2001-09-01

    Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents. PMID:11570510

  17. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    PubMed Central

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  18. Rapid discrimination of Mycobacterium avium strains from AIDS patients by randomly amplified polymorphic DNA analysis.

    PubMed Central

    Matsiota-Bernard, P; Waser, S; Tassios, P T; Kyriakopoulos, A; Legakis, N J

    1997-01-01

    A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform. PMID:9163488

  19. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics

    PubMed Central

    Rose, Sasha J.; Babrak, Lmar M.; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections. PMID:26010725

  20. NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection

    PubMed Central

    Wei, Meili; Wang, Lu; Wu, Tao; Xi, Jun; Han, Yuze; Yang, Xingxiang; Zhang, Ding; Fang, Qiang

    2016-01-01

    Mycobacterium tuberculosis (Mtb) infection activates the NLRP3 inflammasome in macrophages and dendritic cells. Much attention has been paid to the mechanisms for regulation of NLRP3 against Mtb. However, whether epigenetic mechanisms participated in NLRP3 activation is still little known. Here we showed that NLRP3 activation was regulated by DNA methylation modification. Mtb infection promoted NLRP3 activation and inflammatory cytokines expression. NLRP3 promoter was cloned and subsequently identified by Dual-Luciferase Reporter System. The results showed that NLRP3 promoter activity was decreased after methylation by DNA methylase Sss I in vitro. Meanwhile, DNA methyltransferases inhibitor DAC could upregulate the expression of NLRP3. Furthermore, promoter region of NLRP3 gene was demethylated after Mtb H37Rv strain infection. These data revealed that DNA methylation was involved in NLRP3 inflammasome activation during Mtb infection and provided a new insight into the relationship between host and pathogens. PMID:27366746

  1. Non-Replicating Mycobacterium tuberculosis Elicits a Reduced Infectivity Profile with Corresponding Modifications to the Cell Wall and Extracellular Matrix

    PubMed Central

    Bacon, Joanna; Alderwick, Luke J.; Allnutt, Jon A.; Gabasova, Evelina; Watson, Robert; Hatch, Kim A.; Clark, Simon O.; Jeeves, Rose E.; Marriott, Alice; Rayner, Emma; Tolley, Howard; Pearson, Geoff; Hall, Graham; Besra, Gurdyal S.; Wernisch, Lorenz; Williams, Ann; Marsh, Philip D.

    2014-01-01

    A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP) M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease. PMID:24516549

  2. C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding

    PubMed Central

    Kushwaha, Ambuj K.; Grove, Anne

    2012-01-01

    Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins. PMID:23167261

  3. First detection of Mycobacterium ulcerans DNA in environmental samples from South America.

    PubMed

    Morris, Aaron; Gozlan, Rodolphe; Marion, Estelle; Marsollier, Laurent; Andreou, Demetra; Sanhueza, Daniel; Ruffine, Rolland; Couppié, Pierre; Guégan, Jean-François

    2014-01-01

    The occurrences of many environmentally-persistent and zoonotic infections are driven by ecosystem changes, which in turn are underpinned by land-use modifications that alter the governance of pathogen, biodiversity and human interactions. Our current understanding of these ecological changes on disease emergence however remains limited. Buruli ulcer is an emerging human skin disease caused by the mycobacterium, Mycobacterium ulcerans, for which the exact route of infection remains unclear. It can have a devastating impact on its human host, causing extensive necrosis of the skin and underlying tissue, often leading to permanent disability. The mycobacterium is associated with tropical aquatic environments and incidences of the disease are significantly higher on floodplains and where there is an increase of human aquatic activities. Although the disease has been previously diagnosed in South America, until now the presence of M. ulcerans DNA in the wild has only been identified in Australia where there have been significant outbreaks and in western and central regions of Africa where the disease is persistent. Here for the first time, we have identified the presence of the aetiological agent's DNA in environmental samples from South America. The DNA was positively identified using Real-time Polymerase Chain Reaction (PCR) on 163 environmental samples, taken from 23 freshwater bodies in French Guiana (Southern America), using primers for both IS2404 and for the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes (KR). Five samples out of 163 were positive for both primers from three different water bodies. A further nine sites had low levels of IS2404 close to a standard CT of 35 and could potentially harbour M. ulcerans. The majority of our positive samples (8/14) came from filtered water. These results also reveal the Sinnamary River as a potential source of infection to humans. PMID:24498449

  4. A Mycobacterium bovis BCG-naked DNA prime-boost vaccination strategy induced CD4⁺ and CD8⁺ T-cell response against Mycobacterium tuberculosis immunogens.

    PubMed

    Lu, Miao; Xia, Zhi Yang; Bao, Lang

    2014-01-01

    Mycobacterium tuberculosis infection is still a major global public health problem. Presently the only tuberculosis (TB) vaccine available is Bacille Calmette-Guérin (BCG), although it fails to adequately protect against pulmonary TB in adults. To solve this problem, the development of a new effective vaccine is urgently desired. BCG-prime DNA-booster vaccinations strategy has been shown to induce greater protection against tuberculosis (TB) than BCG alone. Some studies have demonstrated that the two genes (Rv1769 and Rv1772) are excellent T-cell antigens and could induce T-cell immune responses. In this research, we built BCG-C or BCG-P prime-recombination plasmid PcDNA3.1-Rv1769 or PcDNA3.1-Rv1772 boost vaccinations strategy to immunize BALB/c mice and evaluated its immunogenicity. The data suggests that the BCG-C+3.1-72 strategy could elicit the most long-lasting and strongest Th1-type cellular immune responses and the BCG-C+3.1-69 strategy could induce the high level CD8+ T-cell response at certain time points. These findings support the ideas that the prime-boost strategy as a combination of vaccines may be better than a single vaccine for protection against tuberculosis. PMID:24741595

  5. Mycobacterium tuberculosis DNA Detection Using Surface Plasmon Resonance Modulated by Telecommunication Wavelength

    PubMed Central

    Hsu, Shih-Hsiang; Lin, Yan-Yu; Lu, Shao-Hsi; Tsai, I-Fang; Lu, Yen-Ta; Ho, Hsin-Tsung

    2014-01-01

    A surface plasmon resonance sensor for Mycobacterium tuberculosis (MTB) deoxyribonucleic acid (DNA) is developed using repeatable telecommunication wavelength modulation based on optical fiber communications laser wavelength and stability. MTB DNA concentrations of 1 μg/mL and 10 μg/mL were successfully demonstrated to have the same spectral half-width in the dip for optimum coupling. The sensitivity was shown to be −0.087 dB/(μg/mL) at all applied telecommunication wavelengths and the highest sensitivity achieved was 115 ng/mL without thiolated DNA immobilization onto a gold plate, which is better than the sensor limit of 400 ng/mL possible with commercial biosensor equipment. PMID:24379050

  6. Lsr2 of Mycobacterium leprae and Its Synthetic Peptides Elicit Restitution of T Cell Responses in Erythema Nodosum Leprosum and Reversal Reactions in Patients with Lepromatous Leprosy

    PubMed Central

    Saini, Chaman; Prasad, H. K.; Rani, Rajni; Murtaza, A.; Misra, Namita; Shanker Narayan, N. P.

    2013-01-01

    The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742–752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs. PMID:23446220

  7. Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

    PubMed Central

    Caldarelli-Stefano, R; Vago, L; Bonetto, S; Nebuloni, M; Costanzi, G

    1999-01-01

    Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues. PMID:10621838

  8. Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

    PubMed

    Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

    2016-01-15

    Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair. PMID:26512127

  9. Boosting BCG-primed mice with chimeric DNA vaccine HG856A induces potent multifunctional T cell responses and enhanced protection against Mycobacterium tuberculosis.

    PubMed

    Ji, Ping; Hu, Zhi-Dong; Kang, Han; Yuan, Qin; Ma, Hui; Wen, Han-Li; Wu, Juan; Li, Zhong-Ming; Lowrie, Douglas B; Fan, Xiao-Yong

    2016-02-01

    The tuberculosis pandemic continues to rampage despite widespread use of the current Bacillus Calmette-Guerin (BCG) vaccine. Because DNA vaccines can elicit effective antigen-specific immune responses, including potent T cell-mediated immunity, they are promising vehicles for antigen delivery. In a prime-boost approach, they can supplement the inadequate anti-TB immunological memory induced by BCG. Based on this, a chimeric DNA vaccine HG856A encoding Mycobacterium tuberculosis (M. tuberculosis) immunodominant antigen Ag85A plus two copies of ESAT-6 was constructed. Potent humoral immune responses, as well as therapeutic effects induced by this DNA vaccine, were observed previously in M. tuberculosis-infected mice. In this study, we further evaluated the antigen-specific T cell immune responses and showed that repeated immunization with HG856A gave modest protection against M. tuberculosis challenge infection and significantly boosted the immune protection primed by BCG vaccination. Enhanced protection was accompanied by increased multifunctional Th1 CD4(+) T cell responses, most notably by an elevated frequency of M. tuberculosis antigen-specific IL-2-producing CD4(+) T cells post-vaccination. These data confirm the potential of chimeric DNA vaccine HG856A as an anti-TB vaccine candidate. PMID:26111521

  10. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    SciTech Connect

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  11. Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis.

    PubMed Central

    Linton, C J; Jalal, H; Leeming, J P; Millar, M R

    1994-01-01

    Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis. Images PMID:7814542

  12. Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens.

    PubMed

    Maity, Hemanta Kumar; Dey, Sohini; Mohan, C Madhan; Khulape, Sagar A; Pathak, Dinesh C; Vakharia, Vikram N

    2015-02-18

    Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a 'DNA prime-protein boost' approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and 'DNA prime-protein boost' elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and 'DNA prime-protein boost' groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p<0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to 'DNA prime-protein boost' group. Additionally, chickens immunized with pCIVP2-cHSP70 and 'DNA prime-protein boost' vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens. PMID

  13. Protective Immunity to H7N9 Influenza viruses elicited by synthetic DNA Vaccine

    PubMed Central

    Yan, Jian; Villarreal, Daniel O.; Racine, Trina; Chu, Jaemi S.; Walters, Jewell N.; Morrow, Matthew P.; Khan, Amir S.; Sardesai, Niranjan Y.; Kim, J. Joseph; Kobinger, Gary P.; Weiner, David B.

    2014-01-01

    Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses’ ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8 T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases. PMID:24631084

  14. Protective immunity to H7N9 influenza viruses elicited by synthetic DNA vaccine.

    PubMed

    Yan, Jian; Villarreal, Daniel O; Racine, Trina; Chu, Jaemi S; Walters, Jewell N; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Kim, J Joseph; Kobinger, Gary P; Weiner, David B

    2014-05-19

    Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses' ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases. PMID:24631084

  15. Mycobacterium bovis DNA detection in colostrum as a potential indicator of vaccination effectiveness against bovine tuberculosis.

    PubMed

    Herrera-Rodríguez, Sara E; Gordiano-Hidalgo, María Alejandra; López-Rincón, Gonzálo; Bojorquez-Narváez, Luis; Padilla-Ramírez, Francisco Javier; Pereira-Suárez, Ana Laura; Flores-Valdez, Mario Alberto; Estrada-Chávez, Ciro

    2013-04-01

    Bovine tuberculosis (bTB) remains a problem on many dairy farms in Mexico, as well as a public health risk. We previously found a high frequency of Mycobacterium bovis DNA in colostrum from dairy cows using a nested PCR to detect mpb70. Since there are no reliable in vivo tests to determine the effectiveness of booster Mycobacterium bovis BCG vaccination against bTB, in this work we monitored M. bovis DNA in colostrum by using this nested PCR. In order to decrease the risk of adverse reactions in animals likely containing viable M. bovis, a single application of BCG and a subunit vaccine (EEP-1) formulated with M. bovis culture filtrate proteins (CFP) and a copolymer as the adjuvant was performed in tuberculin skin test-negative cattle (TST(-)), while TST reactor animals (TST(+)) received EEP-1 only. Booster immunization using EEP-1 was applied to both groups, 2 months after primary vaccination to whole herds and 12 months later to lactating cows. Colostrum samples were collected from 6 farms where the cows were vaccinated over a 12-month period postvaccination and, for comparison, from one control farm where the cows were not vaccinated with comparable bTB prevalence. We observed an inverse relationship between the frequency of M. bovis DNA detection and time postvaccination at the first (P < 0.001) and second (P < 0.0001) 6-month periods. Additionally, the concentration of gamma interferon (IFN-γ) was higher in mpb70 PCR-positive colostrum samples (P = 0.0003). These results suggest that M. bovis DNA frequency in colostrum could be a potentially useful biomarker for bTB vaccine efficacy on commercial dairy farms. PMID:23425597

  16. A combined DNA vaccine provides protective immunity against Mycobacterium bovis and Brucella abortus in cattle.

    PubMed

    Hu, Xi-Dan; Yu, Da-Hai; Chen, Su-Ting; Li, Shu-Xia; Cai, Hong

    2009-04-01

    We evaluated the immunogenicity and protective efficacy of a combined DNA vaccine containing six genes encoding immunodominant antigens from Mycobacterium bovis and Brucella abortus. The number of lymph node and spleen cultures positive for M. bovis and B. abortus from calves immunized with the combined DNA vaccine was significantly reduced (p < 0.01) compared with unvaccinated calves after challenge with virulent M. bovis and B. abortus 544. The combined DNA vaccine group displayed stronger antigen-specific interferon-gamma (IFN-gamma) responses and antigen-specific IFN-gamma ELISPOT activities 2 months after final immunization and after challenge. Antigen-specific CD4(+) and CD8(+) T cell responses in the combined DNA vaccine group were higher than either the Bacillus Calmette-Guerin (BCG)-positive or S19-positive control group. Likewise, more calves in the DNA vaccine group exhibited antigen-specific IgG titers and had higher IgG titers than those in the BCG- or S19-immunized groups 2 months after the final immunization. Moreover, two antigens in the combined DNA vaccine induced significant antigen-specific IFN-gamma responses 6 months after challenge (p < 0.05). Bacterial counts and pathological analyses of the challenged animals indicated that the combined DNA vaccine provided significantly better protection than the BCG vaccine against M. bovis, and the protection level induced by the combined DNA vaccine was comparable to S19 against B. abortus. This is the first report to demonstrate that a single combined DNA vaccine protects cattle against two infectious diseases. PMID:19364278

  17. Aerosol Vaccination with AERAS-402 Elicits Robust Cellular Immune Responses in the Lungs of Rhesus Macaques but Fails to Protect Against High-Dose Mycobacterium tuberculosis Challenge

    PubMed Central

    Darrah, Patricia A.; Bolton, Diane L.; Lackner, Andrew A.; Kaushal, Deepak; Aye, Pyone Pyone; Mehra, Smriti; Blanchard, James L.; Didier, Peter J.; Roy, Chad J.; Rao, Srinivas S.; Hokey, David A.; Scanga, Charles A.; Sizemore, Donata R.; Sadoff, Jerald C.; Roederer, Mario; Seder, Robert A.

    2014-01-01

    Development of a vaccine against pulmonary tuberculosis (TB) may require immunization strategies that induce a high frequency of antigen-specific CD4 and CD8 T cells in the lung. The nonhuman primate (NHP) model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. Here, we used an aerosol (AE) vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis (M.tb) antigens Ag85A, Ag85B, and TB10.4, in bacille Calmette-Guerin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative (PPD)-specific CD4 T cell responses in blood and bronchoalveolar lavage (BAL). In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in BAL that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFU of M.tb Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M.tb challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of AE rAd immunization to generate potent cellular immunity in the lung by AE rAd immunization suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M.tb. PMID:25024382

  18. Aerosol vaccination with AERAS-402 elicits robust cellular immune responses in the lungs of rhesus macaques but fails to protect against high-dose Mycobacterium tuberculosis challenge.

    PubMed

    Darrah, Patricia A; Bolton, Diane L; Lackner, Andrew A; Kaushal, Deepak; Aye, Pyone Pyone; Mehra, Smriti; Blanchard, James L; Didier, Peter J; Roy, Chad J; Rao, Srinivas S; Hokey, David A; Scanga, Charles A; Sizemore, Donata R; Sadoff, Jerald C; Roederer, Mario; Seder, Robert A

    2014-08-15

    Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette-Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative-specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis. PMID:25024382

  19. Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis.

    PubMed

    Ahmed, Wareed; Menon, Shruti; Karthik, Pullela V; Nagaraja, Valakunja

    2015-02-01

    The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (TopoI) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. TopoI is essential for Mtb survival. However, the necessity of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the TopoI-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects. PMID:25516959

  20. Analysis of a genomic DNA expression library of Mycobacterium tuberculosis using tuberculosis patient sera: evidence for modulation of host immune response.

    PubMed Central

    Amara, R R; Satchidanandam, V

    1996-01-01

    DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments. The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins. Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. Interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, other whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients. This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression. PMID:8751927

  1. Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

    PubMed

    He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

    2016-01-01

    The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors. PMID:27079268

  2. Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis

    PubMed Central

    He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

    2016-01-01

    The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors. PMID:27079268

  3. [Detection of Mycobacterium ulcerans DNA in water bugs collected outside the aquatic environment in Benin].

    PubMed

    Marion, E; Deshayes, C; Chauty, A; Cassisa, V; Tchibozo, S; Cottin, J; Doannio, J; Marot, A; Marsollier, L

    2011-04-01

    Hosting of Mycobacterium ulcerans by water bugs is now well established and their vectoring role has been demonstrated experimentally. These findings were recently corroborated by detection of viable bacilli in the saliva of wild water bugs. However, the extent of water bug involvement in M. ulcerans ecology remains unclear and difficult to evaluate due to lack of understanding about water bug biology. The purpose of this study is to describe the first detection of M. ulcerans DNA in the tissue of water bugs captured outside the aquatic environment. This finding supports the hypothesis that water bug migratory behavior contributes not only to the spread of M. ulcerans but also to transmission outside the aquatic environment. PMID:21695876

  4. Application of water-soluble nanofilters for collection of airborne Mycobacterium tuberculosis DNA in hospital wards.

    PubMed

    Vladimirsky, M A; Shipina, L K; Makeeva, E S; Alyapkina, Y S; Mikheev, A Y; Morozov, V N

    2016-05-01

    A simple inexpensive technique for collection of airborne biomarkers of nosocomial infections is described. Biomarkers were collected on water-soluble electrospun nanofilters attached to a household vacuum cleaner from 6-10m(3) of air in 10-15min within several wards of a tuberculosis clinic. Filters were then dissolved in water and tested for the presence of the IS6110 and regX3 genes of Mycobacterium tuberculosis (MTB) using real-time polymerase chain reaction. It was demonstrated that trace amounts of airborne MTB DNA (>3gene copies/m(3)) were always present in the air and on the surfaces in the wards occupied with tuberculosis patients having positive smear tests. PMID:27021397

  5. Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B

    PubMed Central

    Jin, Ke; Wang, Shixia; Zhang, Chunhua; Xiao, Yanling; Lu, Shan; Huang, Zuhu

    2013-01-01

    A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice. PMID:23143772

  6. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  7. The role of Ca²⁺ in the activity of Mycobacterium tuberculosis DNA gyrase.

    PubMed

    Karkare, Shantanu; Yousafzai, Faridoon; Mitchenall, Lesley A; Maxwell, Anthony

    2012-10-01

    DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding. PMID:22844097

  8. Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins*

    PubMed Central

    Khanduja, Jasbeer Singh; Muniyappa, K.

    2012-01-01

    Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins. PMID:22094465

  9. Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity.

    PubMed

    Liu, Pei; Wang, Zhong Quan; Liu, Ruo Dan; Jiang, Peng; Long, Shao Rong; Liu, Li Na; Zhang, Xin Zhuo; Cheng, Xiang Chao; Yu, Chuan; Ren, Hui Jun; Cui, Jing

    2015-06-01

    We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice. PMID:25733024

  10. Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk.

    PubMed

    Odumeru, J; Gao, A; Chen, S; Raymond, M; Mutharia, L

    2001-10-01

    Mycobacterium paratuberculosis is a recognized chronic enteric pathogen that can affect many different species of animals, including primates. It has been suggested that this organism is associated with Crohn's disease in humans, and that milk is a potential source of human exposure to this organism. The limit of the detection of M. paratuberculosis in milk samples by direct PCR was 10(5) cfu/mL if the traditional boiling method was used for template DNA preparation. In this study, an improved method for template DNA preparation was examined. The method involves the use of a bead beater, which breaks up bacterial cell wall mechanically by vibrating bacteria with microbeads at high speed. The effectiveness of this method for lysing M. paratuberculosis cells was compared to that of the freeze-thaw method, and use of commercial kits such as the InstaGene Matrix and the QIAamp Tissue Kit. The bead beater procedure was tested in combination with various cell lysis and template DNA preparation procedures to determine which of these steps improved the limit of detection of PCR assay that amplifies a 413 bp fragment of the IS900 gene. Results showed that the use of the bead beater, in combination with the use of lysis buffer, boiling, and isopropanol precipitation, decreased the limit of detection of M. paratuberculosis in milk by the PCR to 10(2) cfu/mL. The limit of detection was further decreased to 10 cfu/mL when 0.0037% bovine serum albumin was included in the PCR reaction mixtures. The improved assay was 10- to 10(4)-fold more sensitive than the PCR assays using template DNA prepared by other lysis procedures including boiling alone, freeze-thaw plus boiling, or use of commercial kits for lysis. PMID:11768125

  11. MPT-51/CpG DNA vaccine protects mice against Mycobacterium tuberculosis.

    PubMed

    Silva, Bruna Daniella de Souza; da Silva, Ediane Batista; do Nascimento, Ivan Pereira; Dos Reis, Michelle Cristina Guerreiro; Kipnis, André; Junqueira-Kipnis, Ana Paula

    2009-07-16

    Tuberculosis (TB) is a severe infectious disease that kills approximately two million people worldwide every year. Because BCG protection is variable and does not protects adults, there is a great need for a new vaccine against TB that does not represent a risk for immunocompromised patients and that is also capable of protecting adult individuals. MPT-51 is a protein found in the genome of mycobacteria and binds to the fibronectin of the extracellular matrix, which may have a role in host tissue attachment and virulence. In order to test the usefulness of MPT-51 as a subunit vaccine, BALB/c were vaccinated and challenged with Mycobacterium tuberculosis. The infection of BALB/c with M. tuberculosis increased the number of IFN-gamma(+) T lymphocytes specific to MPT-51 in the spleen and lungs. Inoculation with rMPT-51/FIA and with rMPT-51/CpG DNA in non-infected BALB/c increased the amounts of IFN-gamma(+) T lymphocytes. Inoculation with rMPT-51/FIA also induced a humoral response specific to MPT-51. CFU counts of lung tissues done 60 days after infection showed a reduction of about 2 log in the bacteria load in the group of animals inoculated with rMPT-51/CpG DNA. These results make MPT-51 a valuable component to be further evaluated in the development of other subunit vaccines. PMID:19500525

  12. Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs.

    PubMed

    Grubor-Bauk, B; Yu, W; Wijesundara, D; Gummow, J; Garrod, T; Brennan, A J; Voskoboinik, I; Gowans, E J

    2016-01-01

    Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter. Compared with a canonical DNA vaccine and a bicistronic DNA vaccine encoding NS3 and the proapoptotic gene NSP4, the perforin-containing vaccine elicited enhanced cell-mediated immune responses against the NS3 protein in vaccinated mice and pigs, as determined by ELISpot and intracellular cytokine staining, whereas a mouse challenge model suggested that the immunity was CD8(+) T-cell-dependent. The results of the study showed that the inclusion of perforin in the DNA vaccine altered the fate of NS3-positive cells from apoptosis to necrosis, and this resulted in more robust immune responses in mice and pigs, the latter of which represents an accepted large animal model in which to test vaccine efficacy. PMID:26262584

  13. Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex.

    PubMed

    Ghodousi, Arash; Arash, Ghodousi A; Vatani, S; Darban-Sarokhalil, Davood; Omrani, Maryam; Fooladi, A; Fooladi, Aa; Khosaravi, A; Khosaravi, Ad; Feizabadi, Mohammad Mehdi

    2012-03-01

    A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Mycobacterium tuberculosis complex to reduce the cost of using lyticase. This protocol reduces the expense of PFGE typing of Mycobacterium tuberculosis complex as it removes the use of lyticase during the spheroplast formation from these bacteria. PMID:22783461

  14. Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex

    PubMed Central

    Arash, Ghodousi A; Vatani, S; Darban-Sarokhalil, Davood; Omrani, Maryam; Fooladi, AA; Khosaravi, AD; Feizabadi, Mohammad Mehdi

    2012-01-01

    A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Mycobacterium tuberculosis complex to reduce the cost of using lyticase. This protocol reduces the expense of PFGE typing of Mycobacterium tuberculosis complex as it removes the use of lyticase during the spheroplast formation from these bacteria. PMID:22783461

  15. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

    PubMed

    Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

    2015-09-01

    Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

  16. Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

    PubMed

    Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

    2011-02-01

    Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens. PMID:21159933

  17. Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains in Japan Using Whole Genome Amplification and Polymerase Chain Reaction

    PubMed Central

    Suzuki, Koichi; Takigawa, Wataru; Tanigawa, Kazunari; Nakamura, Kazuaki; Ishido, Yuko; Kawashima, Akira; Wu, Huhehasi; Akama, Takeshi; Sue, Mariko; Yoshihara, Aya; Mori, Shuichi; Ishii, Norihisa

    2010-01-01

    Background Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia. Methodology/Principal Findings Here we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials. Conclusion/Significance We have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification. PMID:20865042

  18. Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant.

    PubMed

    Zhou, Huaiyu; Min, Juan; Zhao, Qunli; Gu, Qinmin; Cong, Hua; Li, Ying; He, Shenyi

    2012-02-27

    Previous immunological studies from our laboratory have demonstrated the potential role of Toxoplasma gondii antigens SAG1 and GRA2 as vaccine candidates. To further evaluate the vaccine's effects, a series of recombinant DNA vaccines pVAX1-SAG1, pVAX1-GRA2 and pVAX1-SAG1-GRA2, termed pSAG1, pGRA2 and pSAG1-GRA2, respectively, were constructed. A plasmid pVAX1-S/PreS2, termed pSPreS2 encoding hepatitis B virus (HBV) surface antigen (HBsAg) S and PreS2 as a novel genetic adjuvant, was also constructed. The expression abilities of those DNA plasmids were examined in HFF cells by Western blotting. Then BALB/c mice were intramuscularly immunized with DNA plasmids and followed by challenging with the highly virulent T. gondii RH strain. The results demonstrated that the recombinant DNA vaccine pSAG1-GRA2 was capable of eliciting high levels of antibodies, a Th1 type of immune response with significant production of IFN-γ and low levels of IL-4 or IL-10 in BALB/c mice, and partial protection against the acute phase of toxoplasmosis as compared to pSAG1, pGRA2 and controls. In addition, the adjuvant pSPreS2 formulated with DNA vaccine induced a Th1 type of immune response and therefore might be a novel genetic adjuvant to DNA vaccine for further investigation. PMID:22240340

  19. DNA vaccine constructs against enterovirus 71 elicit immune response in mice

    PubMed Central

    2007-01-01

    Background Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. Methods The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. Results The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. Conclusion The promising results obtained in the present study have prompted further testing

  20. Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

    PubMed Central

    Chaves, F; Yang, Z; el Hajj, H; Alonso, M; Burman, W J; Eisenach, K D; Dronda, F; Bates, J H; Cave, M D

    1996-01-01

    A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns. PMID:8727887

  1. Ocular delivery of compacted DNA-nanoparticles does not elicit toxicity in the mouse retina.

    PubMed

    Ding, Xi-Qin; Quiambao, Alexander B; Fitzgerald, J Browning; Cooper, Mark J; Conley, Shannon M; Naash, Muna I

    2009-01-01

    Subretinal delivery of polyethylene glycol-substituted lysine peptide (CK30PEG)-compacted DNA nanoparticles results in efficient gene expression in retinal cells. This work evaluates the ocular safety of compacted DNA nanoparticles. CK30PEG-compacted nanoparticles containing an EGFP expression plasmid were subretinally injected in adult mice (1 microl at 0.3, 1.0 and 3.0 microg/microl). Retinas were examined for signs of inflammation at 1, 2, 4 and 7 days post-injection. Neither infiltration of polymorphonuclear neutrophils or lymphocytes was detected in retinas. In addition, elevation of macrophage marker F4/80 or myeloid marker myeloperoxidase was not detected in the injected eyes. The chemokine KC mRNA increased 3-4 fold in eyes injected with either nanoparticles or saline at 1 day post-injection, but returned to control levels at 2 days post-injection. No elevation of KC protein was observed in these mice. The monocyte chemotactic protein-1, increased 3-4 fold at 1 day post-injection for both nanoparticle and saline injected eyes, but also returned to control levels at 2 days. No elevations of tumor necrosis factor alpha mRNA or protein were detected. These investigations show no signs of local inflammatory responses associated with subretinal injection of compacted DNA nanoparticles, indicating that the retina may be a suitable target for clinical nanoparticle-based interventions. PMID:19823583

  2. Induction of in vivo resistance to Mycobacterium avium infection by intramuscular injection with DNA encoding interleukin-18

    PubMed Central

    Kim, S H; Cho, D; Kim, T S

    2001-01-01

    Interferon-gamma (IFN-γ) is closely associated with the generation of cell-mediated immunity and resistance to intracellular parasites. Interleukin-18 (IL-18) was known to strongly induce IFN-γ production by T cells and natural killer (NK) cells. In order to determine whether injection with DNA encoding IL-18 can stimulate the resistance to Mycobacterium avium complex (MAC) infection, the mature IL-18 cDNA with κ leader sequence was cloned under control of the cytomegalovirus (CMV) promoter (TcCMVIL-18) and its effect on MAC infection was investigated in genetically susceptible BALB/c mice. Injection with the TcCMVIL-18 DNA during intranasal infection with MAC resulted in a significant decrease in bacterial load of lung during the entire 8-week observation period, while injection with the TcCMV control DNA did not. Lung cells in mice injected with the TcCMVIL-18 DNA showed persistent production of IFN-γ throughout the 8-week period. Furthermore, immunization with the TcCMVIL-18 DNA induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by lung cells than immunization with the TcCMV control vector. This work suggests that IL-18 DNA vaccination may be useful in the immunotherapeutic or immunoprotection approaches of infections by intracellular parasites such as mycobacteria. PMID:11260329

  3. Design and synthesis of novel quinoline-aminopiperidine hybrid analogues as Mycobacterium tuberculosis DNA gyraseB inhibitors.

    PubMed

    Medapi, Brahmam; Renuka, Janupally; Saxena, Shalini; Sridevi, Jonnalagadda Padma; Medishetti, Raghavender; Kulkarni, Pushkar; Yogeeswari, Perumal; Sriram, Dharmarajan

    2015-05-01

    Antibiotics with good therapeutic value and novel mechanism of action are becoming increasingly important in today's battle against bacterial resistance. One of the popular targets being DNA gyrase, is currently becoming well-established and clinically validated for the development of novel antibacterials. In the present work, a series of forty eight quinoline-aminopiperidine based urea and thiourea derivatives were synthesized as pharmacophoric hybrids and evaluated for their biological activity. Compound, 1-(4-chlorophenyl)-3-(1-(6-methoxy-2-methylquinolin-4-yl)piperidin-4-yl)thiourea (45) was found to exhibit promising in vitro Mycobacterium smegmatis GyrB IC₅₀ of 0.95 ± 0.12 μM and a well correlated Mycobacterium tuberculosis (MTB) DNA gyrase supercoiling IC₅₀ of 0.62 ± 0.16 μM. Further, compound 45 also exhibited commendable MTB MIC, safe eukaryotic cytotoxic profile with no signs of cardiotoxicity in zebrafish ether-a-go-go-related gene (zERG). PMID:25801151

  4. Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction*

    PubMed Central

    Sharadamma, Narayanaswamy; Harshavardhana, Yadumurthy; Ravishankar, Apoorva; Anand, Praveen; Chandra, Nagasuma; Muniyappa, K.

    2014-01-01

    The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins. PMID:25324543

  5. In Silico Screening for Novel Inhibitors of DNA Polymerase III Alpha Subunit of Mycobacterium tuberculosis (MtbDnaE2, H37Rv)

    PubMed Central

    Jadaun, Alka; Sudhakar D, Raja; Subbarao, N.; Dixit, Aparna

    2015-01-01

    Tuberculosis, a pandemic disease is caused by Mycobacterium tuberculosis (Mtb). DNA polymerase III encoded by DnaE2 of Mtb is specifically required for its survival in vivo, and hence can be considered to be a potential drug target. Amino acid sequence analysis of the MtbDnaE2 and its human counterpart does not show any significant similarity. Therefore, a 3D model of the MtbDnaE2 was generated using Modeller 9v10 with the template structure of E. Coli DNA polymerase III alpha subunit (2HNH_A). The generated models were validated using a number of programmes such as RAMPAGE/PROCHECK, VERIFY_3D, and ProSA. MtbDnaE2 has few conserved residues and four conserved domains similar to that present in DNA polymerase III of E. coli. In silico screening was performed with bioactive anti-tuberculosis compounds and 6-AU (a known inhibitor of DNA polymerase III of Bacillus subtilis) and its analogues against the modeled MtbDnaE2 structure. Docking was performed using GOLD v5.2 software which resulted in the identification of top ten compounds with high GOLD fitness scores and binding affinity (X-Score). To further evaluate the efficacy of these compounds, in silico ADMET analysis was performed using MedChem Designer v3. Given their high binding affinity to the targeted MtbDnaE2, which is essential for DNA replication in the Mtb and good ADMET properties, these compounds are promising candidates for further evaluation and development as anti-tubercular agents. PMID:25811866

  6. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fecal culture is considered the gold standard for the diagnostics of paratuberculosis, however, PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal material is widely used today, having demonstrated great sensitivity and specificity. To insure the most efficient and r...

  7. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fecal culture is considered the gold standard for the diagnosis of paratuberculosis, however, PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal material is widely used today, having demonstrated great sensitivity and specificity. To insure the most efficient and rep...

  8. Protective Immunity Elicited by a Divalent DNA Vaccine Encoding Both the L7/L12 and Omp16 Genes of Brucella abortus in BALB/c Mice

    PubMed Central

    Luo, Deyan; Ni, Bing; Li, Peng; Shi, Wei; Zhang, Songle; Han, Yue; Mao, Liwei; He, Yangdong; Wu, Yuzhang; Wang, Xiliang

    2006-01-01

    This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can. PMID:16622210

  9. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

    PubMed

    Aldous, Wade K; Pounder, June I; Cloud, Joann L; Woods, Gail L

    2005-05-01

    Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction. PMID:15872286

  10. Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif

    PubMed Central

    Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-Pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-01-01

    Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations. PMID:15650171

  11. Label-free DNA-based detection of Mycobacterium tuberculosis and rifampicin resistance through hydration induced stress in microcantilevers.

    PubMed

    Domínguez, Carmen M; Kosaka, Priscila M; Sotillo, Alma; Mingorance, Jesús; Tamayo, Javier; Calleja, Montserrat

    2015-02-01

    We have developed a label-free assay for the genomic detection of Mycobacterium tuberculosis and rifampicin resistance. The method relies on the quantification of the hydration induced stress on microcantilever biosensors functionalized with oligonucleotide probes, before and after hybridization with specific targets. We have found a limit of detection of 10 fg/mL for PCR amplified products of 122 bp. Furthermore, the technique can successfully target genomic DNA (gDNA) fragments of length >500 bp, and it can successfully discriminate single mismatches. We have used both loci IS6110 and rpoB as targets to detect the mycobacteria and the rifampicin resistance from gDNA directly extracted from bacterial culture and without PCR amplification. We have been able to detect 2 pg/mL target concentration in samples with an excess of interfering DNA and in a total analysis time of 1 h and 30 min. The detection limit found demonstrates the capability to develop direct assays without the need for long culture steps or PCR amplification. The methodology can be easily translated to different microbial targets, and it is suitable for further development of miniaturized devices and multiplexed detection. PMID:25599922

  12. DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China

    PubMed Central

    Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2012-01-01

    Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

  13. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    PubMed

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. PMID:26103519

  14. Effects of pretreatment with pyrazole and inducers of mixed function oxidases on DNA repair elicited by dimethylnitrosamine in rat hepatocytes in vivo and in vitro.

    PubMed

    Kornbrust, D; Dietz, D

    1985-12-17

    Experiments were performed to investigate the relationship between the rate of oxidative metabolism of dimethylnitrosamine (DMN) by rat liver microsomes (i.e., DMN demethylase activity, DMNd) and its genotoxicity in liver, as assessed by the in vitro and in vivo/in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assays. Pretreatment of rats with pyrazole (PYR) resulted in a 4-fold increase in DMNd and a 3-fold greater DNA repair response to in vivo administration of 5 mg DMN/kg body weight. Pretreatment with phenobarbital (PB), dichlorodiphenyltrichloroethane (DDT), 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF) or Aroclor 1254 (ARO) produced a variable degree of inhibition of DMNd and had no significant effects on the response to DMN in the in vivo/in vitro HPC/DR assay. DNA repair elicited by DMN in vitro was decreased in hepatocytes from rats pretreated with 3-MC, while PB, DDT, beta-NF and ARO pretreatments had little effect on the response. In contrast, PYR pretreatment produced a 4.5-6.7-fold increase in the in vitro DNA repair response to DMN, and extended detection of positive responses to lower concentrations. Most of the inducers had no effect on DNA repair elicited by the direct acting alkylator, methyl methanesulfonate (MMS). Thus, the pretreatment-related changes in DMN-induced DNA repair were probably due to alterations in DMNd rather than to effects on the DNA repair capacity of the hepatocytes. PMID:4075445

  15. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    SciTech Connect

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M. . E-mail: tmr15@pitt.edu

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, both sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

  16. A novel dendritic cell-targeted lentiviral vector, encoding Ag85A-ESAT6 fusion gene of Mycobacterium tuberculosis, could elicit potent cell-mediated immune responses in mice.

    PubMed

    Shakouri, Mehdi; Moazzeni, Seyed Mohammad; Ghanei, Mostafa; Arashkia, Arash; Etemadzadeh, Mohammad Hossein; Azadmanesh, Kayhan

    2016-07-01

    Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to high mortality worldwide. It is well-established that cellular immunity plays a critical role to control Mtb infection. Dendritic Cells (DCs) are potent antigen presenting cells, which play an important role to prime cell-mediated immune responses. In vivo targeting of DCs has been shown to induce both strong cellular immunity and protection against tumor challenges. The aim of the present study was not only to assess the immunizing potential of a novel DC-targeted recombinant lentivirus expressing fusion antigen Ag85A-ESAT6 of Mtb, but also to compare it with a recombinant lentivirus with broad cellular tropism expressing the same antigen in mice. The findings demonstrated that our novel recombinant DC-targeted lentivector was able to successfully transduce and express the fusion antigen Ag85A-E6 in vitro and in vivo. Moreover, a single footpad injection of targeted lentivectors could elicit strong T-helper 1 (Th1) immunity against the above mentioned antigen, as indicated by the specific high-level production of IFN-γ and IL-2 using spleen lymphocytes and lymphoproliferative responses. Despite of these promising results, more attempts are required to elucidate the protective and therapeutic efficacy of this approach in future. PMID:27267270

  17. Mycobacterium avium complex in day care hot water systems, and persistence of live cells and DNA in hot water pipes.

    PubMed

    Bukh, Annette S; Roslev, Peter

    2014-04-01

    The Mycobacterium avium complex (MAC) is a group of opportunistic human pathogens that may thrive in engineered water systems. MAC has been shown to occur in drinking water supplies based on surface water, but less is known about the occurrence and persistence of live cells and DNA in public hot water systems based on groundwater. In this study, we examined the occurrence of MAC in hot water systems of public day care centers and determined the persistence of live and dead M. avium cells and naked DNA in model systems with the modern plumbing material cross-linked polyethylene (PEX). The occurrence of MAC and co-occurrence of Legionella spp. and Legionella pneumophila were determined using cultivation and qPCR. Co-occurrences of MAC and Legionella were detected in water and/or biofilms in all hot water systems at temperatures between 40 and 54 °C. Moderate correlations were observed between abundance of culturable MAC and that of MAC genome copies, and between MAC and total eubacterial genome copies. No quantitative relationship was observed between occurrence of Legionella and that of MAC. Persistence in hot water of live and dead M. avium cells and naked DNA was studied using PEX laboratory model systems at 44 °C. Naked DNA and DNA in dead M. avium cells persisted for weeks. Live M. avium increased tenfold in water and biofilms on PEX. The results suggest that water and biofilms in groundwater-based hot water systems can constitute reservoirs of MAC, and that amplifiable naked DNA is relatively short-lived, whereas PEX plumbing material supports persistence and proliferation of M. avium. PMID:24272032

  18. Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients

    PubMed Central

    Vemula, Mani H.; Ganji, Rakesh; Sivangala, Ramya; Jakkala, Kiran; Gaddam, Sumanlatha; Penmetsa, Sitaramaraju; Banerjee, Sharmistha

    2016-01-01

    Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4+ T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can

  19. Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients.

    PubMed

    Vemula, Mani H; Ganji, Rakesh; Sivangala, Ramya; Jakkala, Kiran; Gaddam, Sumanlatha; Penmetsa, Sitaramaraju; Banerjee, Sharmistha

    2016-01-01

    Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins

  20. E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM).

    PubMed

    Miodek, Anna; Mejri, Nawel; Gomgnimbou, Michel; Sola, Christophe; Korri-Youssoufi, Hafsa

    2015-09-15

    Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose. PMID:26313137

  1. Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears.

    PubMed

    Kamble, R R; Shinde, V S; Madhale, S P; Kamble, A A; Ravikumar, B P; Jadhav, R S

    2010-01-01

    Abstract Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases. PMID:20061767

  2. The seal tuberculosis agent, Mycobacterium pinnipedii, infects domestic cattle in New Zealand: epidemiologic factors and DNA strain typing.

    PubMed

    Loeffler, Scott H; de Lisle, Geoffrey W; Neill, Mark A; Collins, Desmond M; Price-Carter, Marian; Paterson, Brent; Crews, Kevin B

    2014-04-01

    The fur seal (Arctocephalus forsteri), which is abundant in coastal areas of New Zealand, harbors several zoonotic pathogens, including Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex. We describe the microbiology and epidemiology of seven cases of M. pinnipedii infection in beef cattle (Bos primigenius) in coastal areas of New Zealand in 1991-2011. Epidemiologic factors were analyzed on six case farms and a telephone survey of 55 neighboring farms. A DNA-strain typing, using analysis of variable number tandem repeats and the direct repeats (VNTR/DR) of those isolates, was used to compare them to M. bovis isolates commonly found in New Zealand cattle and wildlife. In all cases of M. pinnipedii in cattle, only one animal in the herd was found to be infected. In six of seven cases, the lesions were in the thoracic lymph nodes, indicating a likely aerosol pathway. The lack of multiple cases within a herd suggests that cow-to-cow transmission is uncommon, if it occurs at all. There was no significant difference between case and control farms in distance to sea, herd size, herd type, or farming practice. The odds ratio for access to the beach for cattle on the Chatham Islands was significantly higher than it was for farms on the mainland coastal areas (odds ratio [OR] = 3.6, 95% CI = 1.1-11.4) Likewise, the odds ratio for acquiring tuberculosis was increased when farmers had seen seals on the property (OR =  9, 95% CI = 1.4-56.1 ). In all case farms, cattle had access to seals by beach grazing areas or waterways connecting directly with the ocean. The VNTR/DR typing of the isolates showed some variation in the M. pinnipedii isolates, with only two being identical; all isolates were easily distinguishable from M. bovis isolates. PMID:24484478

  3. Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist.

    PubMed Central

    Movahedzadeh, F; Colston, M J; Davis, E O

    1997-01-01

    The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence. PMID:9171394

  4. Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: preliminary study

    PubMed Central

    2013-01-01

    Background Developments in molecular detection and strain differentiation of members of Mycobacterium tuberculosis complex have proved to be useful. The DNA extraction method influences the amplification efficiency, causing interference on the sensitivity and respective inhibitors. The aim of this study was to standardize a simple and fast DNA extraction method, providing DNA amplification by IS6110-PCR effectively free from undue interferences. Findings The efficiency of the six different protocols tested in M. tuberculosis cultures has varied from 75% to 92.5%. This preliminary study evaluating the IS6110 PCR sensitivity and specificity was developed in DNA extracted from microscope slides, and achieved 100% of efficiency. Conclusions DNA extraction by Chelex + NP-40 method from both, cultures of M. tuberculosis and smear slides, resulted in good quantity of interference free DNA, especially in samples with low concentrations of genetic material; therefore, such technique may be used for the molecular diagnosis of tuberculosis. PMID:24373461

  5. Immunogenicity of Eight Dormancy Regulon-Encoded Proteins of Mycobacterium tuberculosis in DNA-Vaccinated and Tuberculosis-Infected Mice▿

    PubMed Central

    Roupie, Virginie; Romano, Marta; Zhang, Lei; Korf, Hannelie; Lin, May Young; Franken, Kees L. M. C.; Ottenhoff, Tom H. M.; Klein, Michèl R.; Huygen, Kris

    2007-01-01

    Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F1 mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB. PMID:17145953

  6. A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.

    PubMed

    Biswas, Tapan; Resto-Roldán, Esteban; Sawyer, Sean K; Artsimovitch, Irina; Tsodikov, Oleg V

    2013-02-01

    Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase-pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z' = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery. PMID:23267008

  7. A novel non-radioactive primase–pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

    PubMed Central

    Biswas, Tapan; Resto-Roldán, Esteban; Sawyer, Sean K.; Artsimovitch, Irina; Tsodikov, Oleg V.

    2013-01-01

    Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase–pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z′ = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery. PMID:23267008

  8. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling.

    PubMed

    Sinha, Krishna Murari; Glickman, Michael S; Shuman, Stewart

    2009-05-19

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3' to 5' DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound Escherichia coli UvrD-ADP-MgF(3) transition state mimetic. Alanine scanning and conservative substitutions identified amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphohydrolase chemistry via transition state stabilization (Arg308, Arg648, Gln275), divalent cation coordination (Glu236), or activation of the nucleophilic water (Glu236, Gln275). Other residues important for ATPase/helicase activity include Phe280 and Phe72, which interact with the DNA 3' single strand tail. ATP hydrolysis was uncoupled from duplex unwinding by mutations at Glu609 (in helicase motif V), which contacts the ATP ribose sugar. Introducing alanine in lieu of the adenine-binding "Q motif" glutamine (Gln24) relaxed the substrate specificity in NTP hydrolysis, e.g., eliciting a gain of function as a UTPase/TTPase, although the Q24A mutant still relied on ATP/dATP for duplex unwinding. Our studies highlight the role of the Q motif as a substrate filter and the contributions of adenosine-binding residues as couplers of NTP hydrolysis to motor activity. The Ku-binding function of UvrD1 lies within its C-terminal 270 amino acid segment. Here we found that deleting the 90 amino acid C-terminal domain, which is structurally uncharacterized, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate, apparently by affecting the strength of the UvrD1-Ku interaction. PMID:19317511

  9. Structures of Mycobacterium tuberculosis DosR and DosR-DNA Complex Involved in Gene Activation during Adaptation to Hypoxic Latency

    SciTech Connect

    Wisedchaisri, Goragot; Wu, Meiting; Rice, Adrian E; Roberts, David M; Sherman, David R; Hol, Wim G.J.

    2010-07-20

    On encountering low oxygen conditions, DosR activates the transcription of 47 genes, promoting long-term survival of Mycobacterium tuberculosis in a non-replicating state. Here, we report the crystal structures of the DosR C-terminal domain and its complex with a consensus DNA sequence of the hypoxia-induced gene promoter. The DosR C-terminal domain contains four {alpha}-helices and forms tetramers consisting of two dimers with non-intersecting dyads. In the DNA-bound structure, each DosR C-terminal domain in a dimer places its DNA-binding helix deep into the major groove, causing two bends in the DNA. DosR makes numerous protein-DNA base contacts using only three amino acid residues per subunit: Lys179, Lys182, and Asn183. The DosR tetramer is unique among response regulators with known structures.

  10. Designing and Construction of a DNA Vaccine Encoding Tb10.4 Gene of Mycobacterium tuberculosis

    PubMed Central

    Rashidian, Samira; Teimourpour, Roghayeh; Meshkat, Zahra

    2016-01-01

    Background: Tuberculosis (TB) remains as a major cause of death. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid and evaluation of its expression in eukaryotic cells. Methods: Firstly, tb10.4 fragment was amplified by PCR and the PCR product was digested with restriction enzymes. Next, it was cloned into pcDNA3.1+ plasmid. Following that, pcDNA3.1+/tb10.4 recombinant plasmid was transfected into eukaryotic cells. Results: 5700 bp band for pcDNA3.1+/tb10.4 recombinant plasmid and 297 bp fragment for tb10.4 were observed. Cloning and transfection were successful. Conclusion: Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis.

  11. Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65 (Hsp65) with human interleukin-2 against Mycobacterium tuberculosis in BALB/c mice.

    PubMed

    Wang, Li-Mei; Bai, Yin-Lan; Shi, Chang-Hong; Gao, Hui; Xue, Ying; Jiang, Hong; Xu, Zhi-Kai

    2008-12-01

    Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin-2 (hIL-2) in BALB/c mice. We showed that the DNA vaccine pcDNA-Hsp65-hIL-2 could induce high levels of antigen-specific antibody, IFN-gamma, CD4(+) and CD8(+) T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG. PMID:19133010

  12. Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

    PubMed

    Virginio, Veridiana Gomes; Gonchoroski, Taylor; Paes, Jéssica Andrade; Schuck, Desirée Cigaran; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2014-10-01

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP. PMID:25148775

  13. Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

    SciTech Connect

    Cherney, L.T.; Cherney, M.M.; Garen, C.R.; Lu, G.J.; James, M.N.G.

    2009-05-12

    The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

  14. Eliciting specific humoral immunity from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene.

    PubMed

    Mosley, Yung-Yi C; Hsieh, Ming Kun; Wu, Ching Ching; Lin, Tsang Long

    2015-01-01

    DNA vaccine coding for infectious bursal disease virus (IBDV) polyprotein gene and that for avian influenza virus (AIV) hemagglutinin (HA) gene have been shown to induce immunity and provide protection against the respective disease. The present study was carried out to determine whether an IBDV polyprotein gene-based DNA fused with AIV HA gene could trigger immune response to both IBDV and AIV. After transfection, VP2 and HA were detected in the cytoplasm and at cell membrane, respectively, by immunofluorescent antibody double staining method, suggesting the fusion strategy did not affect the location of protein expression. VP4 cleavage between VP2 and HA was confirmed by Western blot, indicating the fusion strategy did not affect VP4 function in transfected cells. After vaccination in chickens, the DNA construct VP24-HA/pcDNA induced ELISA and virus neutralizing antibodies against VP2 and hemagglutination inhibition antibody against the HA subtype. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination. PMID:25445883

  15. Combination of DNA prime--adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model.

    PubMed

    Kosinska, Anna D; Zhang, Ejuan; Johrden, Lena; Liu, Jia; Seiz, Pia L; Zhang, Xiaoyong; Ma, Zhiyong; Kemper, Thekla; Fiedler, Melanie; Glebe, Dieter; Wildner, Oliver; Dittmer, Ulf; Lu, Mengji; Roggendorf, Michael

    2013-01-01

    A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients. PMID:23785279

  16. Molecular Identification of Clinical Isolates of Mycobacterium fortuitum by Random Amplified Polymorphic DNA (RAPD) Polymerase Chain Reaction and ERIC PCR

    PubMed Central

    Khosravi, Azar Dokht; Farahani, Abbas; Jamali, Hooshang

    2015-01-01

    Backgrounds Non tuberculous mycobacteria (NTM) are of importance now-a-days due to their increasing virulence outbreaks and emerging antibiotic resistance. Since the most common NTM in Iran is reportedly Mycobacterium fortuitum, the present study was designed with the aim of molecular identification of clinical isolates of M. foruitum to analyse their heterogeneity. Materials and Methods A total of 81 isolates of NTM isolated from various samples were collected. The clinical isolates were assigned to species M. fortuitum by using conventional and molecular methods. The DNA banding patterns of ERIC- PCR and RAPD- PCR were analysed by using Bionumeric 7.5 software. Results Out of 81 tested NTM, 36 strains of M. fortuitum were identified. 33 isolates were selected for molecular typing in this study. Based on RAPD and ERIC analysis, M. fortuitum isolates were divided into 3 and 6 clusters, respectively. Most of the isolates were distributed into types of II RAPD (20 members/ 60.6 %) and V (14 members/ 42.4% with sub cluster I & II) of ERIC. In RAPD analysis, the major fragments were 300 bp, followed by fragment 1000. In ERIC analysis, the major fragments were 280 bp followed by fragment 1200 bp. Conclusion In conclusion, though the results from this study represented higher discriminatory power of ERIC, however the combination of RAPD and ERIC analysis were able to sufficiently discriminate the genotypic diversity, infection control, and gain useful epidemiological information regarding M. fortuitum isolates. PMID:26816886

  17. Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis.

    PubMed Central

    Yang, Z; Chaves, F; Barnes, P F; Burman, W J; Koehler, J; Eisenach, K D; Bates, J H; Cave, M D

    1996-01-01

    Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments. PMID:8940446

  18. Mycobacterium tuberculosis DNA fingerprint clusters and its relationship with RDRio genotype in Brazil

    PubMed Central

    Vinhas, Solange Alves; Palaci, Moisés; Marques, Hebert Silva; de Aguiar, Paola Poloni Lobo; Ribeiro, Fabíola Karla; Peres, Renata Lyrio; Dietze, Reynaldo; Gomes, Harrison Magdinier; Suffys, Philip Noel; Golub, Jonathan E.; Riley, Lee W.; Maciel, Ethel Leonor Noia

    2013-01-01

    SUMMARY Mycobacterium tuberculosis (Mtb) strains designated as RDRio are responsible for a large cluster of new cases of tuberculosis (TB) in Rio de Janeiro. They were previously shown to be associated with severe manifestations of TB. Here, we used three genotyping methods (IS6110 RFLP, spoligotyping, and multiplex PCR) to characterize RDRio and non-RDRio strains from the metropolitan area of Vitória, State of Espirito Santo in southeast Brazil to determine strain diversity and transmission patterns. Strains with identical IS6110 RFLP patterns were considered to belong to a cluster indicative of recent transmission. Between 2000 and 2010, we identified 5470 new TB patients and genotyped 981 Mtb strains. Of these, 376 (38%) were RDRio. By RFLP, 180 (48%) of 376 RDRio strains and 235 (40%) of 593 non-RDRio strains belonged to RFLP cluster pattern groups (p = 0.023). Simpson’s diversity index based on RFLP patterns was 0.96 for RDRio and 0.98 for non-RDRio strains. Thus, although RDRio strains appear to be comprised of a fewer number of RFLP genotypes, they represent a heterogeneous group. While TB cases caused by RDRio appear more likely to be due to recent transmission than cases caused by non-RDRio strains, the difference is small. These observations suggest that factors other than inherent biological characteristic of RDRio lineages are more important in determining recent transmission, and that public health measures to interrupt new transmissions need to be emphasized for TB control in Vitória. PMID:23232111

  19. Mitochondrial DNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression in cultured cells of patients with MERRF syndrome.

    PubMed

    Wu, Shi-Bei; Ma, Yi-Shing; Wu, Yu-Ting; Chen, Yin-Chiu; Wei, Yau-Huei

    2010-06-01

    Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a rare disorder characterized by myoclonus, muscle weakness, cerebellar ataxia, heart conduction block, and dementia. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). We and other investigators have reported that the mtDNA mutation results in not only inefficient generation of adenosine triphosphate but also increased production of reactive oxygen species (ROS) in cultured cells harboring A8344G mutation of mtDNA. In addition, we found an imbalance in the gene expression of antioxidant enzymes in the skin fibroblasts of MERRF patients. The mRNA, protein, and enzyme activity levels of manganese-superoxide dismutase were increased, but those of Cu,Zn-SOD, catalase, and glutathione peroxidase did not show significant changes. Recently, we showed that the excess ROS could damage voltage-dependent anion channel, prohibitin, Lon protease, and aconitase in the MERRF cells. Moreover, there was a dramatic increase in the gene expression and activity of matrix metalloproteinase 1, which may contribute to the cytoskeleton remodeling involved in the weakness and atrophy of muscle commonly seen in MERRF patients. Taken together, we suggest that mtDNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression are involved in the pathogenesis and progression of MERRF syndrome. PMID:20411357

  20. [DNA vaccination via in vivo electroporation can elicit specific immune response against highly pathogenic H5N1 influenza viral structural antigens in mice].

    PubMed

    Wang, Wen; Chen, Hong; Tan, Wen-jie; Deng, Yao; Wang, Min; Liu, Yuan; Yin, Xiao; Zhang, Ke; Guan, Jie; Zhou, Jian-fang; Shu, Yue-long; Ruan, Li

    2010-05-01

    This study aims to develop inexpensive and effective experimental vaccines against highly pathogenic H5N1 Avian Influenza (HPAI) virus and to optimize their immunization programs. To this end, we first synthesized the codon-optimized hemagglutinin gene (HAop) and neuraminidase gene (NAop), both of which were derived from a H5N1 virus (Anhui strain), and constructed successfully the DNA vaccines containing a single cistronic construct (HAwt, HAop, or NAop) or a bicistronic construct (HAop/M2 or NAop/M1) of H5N1 influenza virus origin. Their expression was confirmed by indirect immunofluorescent assay (IFA) and Western blotting. Then twice vaccination of mice with the DNA vaccines by injection intramuscularly or in vivo electroporation (EP) via two different routes was evaluated and analyzed by hemagglutination inhibition (HI) assay, NA-specific antibody detection, micro-neutralizing antibody test and IFN-gamma ELISpot assay. Our results showed that the DNA vaccines with coden-optimized HAop and NAop constructs could quickly elicit a strong immune response by in vivo EP, especially the cellular immune response against HA and NA; the in vivo EP via intradermal route induced stronger humoral immune responses than those via intramuscular route. Our findings will pave a way for further development of novel DNA-based H5N1 vaccine and for the optimization of the immunization programs of DNA vaccine. PMID:20572336

  1. Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross-protective antibody responses.

    PubMed

    Wang, Shixia; Hackett, Anthony; Jia, Na; Zhang, Chunhua; Zhang, Lu; Parker, Chris; Zhou, An; Li, Jun; Cao, Wu-Chun; Huang, Zuhu; Li, Yan; Lu, Shan

    2011-01-01

    Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic. PMID:22205966

  2. Comparative evaluation of PCR and commercial DNA probes for detection and identification to species level of Mycobacterium avium and Mycobacterium intracellulare.

    PubMed Central

    Devallois, A; Picardeau, M; Goh, K S; Sola, C; Vincent, V; Rastogi, N

    1996-01-01

    Selective amplification of a 187-bp fragment within the DT6 sequence using the AV6 and AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of Mycobacterium intracellulare using the IN38 and IN41 primers was performed for 69 clinical isolates identified as M. avium complex by conventional methods. The results were compared in parallel with results with commercial M. avium and M. intracellulare probes. A positive response to either of the two PCRs or M. avium-M. intracellulare AccuProbes constituted positive detection as M. avium complex; this cumulative detection limit was 94.2% for PCR, compared with 90% for AccuProbe. Concordance, on the other hand, was considered an identical species identification using either DT1 PCR and the M. intracellulare probe or DT6 and DT1 PCRs are inexpensive and at least equally sensitive, in-house options to the AccuProbe system for species identification of M. avium and M. intracellulare. PMID:8897178

  3. The ability of Hepatitis B surface antigen DNA vaccine to elicit cell-mediated immune responses, but not antibody responses, was affected by the deglysosylation of S antigen.

    PubMed

    Xing, Yiping; Huang, Zuhu; Lin, Yan; Li, Jun; Chou, Te-Hui; Lu, Shan; Wang, Shixia

    2008-09-19

    Hepatitis B Virus (HBV) infection remains a major worldwide infectious disease with serious long-term morbidity and mortality. The limited selections of drug treatment are not able to control the progress of disease in people with active and persistent HBV infection. Immunotherapy to control the degree of viral infection is one possible alternative solution to this challenge. HBV DNA vaccines, with their strong ability to induce cell-mediated immune responses, offer an attractive option. HBV surface protein is important in viral immunity. Re-establishing anti-S immunity in chronic HBV infected patients will bring significant benefit to the patients. Previous studies have shown that HBV S DNA vaccines are immunogenic in a number of animal studies. In the current study, we further investigated the effect of glycosylation to the expression and immunogenicity of S DNA vaccines. Our results demonstrate that deglycosylation at the two potential N-linked glycosylation sites in S protein resulted in a significant decrease of S-specific cell-mediated immune responses, but did not affect anti-S antibody responses. This finding provides important direction to the development of S DNA vaccines to elicit the optimal and balanced antibody and cell-mediated immune responses to treat people with HBV chronic infections. PMID:18462847

  4. Detection of Mycobacterium bovis DNA in nasal swabs from tuberculous cattle by a multiplex PCR

    PubMed Central

    de Souza Figueiredo, Eduardo Eustáquio; Carvalho, Ricardo Cezar Tavares; Silvestre, Flávia Galindo; Lilenbaum, Walter; Fonseca, Leila Sousa; Silva, Joab Trajano; Paschoalin, Vânia Margaret Flosi

    2010-01-01

    Detection of tuberculosis in cattle relies on the intradermal tuberculin test (ITT), but a definitive diagnosis requires identification of the pathogen after the animal is slaughtered. DNA in nasal swabs from 50 cows was analyzed by m-PCR, targeting for the RvD1-Rv2031c and IS6110 sequences. M. bovis was identified in two of 34 tuberculous cows (5.9%). The use of mPCR of nasal swabs as an in vivo diagnostic tool for bovine tuberculosis is suggested. PMID:24031509

  5. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery.

    PubMed

    Best, Simon R; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R; Wu, T-C; Pai, Sara I

    2009-09-01

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination--conventional intramuscular injection, electroporation-mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery--in the ability to generate antigen-specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin's role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

  6. Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yin, Xiao; Yang, Yang; Tan, Wenjie

    2015-10-01

    Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection. PMID:26215441

  7. A novel vaccine p846 encoding Rv3615c, Mtb10.4, and Rv2660c elicits robust immune response and alleviates lung injury induced by Mycobacterium infection.

    PubMed

    Kong, Hongmei; Dong, Chunsheng; Xiong, Sidong

    2014-01-01

    Development of effective anti-tuberculosis (TB) vaccines is one of the important steps to improve control of TB. Cell-mediated immune response significantly affects the control of M. tuberculosis infection. Thus, vaccines able to elicit strong cellular immune response hold special advantages against TB. In this study, three well-defined mycobacterial antigens (Rv3615c, Mtb10.4 [Rv0228], and Rv2660c) were engineered as a novel triple-antigen fusion DNA vaccine p846. The p846 vaccine consists of a high density of CD4(+) and CD8(+) T-cell epitopes. Intramuscular immunization of p846 induced robust T cells mediated immune response comparable to that of bacillus Calmette-Guérin (BCG) vaccination but more effective than that of individual antigen vaccination. After mycobacterial challenge, p846 immunization decreased bacterial burden at least 15-fold compared with individual antigen-based vaccination. Notably, the lungs of mice immunized with p846 exhibited fewer inflammatory cell infiltrates and less damage than those of control group mice. Our data demonstrate that the potential of p846 vaccine to protect against TB and the feasibility of this design strategy for further TB vaccine development. PMID:24280763

  8. A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered using a spring-powered jet injector elicits a potent neutralizing antibody response in rabbits and nonhuman primates.

    PubMed

    Kwilas, Steve; Kishimori, Jennifer M; Josleyn, Matthew; Jerke, Kurt; Ballantyne, John; Royals, Michael; Hooper, Jay W

    2014-01-01

    Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis(®) (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis(®) and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found

  9. Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide-sensitive iron-sulfur cluster.

    PubMed

    Smith, Laura J; Stapleton, Melanie R; Fullstone, Gavin J M; Crack, Jason C; Thomson, Andrew J; Le Brun, Nick E; Hunt, Debbie M; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S; Green, Jeffrey

    2010-12-15

    Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442

  10. Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis

    PubMed Central

    Moradi, Bagher; Sankian, Mojtaba; Amini, Yousef; Meshkat, Zahra

    2016-01-01

    Background: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette-Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis. Methods: A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 (+)/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis. Results: A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxV in eukaryotic cells. Conclusion: A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate. PMID:27536702

  11. The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

    PubMed Central

    Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

    2016-01-01

    DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

  12. Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

    SciTech Connect

    Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia; Piubelli, Orlando; Alves de Brito, Cyro; Fusaro, Ana Elisa; Eurico de Alencar, Liciana Xavier; August, Thomas; Torres Azevedo Marques, Ernesto; Silva Duarte, Alberto Jose da; Sato, Maria Notomi

    2010-10-10

    Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

  13. A microfluidic electrochemical biosensor based on multiwall carbon nanotube/ferrocene for genomic DNA detection of Mycobacterium tuberculosis in clinical isolates.

    PubMed

    Zribi, B; Roy, E; Pallandre, A; Chebil, S; Koubaa, M; Mejri, N; Magdinier Gomez, H; Sola, C; Korri-Youssoufi, H; Haghiri-Gosnet, A-M

    2016-01-01

    Herein we present a microfluidic-multiplexed platform that integrates electrochemical sensors based on carbon nanotubes associated with ferrocene as redox marker (carbon nanotube (CNT)/ferrocene) for direct detection of pathogenic viral DNA from Hepatitis C and genomic DNA from Mycobacterium tuberculosis in clinical isolates. By operating the fluidic device under high flow (150 μl/min), the formation of a very thin depletion layer at the sensor surface (δS = 230 nm) enhances the capture rate up to one DNA strand per second. By comparison, this capture rate is only 0.02 molecule/s in a static regime without flow. This fluidic protocol allows thus enhancing the limit of detection of the electrochemical biosensor from picomolar in bulk solution to femtomolar with a large dynamic range from 0.1 fM to 1 pM. Kinetics analysis also demonstrates an enhancement of the rate constant of electron transfer (kS) of the electrochemical process from 1 s(-1) up to 6 s(-1) thanks to the geometry of the miniaturized fluidic electrochemical cell. This microfluidic device working under high flow allows selective direct detection of a Mycobacterium tuberculosis (H37Rv) rpoB allele from clinical isolate extracted DNA. We envision that a microfluidic approach under high flow associated with a multiwall CNT/ferrocene sensor could find useful applications as the point-of-care for multi-target diagnostics of biomarkers in real samples. PMID:26865908

  14. Failure of a Mycobacterium tuberculosis DeltaRD1 DeltapanCD Double Deletion Mutant in a Neonatal Calf Aerosol M. bovis Challenge model: Comparisons to Responses Elicited by M. bovis bacille Calmette Guerin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An attenuated Mycobacterium tuberculosis RD1 knockout x pantothenate auxotroph (mc**2 6030) vaccine failed to protect neonatal calves from a low dose, aerosol M. bovis challenge. In contrast, M. bovis bacille Calmette Guerin (BCG)-vaccinates had reduced tuberculosis-associated pathology as compared ...

  15. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    PubMed Central

    Ferreira, Fabiana F.; Ammar, Dib; Bourckhardt, Gilian F.; Kobus-Bianchini, Karoline; Müller, Yara M. R.; Nazari, Evelise M.

    2015-01-01

    The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation. PMID:26793240

  16. Motif-Optimized Subtype A HIV Envelope-based DNA Vaccines Rapidly Elicit Neutralizing Antibodies When Delivered Sequentially

    PubMed Central

    Pissani, Franco; Malherbe, Delphine C.; Robins, Harlan; DeFilippis, Victor R.; Park, Byung; Sellhorn, George; Stamatatos, Leonidas; Overbaugh, Julie; Haigwood, Nancy L.

    2012-01-01

    HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants. PMID:22749601

  17. Immunization with a DNA Vaccine Cocktail Induces a Th1 Response and Protects Mice Against Mycobacterium avium subsp. paratuberculosis Challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several novel antigens of Mycobacterium avium subsp. paratuberculosis have been studied as vaccine components and their immunogenicity has been evaluated. Previously, we reported that 85 antigen complex (85A, 85B, and 85C), superoxide dismutase (SOD), and 35kDa protein could induce significant lymph...

  18. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal PCR is becoming more widely used today, demonstrating similar sensitivity and specificity to culture. To ensure ef...

  19. Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

    PubMed

    Park, Kun Taek; Allen, Andrew J; Davis, William C

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification. PMID:24534783

  20. A limited autoimmunity to p185neu elicited by DNA and allogeneic cell vaccine hampers the progression of preneoplastic lesions in HER-2/NEU transgenic mice.

    PubMed

    Lo Iacono, M; Cavallo, F; Quaglino, E; Rolla, S; Iezzi, M; Pupa, S M; De Giovanni, C; Lollini, P-L; Musiani, P; Forni, G; Calogero, R A

    2005-01-01

    Prevention of the progression of precancerous lesions by vaccines is virtually uncharted territory. Their potential, however, is being assessed in transgenic mice which develop autochthonous tumors with defined stages of progression. In this paper we show that the DNA micro-array technology significantly helps assessment of the preventive efficacy of a combined DNA and cell vaccine. All female rat Her-2/neu transgenic BALB/c (BALB-neuT) mice develop an invasive carcinoma in each of their mammary glands within 25 weeks of age. This is elicited by the activated transforming rat Her-2/neu oncogene embedded in their genome. We have previously shown that vaccination of mice bearing multiple in situ carcinomas with DNA plasmids which code for the extracellular and transmembrane domain of rat p185neu, the product of the rat Her-2/neu oncogene, followed by a boost with rat p185neu+ allogeneic cells engineered to secrete interferon-gamma, keeps 48% of mice tumor free until week 32. We have now extended our follow-up until mice reach one year of age and show that protection vanishes as time progresses. This observation suggests that the accuracy of the results studying immunotherapy against life-threatening tumors is a function of the length of the follow-up. The application of microarrays, and the concordance of morphologic and gene expression data led us to identify antibody as the main mechanism induced by vaccination. Protection is associated with a break of tolerance and a limited autoimmunity against the endogenous mouse p185neu. PMID:15888257

  1. Immunotherapy of tuberculosis with Mycobacterium leprae Hsp65 as a DNA vaccine triggers cross-reactive antibodies against mammalian Hsp60 but not pathological autoimmunity.

    PubMed

    Doimo, Nayara T S; Zárate-Bladés, Carlos R; Rodrigues, Rodrigo F; Tefé-Silva, Cristiane; Trotte, Marcele N S; Souza, Patrícia R M; Soares, Luana S; Rios, Wendy M; Floriano, Elaine M; Brandão, Izaira T; Masson, Ana P; Coelho, Verônica; Ramos, Simone G; Silva, Celio L

    2014-01-01

    Despite substantial efforts in recent years toward the development of new vaccines and drugs against tuberculosis (TB), success has remained elusive. Immunotherapy of TB with mycobacterial Hsp65 as a DNA vaccine (DNA-hsp65) results in a reduction of systemic bacterial loads and lung tissue damage, but the high homology of Hsp65 with the mammalian protein raises concern that pathological autoimmune responses may also be triggered. We searched for autoimmune responses elicited by DNA-hsp65 immunotherapy in mice chronically infected with TB by evaluating the humoral immune response and comprehensive histopathology using stereology. Cross-reactive antibodies between mycobacterial and mammalian Hsp60/65 were detected; however, no signs of pathological autoimmunity were found up to 60 days after the end of the therapy. PMID:24607935

  2. Immunotherapy of tuberculosis with Mycobacterium leprae Hsp65 as a DNA vaccine triggers cross-reactive antibodies against mammalian Hsp60 but not pathological autoimmunity

    PubMed Central

    Doimo, Nayara TS; Zárate-Bladés, Carlos R; Rodrigues, Rodrigo F; Tefé-Silva, Cristiane; Trotte, Marcele NS; Souza, Patrícia RM; Soares, Luana S; Rios, Wendy M; Floriano, Elaine M; Brandão, Izaira T; Masson, Ana P; Coelho, Verônica; Ramos, Simone G; Silva, Celio L

    2014-01-01

    Despite substantial efforts in recent years toward the development of new vaccines and drugs against tuberculosis (TB), success has remained elusive. Immunotherapy of TB with mycobacterial Hsp65 as a DNA vaccine (DNA-hsp65) results in a reduction of systemic bacterial loads and lung tissue damage, but the high homology of Hsp65 with the mammalian protein raises concern that pathological autoimmune responses may also be triggered. We searched for autoimmune responses elicited by DNA-hsp65 immunotherapy in mice chronically infected with TB by evaluating the humoral immune response and comprehensive histopathology using stereology. Cross-reactive antibodies between mycobacterial and mammalian Hsp60/65 were detected; however, no signs of pathological autoimmunity were found up to 60 days after the end of the therapy. PMID:24607935

  3. Pilot Study on the Use of DNA Priming Immunization to Enhance Y. pestis LcrV-Specific B Cell Responses Elicited by a Recombinant LcrV Protein Vaccine

    PubMed Central

    Li, Wei; Wang, Shixia; Lu, Shan

    2013-01-01

    Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis). Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems. PMID:26344467

  4. Comparison of C18-Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of Mycobacterium bovis in Bovine Milk Samples and Analysis of Samples by PCR

    PubMed Central

    Cornejo, Brandon J.; Sahagún-Ruiz, Alfredo; Suárez-Güemes, Francisco; Thornton, Charles G.; Ficht, Thomas A.; Adams, L. Garry

    1998-01-01

    The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence of Mycobacterium bovis by PCR. Detection by a C18-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS6110-based PCR was 94.1 and 58.8%, respectively (P < 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis. PMID:9687483

  5. Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system.

    PubMed

    Sweeney, Raymond W; Whitlock, Robert H; McAdams, Susan C

    2006-11-01

    Three methods of harvesting DNA from broth culture tubes for quantitative real-time polymerase chain reaction (qrtPCR) confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) were evaluated. A commercial DNA extraction kit, the boil method (boiling for 5 minutes), or direct addition of broth culture media to the PCR reaction mix were tested. Samples were evaluated at 8 or 11 days of incubation and at the time of instrument-signal culture-positive. In total, when tested at time to instrument signal positive, 10/10 (100%) of samples extracted by the commercial method were positive on qrtPCR, whereas 9/10 (90%) were positive after the boil method, and 6/10 (60%) were positive after the direct method. Increased volumes of egg-yolk emulsion added to the culture tubes prolonged the number of cycles to threshold positive for the samples that were not subjected to commercial extraction or boiling. Samples were not reliably positive when tested at 8 or 11 days of incubation. The boil method appears to represent a reasonable time- and money-saving method to harvest DNA for qrtPCR confirmation of MAP in broth culture at time to instrument signal positive. PMID:17121088

  6. Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

    PubMed

    Puri, Rupangi Verma; Reddy, P Vineel; Tyagi, Anil K

    2014-01-01

    In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed. PMID:24800740

  7. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.

    PubMed

    Thorne, N; Evans, J T; Smith, E G; Hawkey, P M; Gharbia, S; Arnold, C

    2007-10-01

    A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events. PMID:17803750

  8. Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood of individuals, eight years after completion of anti-leprosy therapy.

    PubMed

    Santos, A R; Balassiano, V; Oliveira, M L; Pereira, M A; Santos, P B; Degrave, W M; Suffys, P N

    2001-11-01

    Thirty eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status. PMID:11784934

  9. Mycobacterium RbpA cooperates with the stress-response σB subunit of RNA polymerase in promoter DNA unwinding

    PubMed Central

    Hu, Yangbo; Morichaud, Zakia; Sudalaiyadum Perumal, Ayyappasamy; Roquet-Baneres, Françoise; Brodolin, Konstantin

    2014-01-01

    RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σA subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σA and stress-response σB, but not the alternative σ subunits from the groups 3 and 4. In contrast to σA, the σB subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response. PMID:25122744

  10. Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

    PubMed

    Nogueira, Christiane Lourenço; Whipps, Christopher M; Matsumoto, Cristianne Kayoko; Chimara, Erica; Droz, Sara; Tortoli, Enrico; de Freitas, Denise; Cnockaert, Margo; Palomino, Juan Carlos; Martin, Anandi; Vandamme, Peter; Leão, Sylvia Cardoso

    2015-12-01

    Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T). PMID:26358475

  11. The Mycobacterium bovis BCG prime-Rv0577 DNA boost vaccination induces a durable Th1 immune response in mice.

    PubMed

    Gu, Dongqing; Chen, Wei; Mi, Youjun; Gong, Xueli; Luo, Tao; Bao, Lang

    2016-04-01

    Tuberculosis remains a major global health problem and effective vaccines are urgently needed. In this study, we used the combined DNA- and protein-based vaccines of immunodominant antigen Rv0577 to boost BCG and evaluated their immunogenicity in BALB/c mice. Our data suggest that the booster vaccine may substantially enhance the immunogenicity of BCG and strengthen both CD4+ T cell-mediated Th1 and CD8+ T cell-mediated cytolytic responses. Compared with the protein-based vaccine, the DNA-based vaccine can induce more durable Th1 immune response, characterized by high levels of antibody response, proliferation response, percentages of CD4+/CD8+ and cytokine secretion in antigen-stimulated splenocyte cultures. In conclusion, we for the first time, developed a protein- and plasmid DNA-based booster vaccine based on Rv0577. Our findings suggest that antigen Rv0577-based DNA vaccine is immunogenic and can efficiently boost BCG, which could be helpful in the design of an efficient vaccination strategy against TB. PMID:26922320

  12. Genetic Diversity of Mycobacterium africanum Clinical Isolates Based on IS6110-Restriction Fragment Length Polymorphism Analysis, Spoligotyping, and Variable Number of Tandem DNA Repeats

    PubMed Central

    Viana-Niero, Cristina; Gutierrez, Cristina; Sola, Christophe; Filliol, Ingrid; Boulahbal, Fadila; Vincent, Véronique; Rastogi, Nalin

    2001-01-01

    A collection of 105 clinical isolates originally identified as Mycobacterium africanum were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the M. tuberculosis complex, whereas genotypic characterization was based on IS6110-restriction fragment length polymorphism (IS6110-RFLP) analysis, IS1081-RFLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyping), variable number of tandem DNA repeats (VNTR), and the polymorphism of the oxyR, pncA, and mtp40 loci. The results obtained showed that a majority of M. africanum isolates were characterized by a specific spoligotyping pattern that was intermediate between those of M. tuberculosis and M. bovis, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respectively. A tentative M. africanum-specific spoligotyping signature appeared to be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as the polymorphism of oxyR and pncA, a total of 24 isolates were excluded from the final study (19 were identified as M. tuberculosis, 2 were identified as M. canetti, and 3 were identified as M. bovis). The remaining 81 M. africanum isolates were efficiently subtyped in three distinct subtypes (A1 to A3) by IS6110-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of the A2 subtype but showed significant variations for subtypes A1 and A3. A phylogenetic tree based on a selection of isolates representing the three subtypes using VNTR and spoligotyping alone or in combination confirmed the subtypes described as well as the heterogeneity of subtype A3. PMID:11136749

  13. Analysis of the vaccine potential of plasmid DNA encoding nine mycolactone polyketide synthase domains in Mycobacterium ulcerans infected mice.

    PubMed

    Roupie, Virginie; Pidot, Sacha J; Einarsdottir, Tobba; Van Den Poel, Christophe; Jurion, Fabienne; Stinear, Timothy P; Huygen, Kris

    2014-01-01

    There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an

  14. A triclade DNA vaccine designed on the basis of a comprehensive serologic study elicits neutralizing antibody responses against all clades and subclades of highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Zhou, Fan; Wang, Guiqin; Buchy, Philippe; Cai, Zhipeng; Chen, Honglin; Chen, Zhiwei; Cheng, Genhong; Wan, Xiu-Feng; Deubel, Vincent; Zhou, Paul

    2012-06-01

    Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades 2.1.3.2, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades 2.3.2.1 and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0, 2.3.2.1, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted. PMID:22496212

  15. A Triclade DNA Vaccine Designed on the Basis of a Comprehensive Serologic Study Elicits Neutralizing Antibody Responses against All Clades and Subclades of Highly Pathogenic Avian Influenza H5N1 Viruses

    PubMed Central

    Zhou, Fan; Wang, Guiqin; Buchy, Philippe; Cai, Zhipeng; Chen, Honglin; Chen, Zhiwei; Cheng, Genhong; Wan, Xiu-Feng; Deubel, Vincent

    2012-01-01

    Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades 2.1.3.2, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades 2.3.2.1 and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0, 2.3.2.1, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted. PMID:22496212

  16. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D

    PubMed Central

    Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3’-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotide and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologs of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both, in an M. smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5’-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. PMID:23198659

  17. A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

    PubMed

    Saxena, Sonal; Dahiya, Shyam S; Sonwane, Arvind A; Patel, Chhabi Lal; Saini, Mohini; Rai, A; Gupta, Praveen K

    2008-12-01

    A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies. PMID:18848857

  18. Ludic Elicitation: Using Games for Knowledge Elicitation

    ERIC Educational Resources Information Center

    Cao, Yan

    2014-01-01

    Knowledge elicitation from human beings is important for many fields, such as decision support systems, risk communication, and customer preference studying. Traditional approaches include observations, questionnaires, structured and semi-structured interviews, and group discussions. Many publications have been studying different techniques for a…

  19. Human plasmacytoid dentritic cells elicit a Type I Interferon response by sensing DNA via the cGAS-STING signaling pathway.

    PubMed

    Bode, Christian; Fox, Mario; Tewary, Poonam; Steinhagen, Almut; Ellerkmann, Richard K; Klinman, Dennis; Baumgarten, Georg; Hornung, Veit; Steinhagen, Folkert

    2016-07-01

    Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN-driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self-DNA and has been linked to TLR9-independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS-STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS-STING pathway and that targeting this pathway could be of therapeutic interest. PMID:27125983

  20. Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis.

    PubMed

    Tang, Xiao-Lei; Wu, Shi-Min; Xie, Yan; Song, Neng; Guan, Qing; Yuan, Chunhui; Zhou, Xiang; Zhang, Xiao-Lian

    2016-05-01

    The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. Mannose-capped lipoarabinomannan (ManLAM) is a lipoglycan serving as a major cell wall component. ManLAM is also an early released antigen in the blood circulation system during Mycobacteria tuberculosis (M.tb) infection and is a perfect target antigen for TB diagnosis. In this study, ssDNA aptamers "antibodies" against ManLAM of the predominant clinical epidemic M.tb Beijing genotype strains were generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. The selected single aptamer T9 demonstrated the highest specificity and binding affinity, with an equilibrium dissociation constant (Kd) of 668 ± 159 nmol/L. We further detected ManLAM antigens in serum and sputum samples from active pulmonary tuberculosis (aPTB) patients, extrapulmonary TB (EPTB) patients and healthy donors by using a T9 based enzyme-linked oligonucleotide assay (ELONA). The results showed that the specificity and sensitivity were 95.31% and 83.00% (for 100 aPTB serum samples), 98.70% and 92.71% (for 96 aPTB sputum samples), and 94.44% and 88.71% (for 62 EPTB serum samples), respectively. A good correlation was observed between the T9 aptamer-based ELONA and the clinical T-SPOT.TB. Thus, T9 based ELONA has potentials for diagnosis of TB, including inactive TB, smear-negative TB, EPTB, and TB with immunodeficiency, and assist the diagnosis of LTBI albeit it could not distinguish LTBI and active TB. PMID:26850356

  1. A single dose of a DNA vaccine encoding apa coencapsulated with 6,6'-trehalose dimycolate in microspheres confers long-term protection against tuberculosis in Mycobacterium bovis BCG-primed mice.

    PubMed

    Carlétti, Dyego; Morais da Fonseca, Denise; Gembre, Ana Flávia; Masson, Ana Paula; Weijenborg Campos, Lívia; Leite, Luciana C C; Rodrigues Pires, Andréa; Lannes-Vieira, Joseli; Lopes Silva, Célio; Bonato, Vânia Luiza Deperon; Horn, Cynthia

    2013-08-01

    Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB. PMID:23740922

  2. Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination▿

    PubMed Central

    de Alencar, Bruna C. G.; Persechini, Pedro M.; Haolla, Filipe A.; de Oliveira, Gabriel; Silverio, Jaline C.; Lannes-Vieira, Joseli; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bruna-Romero, Oscar; Rodrigues, Mauricio M.

    2009-01-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  3. Perforin and gamma interferon expression are required for CD4+ and CD8+ T-cell-dependent protective immunity against a human parasite, Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination.

    PubMed

    de Alencar, Bruna C G; Persechini, Pedro M; Haolla, Filipe A; de Oliveira, Gabriel; Silverio, Jaline C; Lannes-Vieira, Joseli; Machado, Alexandre V; Gazzinelli, Ricardo T; Bruna-Romero, Oscar; Rodrigues, Mauricio M

    2009-10-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  4. Lipid exposure elicits differential responses in gene expression and DNA methylation in primary human skeletal muscle cells from severely obese women.

    PubMed

    Maples, Jill M; Brault, Jeffrey J; Shewchuk, Brian M; Witczak, Carol A; Zou, Kai; Rowland, Naomi; Hubal, Monica J; Weber, Todd M; Houmard, Joseph A

    2015-05-01

    The skeletal muscle of obese individuals exhibits an impaired ability to increase the expression of genes linked with fatty acid oxidation (FAO) upon lipid exposure. The present study determined if this response could be attributed to differential DNA methylation signatures. RNA and DNA were isolated from primary human skeletal muscle cells (HSkMC) from lean and severely obese women following lipid incubation. mRNA expression and DNA methylation were quantified for genes that globally regulate FAO [PPARγ coactivator (PGC-1α), peroxisome proliferator-activated receptors (PPARs), nuclear respiratory factors (NRFs)]. With lipid oversupply, increases in NRF-1, NRF-2, PPARα, and PPARδ expression were dampened in skeletal muscle from severely obese compared with lean women. The expression of genes downstream of the PPARs and NRFs also exhibited a pattern of not increasing as robustly upon lipid exposure with obesity. Increases in CpG methylation near the transcription start site with lipid oversupply were positively related to PPARδ expression; increases in methylation with lipid were depressed in HSkMC from severely obese women. With severe obesity, there is an impaired ability to upregulate global transcriptional regulators of FAO in response to lipid exposure. Transient changes in DNA methylation patterns and differences in the methylation signature with severe obesity may play a role in the transcriptional regulation of PPARδ in response to lipid. The persistence of differential responses to lipid in HSkMC derived from lean and obese subjects supports the possibility of stable epigenetic programming of skeletal muscle cells by the respective environments. PMID:25670728

  5. 3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

    SciTech Connect

    Moorthy, Bhagavatula . E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel; Fazili, Inayat S.; Kondraganti, Sudha R.; Wang Lihua; Couroucli, Xanthi I.; Jiang Weiwu

    2007-03-23

    Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 {mu}g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 {sup o}C for 2 h, giving rise to 9 adducts, as determined by {sup 32}P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16 h, followed by MC (1 {mu}M) treatment for 24 h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

  6. Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle.

    PubMed

    Zimmer, K; Dräger, K G; Klawonn, W; Hess, R G

    1999-03-01

    In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease. PMID:10216457

  7. Prime-boost bacillus Calmette-Guérin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice.

    PubMed

    Xu, Ying; Yang, Enzhuo; Wang, Jianguang; Li, Rui; Li, Guanghua; Liu, Guoyuan; Song, Na; Huang, Qi; Kong, Cong; Wang, Honghai

    2014-10-01

    To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB. PMID:24773322

  8. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

    PubMed Central

    Kapur, V; Li, L L; Iordanescu, S; Hamrick, M R; Wanger, A; Kreiswirth, B N; Musser, J M

    1994-01-01

    Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level. PMID:8027320

  9. Detection of Mycobacterium avium in pet birds

    PubMed Central

    Godoy, Silvia Neri; Sakamoto, Sidnei Miyoshi; de Paula, Cátia Dejuste; Catão-Dias, José Luiz; Matushima, Eliana Reiko

    2009-01-01

    The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential. PMID:24031356

  10. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  11. Whole genome analyses of marine fish pathogenic isolate, Mycobacterium sp. 012931.

    PubMed

    Kurokawa, Satoru; Kabayama, Jun; Hwang, Seong Don; Nho, Seong Won; Hikima, Jun-ichi; Jung, Tae Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Mori, Tetsushi; Aoki, Takashi

    2014-10-01

    Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen. PMID:24879010

  12. Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

    PubMed

    Lee, So-Young; Kim, Byoung-Jun; Kim, Hong; Won, Yu-Seop; Jeon, Che Ok; Jeong, Joseph; Lee, Seon Ho; Lim, Ji-Hun; Lee, Seung-Heon; Kim, Chang Ki; Kook, Yoon-Hoh; Kim, Bum-Joon

    2016-08-01

    Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T). PMID:27189351

  13. Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

    PubMed Central

    Panas, Michael W.; Jain, Paras; Yang, Hui; Mitra, Shimontini; Biswas, Debasis; Wattam, Alice Rebecca; Letvin, Norman L.; Jacobs, William R.

    2014-01-01

    Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc2155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC–mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials. PMID:25197070

  14. STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

    PubMed

    de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

    2016-07-15

    Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. PMID:27190175

  15. Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyfunctional T cells simultaneously produce IFN-gamma, IL-2 and TNF-alpha and play relevant roles in several chronic infections, including TB. Mycobacterium bovis infection of cattle elicits ex vivo polyfunctional T cell responses. Vaccine-elicited IFN-gamma Tcm (CD4+ CD45RO+ CCR7+) responses corr...

  16. Rose myrtle (Rhodomyrtus tomentosa) extract and its component, piceatannol, enhance the activity of DNA polymerase and suppress the inflammatory response elicited by UVB‑induced DNA damage in skin cells.

    PubMed

    Shiratake, Sawako; Nakahara, Tatsuo; Iwahashi, Hiroyasu; Onodera, Takefumi; Mizushina, Yoshiyuki

    2015-10-01

    A number of naturally occurring agents are hypothesized to protect against ultraviolet (UV)‑induced skin damage. The present study screened >50 plant extracts for inhibitors of UVB‑induced cytotoxicity, using cultured normal human epidermal keratinocytes (NHEK), and identified that the fruit of rose myrtle (Rhodomyrtus tomentosa) was the most marked inhibitor of cell death. The protective effect of rose myrtle extract and the two key components, piceatannol and piceatannol‑4'‑O‑β‑D‑glucopyranoside, on UVB‑induced damage and inflammation in cultured NHEK was investigated. The 80% ethanol extract from rose myrtle fruit with piceatannol exhibited protection of UVB‑induced cytotoxicity in NHEK; however, piceatannol‑4'‑O‑β‑D‑glucopyranoside exhibited no protection, as determined by a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. This extract and piceatannol reduced the production of UVB‑induced cyclobutane pyrimidine dimers and enhanced the cellular enzyme activity of the DNA polymerases in UVB‑irradiated NHEK, suggesting that UVB‑stimulated DNA damage was repaired by the polymerases. In addition, the secretion of prostaglandin E2, which is an inflammatory mediator, was decreased. These results indicated that rose myrtle fruit extract and its key constituent, piceatannol, are potential photoprotective candidates for UV‑induced skin damage. PMID:26239705

  17. Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

    PubMed Central

    Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C.; Rutten, Victor

    2016-01-01

    The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

  18. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Huntley, J F; Stabel, J R; Paustian, M L; Reinhardt, T A; Bannantine, J P

    2005-10-01

    Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization. PMID:16177367

  19. Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis.

    PubMed

    Kontos, Fanourios; Mavromanolakis, Dimitrios Nikitas; Zande, Marina Chari; Gitti, Zoe Georgios

    2016-01-01

    Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid. PMID:27080783

  20. Mycobacterium marinum infection.

    PubMed

    Cassetty, Christopher T; Sanchez, Miguel

    2004-01-01

    A 49-year-old man presented with nodules on his right hand after a history of Mycobacterium marinum infection recently treated with rifampin and clarithromycin. The patient has an aquarium with Betta fish (Siamese fighting fish). PMID:15748591

  1. Comparative Genomic Hybridizations Reveal Genetic Regions within the Mycobacterium avium Complex That Are Divergent from Mycobacterium avium subsp. paratuberculosis Isolates†

    PubMed Central

    Paustian, Michael L.; Kapur, Vivek; Bannantine, John P.

    2005-01-01

    Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs. PMID:15774884

  2. Molecular cloning and cold shock induced overexpression of the DNA encoding phor sensor domain from Mycobacterium tuberculosis as a target molecule for novel anti-tubercular drugs

    NASA Astrophysics Data System (ADS)

    Langi, Gladys Emmanuella Putri; Moeis, Maelita R.; Ihsanawati, Giri-Rachman, Ernawati Arifin

    2014-03-01

    Mycobacterium tuberculosis (Mtb), the sole cause of Tuberculosis (TB), is still a major global problem. The discovery of new anti-tubercular drugs is needed to face the increasing TB cases, especially to prevent the increase of cases with resistant Mtb. A potential novel drug target is the Mtb PhoR sensor domain protein which is the histidine kinase extracellular domain for receiving environmental signals. This protein is the initial part of the two-component system PhoR-PhoP regulating 114 genes related to the virulence of Mtb. In this study, the gene encoding PhoR sensor domain (SensPhoR) was subcloned from pGEM-T SensPhoR from the previous study (Suwanto, 2012) to pColdII. The construct pColdII SensPhoR was confirmed through restriction analysis and sequencing. Using the construct, SensPhoR was overexpressed at 15°C using Escherichia coli BL21 (DE3). Low temperature was chosen because according to the solubility prediction program of recombinant proteins from The University of Oklahama, the PhoR sensor domain has a chance of 79.8% to be expressed as insoluble proteins in Escherichia coli's (E. coli) cytoplasm. This prediction is also supported by other similar programs: PROSO and PROSO II. The SDS PAGE result indicated that the PhoR sensor domain recombinant protein was overexpressed. For future studies, this protein will be purified and used for structure analysis which can be used to find potential drugs through rational drug design.

  3. Crystal Structure of Mycobacterium tuberculosis H37Rv AldR (Rv2779c), a Regulator of the ald Gene: DNA BINDING AND IDENTIFICATION OF SMALL MOLECULE INHIBITORS.

    PubMed

    Dey, Abhishek; Shree, Sonal; Pandey, Sarvesh Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2016-06-01

    Here we report the crystal structure of M. tuberculosis AldR (Rv2779c) showing that the N-terminal DNA-binding domains are swapped, forming a dimer, and four dimers are assembled into an octamer through crystal symmetry. The C-terminal domain is involved in oligomeric interactions that stabilize the oligomer, and it contains the effector-binding sites. The latter sites are 30-60% larger compared with homologs like MtbFFRP (Rv3291c) and can consequently accommodate larger molecules. MtbAldR binds to the region upstream to the ald gene that is highly up-regulated in nutrient-starved tuberculosis models and codes for l-alanine dehydrogenase (MtbAld; Rv2780). Further, the MtbAldR-DNA complex is inhibited upon binding of Ala, Tyr, Trp and Asp to the protein. Studies involving a ligand-binding site G131T mutant show that the mutant forms a DNA complex that cannot be inhibited by adding the amino acids. Comparative studies suggest that binding of the amino acids changes the relative spatial disposition of the DNA-binding domains and thereby disrupt the protein-DNA complex. Finally, we identified small molecules, including a tetrahydroquinoline carbonitrile derivative (S010-0261), that inhibit the MtbAldR-DNA complex. The latter molecules represent the very first inhibitors of a feast/famine regulatory protein from any source and set the stage for exploring MtbAldR as a potential anti-tuberculosis target. PMID:27006398

  4. Unconsciously elicited perceptual prior

    PubMed Central

    Chang, Raymond; Baria, Alexis T.; Flounders, Matthew W.; He, Biyu J.

    2016-01-01

    Increasing evidence over the past decade suggests that vision is not simply a passive, feed-forward process in which cortical areas relay progressively more abstract information to those higher up in the visual hierarchy, but rather an inferential process with top-down processes actively guiding and shaping perception. However, one major question that persists is whether such processes can be influenced by unconsciously perceived stimuli. Recent psychophysics and neuroimaging studies have revealed that while consciously perceived stimuli elicit stronger responses in higher visual and frontoparietal areas than those that fail to reach conscious awareness, the latter can still drive high-level brain and behavioral responses. We investigated whether unconscious processing of a masked natural image could facilitate subsequent conscious recognition of its degraded counterpart (a black-and-white “Mooney” image) presented many seconds later. We found that this is indeed the case, suggesting that conscious vision may be influenced by priors established by unconscious processing of a fleeting image.

  5. Evaluation of the risk of paratuberculosis in adult cows fed Mycobacterium avium subsp paratuberculosis DNA-positive or -negative colostrum as calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective - Estimate the risk of MAP infection in Holstein calves associated with ingestion of MAP DNA positive (vs negative) colostrum at birth. Animals - 205 Holstein heifer calves born in 12 commercial dairy herds. Procedure - Every calf born was separated from its dam within 30 to 60 minutes ...

  6. Mycobacterium microti: More diverse than previously thought.

    PubMed

    Smith, N H; Crawshaw, T; Parry, J; Birtles, R J

    2009-08-01

    Mycobacterium microti is a member of the Mycobacterium tuberculosis complex of bacteria. This species was originally identified as a pathogen of small rodents and shrews and was associated with limited diversity and a much reduced spoligotype pattern. More recently, specific deletions of chromosomal DNA have been shown to define this group of organisms, which can be identified by the absence of chromosomal region RD1(mic). We describe here the molecular characteristics of 141 strains of the Mycobacterium tuberculosis complex isolated in Great Britain over a 14-year period. All strains have characteristic loss of some spoligotype spacers and characteristic alleles at the ETR-E and ETR-F variable-number tandem-repeat (VNTR) loci, and a sample of these strains was deleted for regions RD7, RD9, and RD1(mic) but intact for regions RD4 and RD12. We therefore identified these strains as M. microti and show that they have much more diverse spoligotype patterns and VNTR types than previously thought. The most common source of these strains was domestic cats, and we show that the molecular types of M. microti are geographically localized in the same way that molecular types of Mycobacterium bovis are geographically localized in cattle in the United Kingdom. We describe the pathology of M. microti infection in cats and suggest that the feline disease is a spillover from a disease maintained in an unknown wild mammal, probably field voles. The location of the cats with M. microti infection suggests that they do not overlap geographically with the strains of Mycobacterium bovis in Great Britain. PMID:19535520

  7. Mycobacterium microti: More Diverse than Previously Thought▿

    PubMed Central

    Smith, N. H.; Crawshaw, T.; Parry, J.; Birtles, R. J.

    2009-01-01

    Mycobacterium microti is a member of the Mycobacterium tuberculosis complex of bacteria. This species was originally identified as a pathogen of small rodents and shrews and was associated with limited diversity and a much reduced spoligotype pattern. More recently, specific deletions of chromosomal DNA have been shown to define this group of organisms, which can be identified by the absence of chromosomal region RD1mic. We describe here the molecular characteristics of 141 strains of the Mycobacterium tuberculosis complex isolated in Great Britain over a 14-year period. All strains have characteristic loss of some spoligotype spacers and characteristic alleles at the ETR-E and ETR-F variable-number tandem-repeat (VNTR) loci, and a sample of these strains was deleted for regions RD7, RD9, and RD1mic but intact for regions RD4 and RD12. We therefore identified these strains as M. microti and show that they have much more diverse spoligotype patterns and VNTR types than previously thought. The most common source of these strains was domestic cats, and we show that the molecular types of M. microti are geographically localized in the same way that molecular types of Mycobacterium bovis are geographically localized in cattle in the United Kingdom. We describe the pathology of M. microti infection in cats and suggest that the feline disease is a spillover from a disease maintained in an unknown wild mammal, probably field voles. The location of the cats with M. microti infection suggests that they do not overlap geographically with the strains of Mycobacterium bovis in Great Britain. PMID:19535520

  8. Molecular Identification of Mycobacterium Species of Public Health Importance in Cattle in Zimbabwe by 16S rRNA Gene Sequencing

    PubMed Central

    Padya, Leah; Chin'ombe, Nyasha; Magwenzi, Marcelyn; Mbanga, Joshua; Ruhanya, Vurayai; Nziramasanga, Pasipanodya

    2015-01-01

    Mycobacterium species are naturally found in the environment as well as in domestic animals such as cattle. So far, more than 150 species of Mycobacterium, some of which are pathogenic, have been identified. Laboratory isolation, detection and identification of Mycobacterium species are therefore critical if human and animal infections are to be controlled. The objective of this study was to identify Mycobacterium species isolated in cattle in Zimbabwe using 16S ribosomal RNA gene amplification and sequencing. A total of 134 cow dung samples were collected throughout Zimbabwe and mycobacteria were isolated by culture. Only 49 culture isolates that were found to be acid-fast bacilli positive by Ziehl-Neelsen staining. The 16S rRNA gene was successfully amplified by PCR in 41 (84%) of the samples. There was no amplification in 8 (16%) of the samples. Out of the 41 samples that showed amplification, 26 (63%) had strong PCR bands and were selected for DNA sequencing. Analysis of the DNA sequences showed that 7 (27%) belonged to Mycobacterium neoaurum, 6 (23%) belonged to Mycobacterium fortuitum, 3 (12%) to Mycobacterium goodii, 2 (1%) to Mycobacterium arupense, 2 (1%) to Mycobacterium peregrinum or M. septicum and 1 isolate (0.04%) to Mycobacterium elephantis. There were 5 (19%) isolates that were non-mycobacteria and identified as Gordonia terrae, a close relative of Mycobacterium. The study therefore provided a molecular basis for detection and identification of Mycobacterium species in animals and humans. PMID:26668660

  9. Mycobacterium fortuitum lipoid pneumonia in a dog.

    PubMed

    Leissinger, M K; Garber, J B; Fowlkes, N; Grooters, A M; Royal, A B; Gaunt, S D

    2015-03-01

    A 1-year old female spayed German Shepherd dog was evaluated for acute onset of dyspnea. Pyogranulomatous inflammation and green globoid structures were present on aspirates of the affected lung. Impression smears and histopathology confirmed pyogranulomatous pneumonia, with large amounts of lipid corresponding to the green structures noted cytologically, and identified poorly staining bacterial rods within lipid vacuoles. Special stains confirmed the presence of acid-fast bacterial rods, and polymerase chain reaction and DNA sequencing identified the organism as Mycobacterium fortuitum. M. fortuitum pneumonia is well described in humans and has previously been reported in 4 dogs and 1 cat. Lipid was a prominent cytologic and histologic feature, as is often described in humans and in the single feline case report. Additionally, this case highlights the variable cytologic appearance of lipid, as well as Mycobacterium spp, which are classically nonstaining with Wright-Giemsa. PMID:24788402

  10. Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime-DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin-2-producing CD4 T-cell frequency post-vaccination.

    PubMed

    Kang, Han; Yuan, Qin; Ma, Hui; Hu, Zhi-Dong; Han, De-Ping; Wu, Kang; Lowrie, Douglas B; Fan, Xiao-Yong

    2014-12-01

    The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. Simple measurement of interferon-γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T-cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette-Guérin (BCG) prime-Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG-vaccinated mice, and the reduction in colony-forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin-2 (IL-2) -producing CD4 T cells and increased IL-2 production when measured as integrated mean fluorescence intensity post-vaccination as well. These data suggest that measurement of elevated frequency of IL-2-producing CD4 T cells or IL-2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime-boost strategies may be a useful approach to the control of M. tuberculosis infection. PMID:24965530

  11. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  12. Mycobacterium avium subspecies Paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome sequence has now defined the complete catalog of genes that make Mycobacterium avium subsp paratuberculosis what it is. Although similarity searches and bioinformatics analyses have assigned potential function to hundreds of genes in this pathogen, the future challenge is to begin to sys...

  13. Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease in cattle and other domesticated and wild ruminant species. The organism and disease were first described over a century ago, and despite the considerable morbidity and mortality associated with Map infection...

  14. Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille Calmette–Guérin (BCG)

    PubMed Central

    Batoni, G; Esin, S; Pardini, M; Bottai, D; Senesi, S; Wigzell, H; Campa, M

    2000-01-01

    In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4+ T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of γδ+ T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of γδ+ T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4+ T cells and CD16+/CD3− (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens. PMID:10632662

  15. Annotated Genome Sequence of Mycobacterium massiliense Strain M154, Belonging to the Recently Created Taxon Mycobacterium abscessus subsp. bolletii comb. nov.

    PubMed Central

    Wong, Yan Ling; Tan, Joon Liang; Ong, Chia Sui; Wong, Guat Jah; Ng, Kee Peng

    2012-01-01

    Mycobacterium massiliense has recently been proposed as a member of Mycobacterium abscessus subsp. bolletii comb. nov. Strain M154, a clinical isolate from the bronchoalveolar lavage fluid of a Malaysian patient presenting with lower respiratory tract infection, was subjected to shotgun DNA sequencing with the Illumina sequencing technology to obtain whole-genome sequence data for comparison with other genetically related strains within the M. abscessus species complex. PMID:22887675

  16. Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia.

    PubMed

    Najiah, M; Lee, K L; Noorasikin, H; Nadirah, M; Lee, S W

    2011-12-01

    Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains. PMID:20971487

  17. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rahman, Arfatur; Sahrin, Mahfuza; Afrin, Sadia; Earley, Keith; Ahmed, Shahriar; Rahman, S. M. Mazidur; Banu, Sayera

    2016-01-01

    Background GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. Methods We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. Results The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. Conclusion Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis. PMID:27054344

  18. Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain.

    PubMed

    Jaafar, Mohammad Maaruf; Halim, Mohd Zakihalani A; Ismail, Mohamad Izwan; Shien, Lee Lian; Kek, Teh Lay; Fong, Ngeow Yun; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Hock, Tang Thean; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-03-01

    Mycobacterium tuberculosis is an acid fast bacterial species in the family Mycobacteriaceae and is the causative agent of most cases of tuberculosis. Here, we report the genomic features of Mycobacterium tuberculosis isolated from the cerebrospinal fluid (CSF) of a patient diagnosed with both pulmonary and extrapulmonary tuberculosis (TB). The isolated strain was identified as Mycobacterium tuberculosis PR08 (MTB PR08). Genomic DNA of the MTB PR08 strain was extracted and subjected to whole genome sequencing using MiSeq (Illumina, CA,USA). The draft genome size of MTB PR08 strain is 4,292,364 bp with a G + C content of 65.2%. This strain was annotated to have 4723 genes and 48 RNAs. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010895. PMID:26981383

  19. Mycobacterium chelonae Is an Ubiquitous Atypical Mycobacterium

    PubMed Central

    Pinto-Gouveia, Miguel; Gameiro, Ana; Ramos, Leonor; Cardoso, José Carlos; Brites, Maria Manuel; Tellechea, Óscar; Figueiredo, Américo

    2015-01-01

    The type of cutaneous infection varies mainly according to the patient's immune status, and the disseminated form is mostly found in the context of immunosuppression. We report the case of a 62-year-old male who was under long-term systemic corticosteroid therapy and presented with a 7-month history of multiple painless cutaneous lesions at various stages of development: papules, nodules, pustules and hemorrhagic crusts, as well as small erosions and ulcers distributed over the limbs and scalp. Cutaneous biopsy showed a suppurative granulomatous infiltrate with abscess formation. Fite stain revealed numerous extracellular bacilli, suggesting mycobacterial infection, particularly by atypical mycobacteria. Culture of a skin sample revealed Mycobacterium chelonae. The patient started multidrug therapy and showed clinical improvement despite of resistance to one of the antibiotics. This striking presentation underlines the role of immunosuppression with corticotherapy as a major risk factor for these infections. Multidrug therapy is advised and antibiogram is essential in directing treatment. PMID:26351432

  20. Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan.

    PubMed

    Imajoh, Masayuki; Sugiura, Hidehiro; Hashida, Yumiko; Hatai, Kishio; Oshima, Syun-ichirou; Daibata, Masanori; Kawai, Kenji

    2013-01-01

    In Japan, a Mycobacterium marinum-like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA-DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β-subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity. PMID:23043488

  1. Evidences for anti-mycobacterium activities of lipids and surfactants.

    PubMed

    Hussain, Afzal; Singh, Sandeep Kumar

    2016-01-01

    Tuberculosis is the most widespread and deadly airborne disease caused by Mycobacterium tuberculosis. The two-pronged lethal effect on the bacteria using lipids/surfactants and anti-tubercular drugs may render the miniaturization of dose owing to synergistic and tandem effect of both. The current research has been focused on screening and evaluating various lipids/surfactants possessing inherent anti-mycobacterium activity that can ferry the anti-tubercular drugs. In vitro anti-mycobacterium activity was evaluated using agar well diffusion method. Furthermore, time-concentration dependent killing and DNA/RNA content release studies were performed to correlate the findings. The exact mechanism of bacterial killing was further elucidated by electron/atomic force microscopy studies. Finally, to negate any toxicity, in vitro hemolysis and toxicity studies were performed. The study revealed that capmul MCM C-8, labrasol and acconon C-80 possessed highest in vitro anti-mycobacterium activity. Electron/atomic force microscopy results confirmed in vitro studies and verified the killing of Mycobacterium owing to the release of cytoplasmic content after cell wall fragmentation and disruption. Moreover, the least hemolysis and hundred percent survivals rate of mice using the excipients demonstrated the safety aspects of explored excipients that can ferry the anti-tubercular drugs. The present study concluded the safe, efficient and synergistic activity of the explored excipients and anti-tubercular drugs in controlling the menace of tuberculosis. PMID:26712622

  2. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  3. Mycobacterium asiaticum infection in a red-handed tamarin (Saguinus midas).

    PubMed

    Siegal-Willott, Jessica; Isaza, Ramiro; Fiorello, Christine; Reinhard, Mary

    2006-09-01

    A 4-yr-old, intact male red-handed tamarin (Saguinus midas) was evaluated because of a 6-mo history of an enlarging axillary mass. Diagnostic findings included a positive intradermal tuberculin test, persistent severe leukocytosis, and hyperglobulinemia. A nontuberculous mycobacterium species isolated from the mass was identified as Mycobacterium asiaticum using 16s ribosomal DNA sequencing and high-performance liquid chromatography. PMID:17319146

  4. Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

    PubMed Central

    Rath, Poonam; Huang, Chengdong; Wang, Tao; Wang, Tianzhi; Li, Huilin; Prados-Rosales, Rafael; Elemento, Olivier; Casadevall, Arturo; Nathan, Carl F.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host–pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named “vesiculogenesis and immune response regulator.” PMID:24248369

  5. Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermalinjection in cattle. In vitro, such antigens stimulate the production of interferon (IFN)-gamma by bovine T-cells in whole blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-g...

  6. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    NASA Astrophysics Data System (ADS)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  7. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    PubMed

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942

  8. THE ISOLATION AND IDENTIFICATION OF MYCOBACTERIUM AVIUM COMPLEX (MAC) RECOVERED FROM LOS ANGELES POTABLE WATER, A POSSIBLE SOURCE OF INFECTION IN AIDS PATIENTS

    EPA Science Inventory

    Los Angeles water was investigated as a possible source of Mycobacterium avium complex (MAC) infection in patients with AIDS. MAC consists of M.avium (MA), M. intracellulare (MI) and Mycobacterium X (MX)(positive for MAC by DNA probe but not MA or MI). The study included 13 reser...

  9. Mycobacterium tuberculosis Infection of Domesticated Asian Elephants, Thailand

    PubMed Central

    Angkawanish, Taweepoke; Sirimalaisuwan, Anucha; Kaewsakhorn, Thattawan; Boonsri, Kittikorn; Rutten, Victor P.M.G.

    2010-01-01

    Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential. PMID:21122228

  10. Prosthetic Valve Endocarditis and Bloodstream Infection Due to Mycobacterium chimaera

    PubMed Central

    Achermann, Yvonne; Rössle, Matthias; Hoffmann, Matthias; Deggim, Vanessa; Kuster, Stefan; Zimmermann, Dieter R.; Hombach, Michael; Hasse, Barbara

    2013-01-01

    Prosthetic valve endocarditis (PVE) due to fast-growing nontuberculous mycobacteria (NTM) has been reported anecdotally. Reports of PVE with slowly growing NTM, however, are lacking. We present here one case of PVE and one case of bloodstream infection caused by Mycobacterium chimaera. Randomly amplified polymorphic DNA (RAPD)-PCR indicated a relatedness of the two M. chimaera strains. Both patients had heart surgery 2 years apart from each other. A nosocomial link was not detected. PMID:23536407

  11. Uric acid utilization by Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates.

    PubMed Central

    Falkinham, J O; George, K L; Parker, B C; Gruft, H

    1983-01-01

    Forty-nine human and environmental isolates of Mycobacterium intracellulare and Mycobacterium scrofulaceum were tested for their ability to grow on uric acid and a number of its degradation products. Nearly all (88 to 90%) strains used uric acid or allantoin as a sole nitrogen source; fewer (47 to 69%) used allantoate, urea, or possibly ureidoglycollate. Enzymatic activities of one representative isolate demonstrated the existence of a uric acid degradation pathway resembling that in other aerobic microorganisms. PMID:6863220

  12. Eliciting expert knowledge in conservation science.

    PubMed

    Martin, Tara G; Burgman, Mark A; Fidler, Fiona; Kuhnert, Petra M; Low-Choy, Samantha; McBride, Marissa; Mengersen, Kerrie

    2012-02-01

    Expert knowledge is used widely in the science and practice of conservation because of the complexity of problems, relative lack of data, and the imminent nature of many conservation decisions. Expert knowledge is substantive information on a particular topic that is not widely known by others. An expert is someone who holds this knowledge and who is often deferred to in its interpretation. We refer to predictions by experts of what may happen in a particular context as expert judgments. In general, an expert-elicitation approach consists of five steps: deciding how information will be used, determining what to elicit, designing the elicitation process, performing the elicitation, and translating the elicited information into quantitative statements that can be used in a model or directly to make decisions. This last step is known as encoding. Some of the considerations in eliciting expert knowledge include determining how to work with multiple experts and how to combine multiple judgments, minimizing bias in the elicited information, and verifying the accuracy of expert information. We highlight structured elicitation techniques that, if adopted, will improve the accuracy and information content of expert judgment and ensure uncertainty is captured accurately. We suggest four aspects of an expert elicitation exercise be examined to determine its comprehensiveness and effectiveness: study design and context, elicitation design, elicitation method, and elicitation output. Just as the reliability of empirical data depends on the rigor with which it was acquired so too does that of expert knowledge. PMID:22280323

  13. Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis

    PubMed Central

    Young, Douglas B.; Comas, Iñaki; de Carvalho, Luiz P. S.

    2015-01-01

    Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms (SNPs) with predicted impact on protein function and transcriptional regulation. Differences in the B12 synthesis pathway, methionine biosynthesis, fatty acid catabolism, and DNA repair and replication are consistent with adaptations to different environmental niches and pathogenic lifestyles. While there is no evidence of further gene acquisition during expansion of the M. tuberculosis complex, the emergence of other forms of genetic diversity provides insights into continuing host-pathogen co-evolution and has the potential to identify novel targets for disease intervention. PMID:25988174

  14. High resolution melting analysis for the differentiation of Mycobacterium species.

    PubMed

    Issa, Rahizan; Abdul, Hatijah; Hashim, Siti Hasmah; Seradja, Valentinus H; Shaili, Nurul 'Aishah; Hassan, Nurul Akma Mohd

    2014-10-01

    A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species. PMID:25038139

  15. Mycobacterium ulcerans disease.

    PubMed Central

    van der Werf, Tjip S.; Stienstra, Ymkje; Johnson, R. Christian; Phillips, Richard; Adjei, Ohene; Fleischer, Bernhard; Wansbrough-Jones, Mark H.; Johnson, Paul D. R.; Portaels, Françoise; van der Graaf, Winette T. A.; Asiedu, Kingsley

    2005-01-01

    Mycobacterium ulcerans disease (Buruli ulcer) is an important health problem in several west African countries. It is prevalent in scattered foci around the world, predominantly in riverine areas with a humid, hot climate. We review the epidemiology, bacteriology, transmission, immunology, pathology, diagnosis and treatment of infections. M. ulcerans is an ubiquitous micro-organism and is harboured by fish, snails, and water insects. The mode of transmission is unknown. Lesions are most common on exposed parts of the body, particularly on the limbs. Spontaneous healing may occur. Many patients in endemic areas present late with advanced, severe lesions. BCG vaccination yields a limited, relatively short-lived, immune protection. Recommended treatment consists of surgical debridement, followed by skin grafting if necessary. Many patients have functional limitations after healing. Better understanding of disease transmission and pathogenesis is needed for improved control and prevention of Buruli ulcer. PMID:16283056

  16. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage

    PubMed Central

    Cañas, María-Alexandra; Giménez, Rosa; Fábrega, María-José; Toloza, Lorena; Badia, Josefa

    2016-01-01

    Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs) are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN) and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether colibactin is secreted

  17. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage.

    PubMed

    Cañas, María-Alexandra; Giménez, Rosa; Fábrega, María-José; Toloza, Lorena; Baldomà, Laura; Badia, Josefa

    2016-01-01

    Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs) are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN) and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether colibactin is secreted

  18. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants

    PubMed Central

    2004-01-01

    Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed. PMID:15560754

  19. Safety and Immunogenicity of the Mycobacterium tuberculosis {Delta}lysA {Delta}panCD Vaccine in Domestic Cats Infected with Feline Immunodeficiency Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feline immunodeficiency virus (FIV)+ and FIV- cats (n = 4/group) received 2 x 10**6 cfu Mycobacterium tuberculosis Delta-lysA Delta-panCD intramuscularly. Vaccination elicited antibody responses; albeit, at lower levels in FIV+ cats as compared to FIV- cats. Delayed-type hypersensitivity responses ...

  20. Advances in molecular diagnostics for Mycobacterium bovis.

    PubMed

    Collins, Desmond M

    2011-07-01

    The two most important molecular diagnostic techniques for bovine tuberculosis are the polymerase chain reaction (PCR) because of its rapid determination of infection, and DNA strain typing because of its ability to answer important epidemiological questions. PCR tests for Mycobacterium bovis have been improved through recent advances in PCR technology, but still lack the sensitivity of good culture methods, and in some situations are susceptible to giving both false negative and false positive results. Therefore, PCR does not usually replace the need for culture, but is used to provide fast preliminary results. DNA typing of M. bovis isolates by restriction endonuclease analysis (REA) was developed 25 years ago in New Zealand, and remains an important tool in the New Zealand control scheme, where the typing results are combined with other information to determine large and expensive possum poisoning operations. A range of other DNA typing systems developed for M. bovis in the 1990 s have assisted epidemiological investigations in some countries but are now less commonly used. Variable number tandem repeat (VNTR) typing and spoligotyping, either alone or together, have now become the preferred approaches as they are robust and amenable to electronic analysis and comparison. Spoligotyping gives only moderate discrimination but can be easily applied to large numbers of isolates, and VNTR typing provides better discrimination than all other methods except for REA. While the current typing techniques are sufficient for most epidemiological purposes, more discriminating methods are likely to become available in the near future. PMID:21420257

  1. Characterization of a Mycobacterium intracellulare Variant Strain by Molecular Techniques

    PubMed Central

    Menendez, M. C.; Palenque, E.; Navarro, M. C.; Nuñez, M. C.; Rebollo, M. J.; Garcia, M. J.

    2001-01-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members. PMID:11724827

  2. Vaccination of mice against Mycobacterium leprae infection.

    PubMed Central

    Singh, N B; Lowe, A C; Rees, R J; Colston, M J

    1989-01-01

    Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae. This protection is long lasting and systemic in that immunization in the left flank results in protection in both the left and right footpads. Immunization with Mycobacterium vaccae was ineffective in protecting mice against M. leprae infection, while Mycobacterium bovis BCG provided partial protection. Mycobacterium habana TMC 5135 (now known as Mycobacterium simiae) was found to be as effective as M. leprae in protecting mice against footpad infection. PMID:2643581

  3. PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

    PubMed Central

    Huard, Richard C.; de Oliveira Lazzarini, Luiz Claudio; Butler, W. Ray; van Soolingen, Dick; Ho, John L.

    2003-01-01

    The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use. PMID:12682155

  4. Endotoxin elicits ambivalent social behaviors.

    PubMed

    Yee, Jason R; Prendergast, Brian J

    2012-07-01

    The acute phase response to infection is reliably accompanied by decreases in social investigation; however, social behavior is commonly assayed in inescapable environments using unfamiliar social stimuli. In this experiment, male Wistar rats were raised from weaning with 2 familiar, same-sex conspecifics. In adulthood, rats were implanted with radiotelemetry devices that permitted localization in space, and were challenged with LPS treatments (150 μg/kg, i.p.) in a novel, semi-natural arena which afforded the treated (Focal) animal exclusive control of social exposure, and the ability to avoid social interactions. LPS reliably elicited thermoregulatory responses (transient hypothermia and fever) during the scotophase following injection, but did not yield changes in the proportion of time spent engaged in social interactions: both LPS- and saline-treated rats spent approximately 10% of the night with their familiar cagemates. Injection treatments markedly altered the spatial distribution of activity: LPS-treated rats exhibited significant increases in the amount of time spent as far as possible from their cagemates. The data suggest that sickness responses to LPS may give rise to a transient state of social ambivalence-characterized by a persistent motivation to engage in social contact, but also by increased avoidance of social environments. Selective maintenance of social motivation illustrates plasticity in the expression of sickness behaviors and may be adaptive in social species. PMID:22172640

  5. Comparative Genomic Analysis of Mycobacterium avium subspecies Obtained from Multiple Host Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium (M. avium) subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. p...

  6. Fatal Granulomatous Meningoencephalitis associated to Mycobacterium Mucogenicum-like Microorganism: a Case Report

    PubMed Central

    SARIOL, CARLOS A.; GALIB, YUSSEF; PANTOJA, PETRALEIGH; COLÓN, LILLIAN; GONZÁLEZ, ANARDA; TORMOS, LEE MARIE; SANTANA, JORGE; LUCIANO, CARLOS A.; GONZÁLEZ-MARTÍNEZ, JANIS; KRAISELBURD, EDMUNDO N.

    2010-01-01

    Mycobacterium mucogenicum is rarely associated to human infections. However, in the last year, a few reports of sepsis and fatal cases of central nervous systems have been documented. Here we report a fatal case of granulomatous meningoencephalitis of three weeks of evolution where DNA from a M. mucogenicum-like microorganism was identified post-mortem in samples of brain tissue. PMID:19715122

  7. Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens.

    PubMed

    Bang, Didi; Bengård Andersen, Ase; Thomsen, Vibeke Østergaard

    2006-07-01

    A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis. PMID:16825393

  8. Outbreak of Mycobacterium haemophilum infections after permanent makeup of the eyebrows.

    PubMed

    Giulieri, Stefano; Morisod, Benoit; Edney, Timothy; Odman, Micaela; Genné, Daniel; Malinverni, Raffaele; Hammann, Catherine; Musumeci, Enrico; Voide, Cathy; Greub, Gilbert; Masserey, Eric; Bille, Jacques; Cavassini, Matthias; Jaton, Katia

    2011-02-15

    We report a Mycobacterium haemophilum outbreak after permanent make-up of the eyebrows performed by the same freelance artist. Twelve patients presented an eyebrow lesion and cervical lymphadenitis. All were treated with antibiotics. Surgery was required in 10 cases. M. haemophilum DNA was identified in the make-up ink. PMID:21258102

  9. Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease

    PubMed Central

    Iakhiaeva, Elena; Williams, Myra D.; Brown-Elliott, Barbara A.; Vasireddy, Sruthi; Vasireddy, Ravikiran; Lande, Leah; Peterson, Donald D.; Sawicki, Janet; Kwait, Rebecca; Tichenor, Wellington S.; Turenne, Christine; Falkinham, Joseph O.

    2013-01-01

    Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC. PMID:23536397

  10. Recombinant BCG Expressing Mycobacterium ulcerans Ag85A Imparts Enhanced Protection against Experimental Buruli ulcer

    PubMed Central

    Hart, Bryan E.; Hale, Laura P.; Lee, Sunhee

    2015-01-01

    Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette–Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4+ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy. PMID:26393347

  11. Recombinant BCG Expressing Mycobacterium ulcerans Ag85A Imparts Enhanced Protection against Experimental Buruli ulcer.

    PubMed

    Hart, Bryan E; Hale, Laura P; Lee, Sunhee

    2015-09-01

    Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette-Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4+ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy. PMID:26393347

  12. Treatment of Mycobacterium abscessus Infection

    PubMed Central

    Beekmann, Susan E.; Polgreen, Philip M.; Mackey, Kate; Winthrop, Kevin L.

    2016-01-01

    Mycobacterium abscessus is often resistant to multiple antimicrobial drugs, and data supporting effective drugs or dosing regimens are limited. To better identify treatment approaches and associated toxicities, we collected a series of case reports from the Emerging Infections Network. Side effects were common and often led to changing or discontinuing therapy. PMID:26890211

  13. Insights into redox sensing metalloproteins in Mycobacterium tuberculosis.

    PubMed

    Chim, Nicholas; Johnson, Parker M; Goulding, Celia W

    2014-04-01

    Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels. PMID:24314844

  14. Recombinant Mycobacterium bovis BCG as an HIV vaccine vector.

    PubMed

    Chapman, Rosamund; Chege, Gerald; Shephard, Enid; Stutz, Helen; Williamson, Anna-Lise

    2010-06-01

    HIV-1 has resulted in a devastating AIDS pandemic. An effective HIV/AIDS vaccine that can be used to either, prevent HIV infection, control infection or prevent progression of the disease to AIDS is needed. In this review we discuss the use of Mycobacterium bovis BCG, the tuberculosis vaccine, as a vaccine vector for an HIV vaccine. Numerous features make BCG an attractive vehicle to deliver HIV antigens. It has a good safety profile, elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable, a necessary consideration for developing countries. In this review we discuss the numerous factors that influence generation of a genetically stable recombinant BCG vaccine for HIV. PMID:20353397

  15. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    PubMed Central

    Mirsamadi, ES; Farnia, P; Jahani Sherafat, S; Esfahani, M; Faramarzi, N

    2010-01-01

    Background and Objectives Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme. Materials and Methods DNA extraction was performed using CTAB (cetyltrimethylammonium bromide) from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping. Results Of 200 specimens, 19 (9.5%) were Beijing strain and 181 (90.5%) were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains (84.2%) were positive for plcA, 17 (89.4%) were positive for plcB and 17 (89.4%) were positive for plcC genes. The standard strain (H37RV) was used as control. Conclusion The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis. PMID:22347572

  16. The Mycobacterium avium complex.

    PubMed Central

    Inderlied, C B; Kemper, C A; Bermudez, L E

    1993-01-01

    Mycobacterium avium complex (MAC) disease emerged early in the epidemic of AIDS as one of the common opportunistic infections afflicting human immunodeficiency virus-infected patients. However, only over the past few years has a consensus developed about its significance to the morbidity and mortality of AIDS. M. avium was well known to mycobacteriologists decades before AIDS, and the MAC was known to cause disease, albeit uncommon, in humans and animals. The early interest in the MAC provided a basis for an explosion of studies over the past 10 years largely in response to the role of the MAC in AIDS opportunistic infection. Molecular techniques have been applied to the epidemiology of MAC disease as well as to a better understanding of the genetics of antimicrobial resistance. The interaction of the MAC with the immune system is complex, and putative MAC virulence factors appear to have a direct effect on the components of cellular immunity, including the regulation of cytokine expression and function. There now is compelling evidence that disseminated MAC disease in humans contributes to both a decrease in the quality of life and survival. Disseminated disease most commonly develops late in the course of AIDS as the CD4 cells are depleted below a critical threshold, but new therapies for prophylaxis and treatment offer considerable promise. These new therapeutic modalities are likely to be useful in the treatment of other forms of MAC disease in patients without AIDS. The laboratory diagnosis of MAC disease has focused on the detection of mycobacteria in the blood and tissues, and although the existing methods are largely adequate, there is need for improvement. Indeed, the successful treatment of MAC disease clearly will require an early and rapid detection of the MAC in clinical specimens long before the establishment of the characteristic overwhelming infection of bone marrow, liver, spleen, and other tissue. Also, a standard method of susceptibility testing

  17. Support from Phylogenomic Networks and Subspecies Signatures for Separation of Mycobacterium massiliense from Mycobacterium bolletii

    PubMed Central

    Tan, Joon Liang; Choo, Siew Woh

    2015-01-01

    Mycobacterium abscessus subspecies classification has important clinical implications. We used phylogenomic network and amino acid analyses to provide evidence for the separation of Mycobacterium bolletii and Mycobacterium massiliense into two distinct subspecies which can potentially be differentiated rapidly by their protein signatures. PMID:26157149

  18. Direct identification and typing of Mycobacterium tuberculosis by PCR.

    PubMed Central

    Neimark, H; Ali Baig, M; Carleton, S

    1996-01-01

    We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints. PMID:8880499

  19. Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa.

    PubMed

    Singh, Bhupender; Nitharwal, Ram Gopal; Ramesh, Malavika; Pettersson, B M Fredrik; Kirsebom, Leif A; Dasgupta, Santanu

    2013-04-01

    Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp. PMID:23387305

  20. A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings.

    PubMed

    Laukkanen, H; Soini, H; Kontunen-Soppela, S; Hohtola, A; Viljanen, M

    2000-07-01

    We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.). The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture. Growth of Scots pine cells had ceased in all of these cultures. Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales. Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species. Its sequence most closely resembled those of Mycobacterium obuense (97%) and M. aichiense (96%). Tissue browning was frequently observed in callus or suspension culture of Scots pine. Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions. Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants. However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M. chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium. Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M. chlorophenolicum or Pseudomotnas fluorescens. We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree. Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study. PMID:11303582

  1. [Coinfection of Mycobacterium malmoense and Mycobacterium tuberculosis in a patient with acquired inmune deficiency syndrome].

    PubMed

    Mederos Cuervo, Lilian María; Reyes Pérez, Angélica; Valdes Alonso, Lidunka; Rodríguez Delgado, Francisco; Sardiñas Aragón, Misleydis; Martínez Romero, María Rosarys; Díaz Romero, Raúl

    2014-01-01

    A case is presented of coinfection with Mycobacterium malmoense and Mycobacterium tuberculosis in a Cuban patient with AIDS which produced respiratory and liver disease respectively. Cultures done from sputum samples showed the presence of a non-pigmented, slow growing mycobacterial strain belonging to Runyon group III and identified as Mycobacterium malmoense. From cultures of liver tissue removed laparoscopically, a strain was isolated and subsequently identified as Mycobacterium tuberculosis. Anatomapathologic examination confirmed the diagnosis of tuberculosis, the patient received specific treatment and had a favorable clinical course. This report of a rare case of coinfection of Mycobacterium describes the first report of hepatic tuberculosis in a patient with AIDS in Cuba. PMID:25597735

  2. Co-infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy

    PubMed Central

    Donoghue, Helen D.; Marcsik, Antónia; Matheson, Carney; Vernon, Kim; Nuorala, Emilia; Molto, Joseph E.; Greenblatt, Charles L.; Spigelman, Mark

    2005-01-01

    Both leprosy and tuberculosis were prevalent in Europe during the first millennium but thereafter leprosy declined. It is not known why this occurred, but one suggestion is that cross-immunity protected tuberculosis patients from leprosy. To investigate any relationship between the two diseases, selected archaeological samples, dating from the Roman period to the thirteenth century, were examined for both Mycobacterium leprae and Mycobacterium tuberculosis DNA, using PCR. The work was carried out and verified in geographically separate and independent laboratories. Several specimens with palaeopathological signs of leprosy were found to contain DNA from both pathogens, indicating that these diseases coexisted in the past. We suggest that the immunological changes found in multi-bacillary leprosy, in association with the socio-economic impact on those suffering from the disease, led to increased mortality from tuberculosis and therefore to the historical decline in leprosy. PMID:15734693

  3. MycoCAP - Mycobacterium Comparative Analysis Platform.

    PubMed

    Choo, Siew Woh; Ang, Mia Yang; Dutta, Avirup; Tan, Shi Yang; Siow, Cheuk Chuen; Heydari, Hamed; Mutha, Naresh V R; Wee, Wei Yee; Wong, Guat Jah

    2015-01-01

    Mycobacterium spp. are renowned for being the causative agent of diseases like leprosy, Buruli ulcer and tuberculosis in human beings. With more and more mycobacterial genomes being sequenced, any knowledge generated from comparative genomic analysis would provide better insights into the biology, evolution, phylogeny and pathogenicity of this genus, thus helping in better management of diseases caused by Mycobacterium spp.With this motivation, we constructed MycoCAP, a new comparative analysis platform dedicated to the important genus Mycobacterium. This platform currently provides information of 2108 genome sequences of at least 55 Mycobacterium spp. A number of intuitive web-based tools have been integrated in MycoCAP particularly for comparative analysis including the PGC tool for comparison between two genomes, PathoProT for comparing the virulence genes among the Mycobacterium strains and the SuperClassification tool for the phylogenic classification of the Mycobacterium strains and a specialized classification system for strains of Mycobacterium abscessus. We hope the broad range of functions and easy-to-use tools provided in MycoCAP makes it an invaluable analysis platform to speed up the research discovery on mycobacteria for researchers. Database URL: http://mycobacterium.um.edu.my. PMID:26666970

  4. MycoCAP - Mycobacterium Comparative Analysis Platform

    PubMed Central

    Choo, Siew Woh; Ang, Mia Yang; Dutta, Avirup; Tan, Shi Yang; Siow, Cheuk Chuen; Heydari, Hamed; Mutha, Naresh V. R.; Wee, Wei Yee; Wong, Guat Jah

    2015-01-01

    Mycobacterium spp. are renowned for being the causative agent of diseases like leprosy, Buruli ulcer and tuberculosis in human beings. With more and more mycobacterial genomes being sequenced, any knowledge generated from comparative genomic analysis would provide better insights into the biology, evolution, phylogeny and pathogenicity of this genus, thus helping in better management of diseases caused by Mycobacterium spp.With this motivation, we constructed MycoCAP, a new comparative analysis platform dedicated to the important genus Mycobacterium. This platform currently provides information of 2108 genome sequences of at least 55 Mycobacterium spp. A number of intuitive web-based tools have been integrated in MycoCAP particularly for comparative analysis including the PGC tool for comparison between two genomes, PathoProT for comparing the virulence genes among the Mycobacterium strains and the SuperClassification tool for the phylogenic classification of the Mycobacterium strains and a specialized classification system for strains of Mycobacterium abscessus. We hope the broad range of functions and easy-to-use tools provided in MycoCAP makes it an invaluable analysis platform to speed up the research discovery on mycobacteria for researchers. Database URL: http://mycobacterium.um.edu.my PMID:26666970

  5. Strategies for Eliciting HIV-1 Inhibitory Antibodies

    PubMed Central

    Tomaras, Georgia D.; Haynes, Barton F.

    2012-01-01

    Purpose of review Major roadblocks persist in the development of vaccines that elicit potent neutralizing antibodies targeting diverse HIV-1 strains, similar to known broadly neutralizing HIV-1 human monoclonal antibodies. Alternatively, other types of anti-HIV-1 envelope antibodies that may not neutralize HIV-1 in traditional neutralization assays but have other anti-HIV-1 activities (hereafter termed HIV-1 inhibitory antibodies) can be elicited by current vaccine strategies, and numerous studies are exploring their roles in preventing HIV-1 acquisition. We review examples of strategies for eliciting potentially protective HIV-1 inhibitory antibodies. Recent Findings Heterologous prime-boost strategies can yield anti-HIV immune responses; although only one (canarypox prime, Env protein boost) has been tested and shown positive results in an efficacy trial (RV144). Although the immune correlates of protection are as yet undefined, the reduced rate of acquisition without a significant effect on initial viral loads or CD4+ T cell counts, have raised the hypothesis of an RV144 vaccine-elicited transient protective B cell response. Summary In light of the RV144 trial, there is a critical need to define the entire functional spectrum of anti-HIV-1 antibodies, how easily each can be elicited, and how effective different types of antibody effector mechanisms can be in prevention of HIV-1 transmission. PMID:20978384

  6. Stereoscopy Amplifies Emotions Elicited by Facial Expressions

    PubMed Central

    Kätsyri, Jari; Häkkinen, Jukka

    2015-01-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants’ gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15–65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions. PMID:27551358

  7. Stereoscopy Amplifies Emotions Elicited by Facial Expressions.

    PubMed

    Hakala, Jussi; Kätsyri, Jari; Häkkinen, Jukka

    2015-12-01

    Mediated facial expressions do not elicit emotions as strongly as real-life facial expressions, possibly due to the low fidelity of pictorial presentations in typical mediation technologies. In the present study, we investigated the extent to which stereoscopy amplifies emotions elicited by images of neutral, angry, and happy facial expressions. The emotional self-reports of positive and negative valence (which were evaluated separately) and arousal of 40 participants were recorded. The magnitude of perceived depth in the stereoscopic images was manipulated by varying the camera base at 15, 40, 65, 90, and 115 mm. The analyses controlled for participants' gender, gender match, emotional empathy, and trait alexithymia. The results indicated that stereoscopy significantly amplified the negative valence and arousal elicited by angry expressions at the most natural (65 mm) camera base, whereas stereoscopy amplified the positive valence elicited by happy expressions in both the narrowed and most natural (15-65 mm) base conditions. Overall, the results indicate that stereoscopy amplifies the emotions elicited by mediated emotional facial expressions when the depth geometry is close to natural. The findings highlight the sensitivity of the visual system to depth and its effect on emotions. PMID:27551358

  8. The 65-kilodalton antigen of Mycobacterium tuberculosis.

    PubMed Central

    Shinnick, T M

    1987-01-01

    The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence. Images PMID:3029018

  9. Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

    PubMed Central

    Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M.

    2015-01-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses. PMID:25588660

  10. Mycobacterium massiliense Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth

    PubMed Central

    Yoon, Yina; Na, Yirang; Kim, Bum-Joon; Seok, Seung Hyeok

    2016-01-01

    Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against Mycobacterium massiliense (M. mass) has yet to be described. In this study, we show that M. mass, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, M. mass-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap M. mass and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of M. mass with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against M. mass infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria. PMID:27191593

  11. Microbial metabolism of polycyclic aromatic hydrocarbons: Isolation and characterization of a pyrene-degrading bacterium. [Mycobacterium sp

    SciTech Connect

    Heitkamp, M.A.; Franklin, W.; Cerniglia, C.E. )

    1988-10-01

    Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G+C content of the DNA, and high-molecular-weight mycolic acids (C{sub 58} to C{sub 64}). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30{degree}C and minimally at 35{degree}C. No growth was observed at 5 or 42{degree}C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl- and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.

  12. Amplicon DNA Melting Analysis for the Simultaneous Detection of Brucella spp and Mycobacterium tuberculosis Complex. Potential Use in Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis

    PubMed Central

    Sanjuan-Jimenez, Rocio; Colmenero, Juan D.; Bermúdez, Pilar; Alonso, Antonio; Morata, Pilar

    2013-01-01

    Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a

  13. Rational elicitation of cold-sensitive phenotypes.

    PubMed

    Baliga, Chetana; Majhi, Sandipan; Mondal, Kajari; Bhattacharjee, Antara; VijayRaghavan, K; Varadarajan, Raghavan

    2016-05-01

    Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes. PMID:27091994

  14. Experimental Validation of a Nested Polymerase Chain Reaction Targeting the Genetic Element ISMAP02 for Detection of Mycobacterium avium subspecies paratuberculosis in Bovine Colostrum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP) (strain K-10) at increasing concentrations between 1×10**1 and 1× 10**9 cells/mL were tested for recovery of MAP DNA using a modified nested ISMAP02 target PCR initially developed for detecting MAP DNA...

  15. Comparative Genetic Analysis of Mycobacterium ulcerans and Mycobacterium marinum Reveals Evidence of Recent Divergence

    PubMed Central

    Stinear, Timothy P.; Jenkin, Grant A.; Johnson, Paul D. R.; Davies, John K.

    2000-01-01

    Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a

  16. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

    PubMed Central

    Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Soares, Paulo Martins; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Ângela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

    2013-01-01

    In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials. PMID:23778657

  17. Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice

    PubMed Central

    Kim, Jong-Seok; Kim, Hongmin; Kwon, Kee Woong; Han, Seung Jung; Eum, Seok-Yong; Cho, Sang-Nae; Shin, Sung Jae

    2015-01-01

    Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner. PMID:26509812

  18. Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice.

    PubMed

    Cha, Seung Bin; Kim, Woo Sik; Kim, Jong-Seok; Kim, Hongmin; Kwon, Kee Woong; Han, Seung Jung; Eum, Seok-Yong; Cho, Sang-Nae; Shin, Sung Jae

    2015-01-01

    Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner. PMID:26509812

  19. Commercial DNA Probes for Mycobacteria Incorrectly Identify a Number of Less Frequently Encountered Species▿ †

    PubMed Central

    Tortoli, Enrico; Pecorari, Monica; Fabio, Giuliana; Messinò, Massimino; Fabio, Anna

    2010-01-01

    Although commercially available DNA probes for identification of mycobacteria have been investigated with large numbers of strains, nothing is known about the ability of these probes to identify less frequently encountered species. We analyzed, with INNO LiPA MYCOBACTERIA (Innogenetics) and with GenoType Mycobacterium (Hein), 317 strains, belonging to 136 species, 61 of which had never been assayed before. INNO LiPA misidentified 20 taxa, the majority of which cross-reacted with the probes specific for Mycobacterium fortuitum and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. GenoType misidentified 28 taxa, most of which cross-reacted with M. intracellulare and M. fortuitum probes; furthermore, eight species were not recognized as members of the genus Mycobacterium. Among 54 strains investigated with AccuProbe (Gen-Probe), cross-reactions were detected for nine species, with the probes aiming at the M. avium complex being most involved in cross-reactions. PMID:19906898

  20. Macrophage Transcriptional Response to Species-Adapted Mycobacterium avium subsp. Paratuberculosis Isolates: The Role of Pathogen Genotype in Host Response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transcriptional response of human and bovine macrophages to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) isolates from cattle and sheep were examined using DNA microarrays. M. paratuberculosis is the etiologic agent of Johne’s Disease, a chronic infection of ruminant anima...

  1. Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissue Using IS6110 Real-Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Current accepted methods of detecting Mycobacterium bovis (M. bovis) in fresh tissues requires months for definitive culture results. It has been proposed that direct detection of M. bovis DNA in fresh tissues could provide more rapid results. These data would complement the current me...

  2. Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium

    PubMed Central

    Jeevarajah, Dharshini; Patterson, John H.; Taig, Ellen; Sargeant, Tobias; McConville, Malcolm J.; Billman-Jacobe, Helen

    2004-01-01

    Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis. PMID:15466031

  3. Eliciting User Requirements Using Appreciative Inquiry

    ERIC Educational Resources Information Center

    Gonzales, Carol Kernitzki

    2010-01-01

    Many software development projects fail because they do not meet the needs of users, are over-budget, and abandoned. To address this problem, the user requirements elicitation process was modified based on principles of Appreciative Inquiry. Appreciative Inquiry, commonly used in organizational development, aims to build organizations, processes,…

  4. Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex.

    PubMed

    Huard, Richard C; Fabre, Michel; de Haas, Petra; Lazzarini, Luiz Claudio Oliveira; van Soolingen, Dick; Cousins, Debby; Ho, John L

    2006-06-01

    In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, long-sequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n = 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for "Mycobacterium canettii," Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC. PMID:16740934

  5. LinkIT: a ludic elicitation game for eliciting risk perceptions.

    PubMed

    Cao, Yan; McGill, William L

    2013-06-01

    The mental models approach, a leading strategy to develop risk communications, involves a time- and labor-intensive interview process and a lengthy questionnaire to elicit group-level risk perceptions. We propose that a similarity ratings approach for structural knowledge elicitation can be adopted to assist the risk mental models approach. The LinkIT game, inspired by games with a purpose (GWAP) technology, is a ludic elicitation tool designed to elicit group understanding of the relations between risk factors in a more enjoyable and productive manner when compared to traditional approaches. That is, consistent with the idea of ludic elicitation, LinkIT was designed to make the elicitation process fun and enjoyable in the hopes of increasing participation and data quality in risk studies. Like the mental models approach, the group mental model obtained via the LinkIT game can hence be generated and represented in a form of influence diagrams. In order to examine the external validity of LinkIT, we conducted a study to compare its performance with respect to a more conventional questionnaire-driven approach. Data analysis results conclude that the two group mental models elicited from the two approaches are similar to an extent. Yet, LinkIT was more productive and enjoyable than the questionnaire. However, participants commented that the current game has some usability concerns. This presentation summarizes the design and evaluation of the LinkIT game and suggests areas for future work. PMID:23078149

  6. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

    PubMed Central

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D.; Pereira, Luiz Cezar M.; Silva, Ita de Oliveira e; Ruiz-Miranda, Carlos R.; Truman, Richard; Stone, Anne C.

    2015-01-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts. PMID:26571269

  7. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    PubMed

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D; Pereira, Luiz Cezar M; Silva, Ita de Oliveira; Ruiz-Miranda, Carlos R; Truman, Richard; Stone, Anne C

    2015-11-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts. PMID:26571269

  8. Vertebral osteomyelitis caused by Mycobacterium abscessus

    PubMed Central

    Garcia, Daniel C; Sandoval-Sus, Jose; Razzaq, Kanwal; Young, Lary

    2013-01-01

    Mycobacterium infection caused by non-tuberculous mycobacterial (NTBM) organisms is becoming more common. Although NTBM osteomyelitis is unusual, it can occur in otherwise healthy individuals, but it is also associated with immunocompromised states, such as steroidal therapy and AIDS, and may be observed following trauma. Mycobacterium avium is reported to be the most common causative agent, and Mycobacterium abscessus has only been described in two cases. We report the case of a 44-year-old man with history of hepatitis C diagnosed with osteomyelitis of the thoracic spine caused by M abscessus. We present a literature review of NTBM osteomyelitis and a discussion of its diagnosis and treatment. PMID:23925676

  9. Mycobacterium arupense in Cancer Patients

    PubMed Central

    Al Hamal, Zainab; Jordan, Mary; Hachem, Ray Y.; Alawami, Hussain M.; Alburki, Abdussalam M.; Yousif, Ammar; Deshmukh, Poonam; Jiang, Ying; Chaftari, Ann-Marie; Raad, Issam I.

    2016-01-01

    Abstract Mycobacterium arupense is a slow-growing, nonchromogenic, acid-fast bacillus. Its clinical spectrum, epidemiology, and frequency of colonization versus true infection remain unknown. We evaluated the clinical significance of M arupense and positive cultures from cancer patients. We retrospectively reviewed records of all cancer patients treated at our institution between 2007 and 2014 to identify those who had positive cultures for M arupense. Mycobacterium arupense was identified by sequencing the 16S rRNA and hsp65 genes. A total of 53patients had positive cultures, 100% of which were isolated from respiratory specimens. Of these, 7 patients met the American Thoracic Society/Infectious Diseases Society of America criteria for a definitive diagnosis of M arupense infection, 14 cases were considered to be probable infections, and 29 cases were considered to be possible infections. Of the included patients, 13 received therapy for M arupense infection and 40 did not. The outcomes of treated and untreated patients did not differ significantly. No relapses of M arupense infection. In addition, there were no M arupense-related deaths in either group. In cancer patients, M arupense appears to be mostly a commensal organism rather than a pathogen. Patients who did or did not receive treatment had similar outcomes. Validation of these findings in a larger prospective trial is warranted. PMID:27057825

  10. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native