Science.gov

Sample records for host cell factors

  1. Host Cell Factors in Filovirus Entry: Novel Players, New Insights

    PubMed Central

    Hofmann-Winkler, Heike; Kaup, Franziska; Pöhlmann, Stefan

    2012-01-01

    Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism. PMID:23342362

  2. Host cell factors as antiviral targets in arenavirus infection.

    PubMed

    Linero, Florencia N; Sepúlveda, Claudia S; Giovannoni, Federico; Castilla, Viviana; García, Cybele C; Scolaro, Luis A; Damonte, Elsa B

    2012-09-01

    Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.

  3. Hierarchies of Host Factor Dynamics at the Entry Site of Shigella flexneri during Host Cell Invasion

    PubMed Central

    Ehsani, Soudeh; Santos, José Carlos; Rodrigues, Cristina D.; Henriques, Ricardo; Audry, Laurent; Zimmer, Christophe; Sansonetti, Philippe; Tran Van Nhieu, Guy

    2012-01-01

    Shigella flexneri, the causative agent of bacillary dysentery, induces massive cytoskeletal rearrangement, resulting in its entry into nonphagocytic epithelial cells. The bacterium-engulfing membrane ruffles are formed by polymerizing actin, a process activated through injected bacterial effectors that target host small GTPases and tyrosine kinases. Once inside the host cell, S. flexneri escapes from the endocytic vacuole within minutes to move intra- and intercellularly. We quantified the fluorescence signals from fluorescently tagged host factors that are recruited to the site of pathogen entry and vacuolar escape. Quantitative time lapse fluorescence imaging revealed simultaneous recruitment of polymerizing actin, small GTPases of the Rho family, and tyrosine kinases. In contrast, we found that actin surrounding the vacuole containing bacteria dispersed first from the disassembling membranes, whereas other host factors remained colocalized with the membrane remnants. Furthermore, we found that the disassembly of the membrane remnants took place rapidly, within minutes after bacterial release into the cytoplasm. Superresolution visualization of galectin 3 through photoactivated localization microscopy characterized these remnants as small, specular, patchy structures between 30 and 300 nm in diameter. Using our experimental setup to track the time course of infection, we identified the S. flexneri effector IpgB1 as an accelerator of the infection pace, specifically targeting the entry step, but not vacuolar progression or escape. Together, our studies show that bacterial entry into host cells follows precise kinetics and that this time course can be targeted by the pathogen. PMID:22526677

  4. Emerging functions as host cell factors - an encyclopedia of annexin-pathogen interactions.

    PubMed

    Kuehnl, Alexander; Musiol, Agnes; Raabe, Carsten A; Rescher, Ursula

    2016-10-01

    Emerging infectious diseases and drug-resistant infectious agents call for the development of innovative antimicrobial strategies. With pathogenicity now considered to arise from the complex and bi-directional interplay between a microbe and the host, host cell factor targeting has emerged as a promising approach that might overcome the limitations of classical antimicrobial drug development and could open up novel and efficient therapeutic strategies. Interaction with and modulation of host cell membranes is a recurrent theme in the host-microbe relationship. In this review, we provide an overview of what is currently known about the role of the Ca2+ dependent, membrane-binding annexin protein family in pathogen-host interactions, and discuss their emerging functions as host cell derived auxiliary proteins in microbe-host interactions and host cell targets.

  5. Cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells.

    PubMed

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-04-01

    Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are "cyclomodulins" that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

  6. Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

    PubMed Central

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-01-01

    Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions. PMID:22069713

  7. Platelet-activating Factor Receptor Initiates Contact of Acinetobacter baumannii Expressing Phosphorylcholine with Host Cells

    PubMed Central

    Smani, Younes; Docobo-Pérez, Fernando; López-Rojas, Rafael; Domínguez-Herrera, Juan; Ibáñez-Martínez, José; Pachón, Jerónimo

    2012-01-01

    Adhesion is an initial and important step in Acinetobacter baumannii causing infections. However, the exact molecular mechanism of such a step between A. baumannii and the host cells remains unclear. Here, we demonstrated that the phosphorylcholine (ChoP)-containing outer membrane protein of A. baumannii binds to A549 cells through platelet-activating factor receptor (PAFR), resulting in activation of G protein and intracellular calcium. Upon A. baumannii expressing ChoP binding to PAFR, clathrin and β-arrestins, proteins involved in the direction of the vacuolar movement, are activated during invasion of A. baumannii. PAFR antagonism restricts the dissemination of A. baumannii in the pneumonia model. These results define a role for PAFR in A. baumannii interaction with host cells and suggest a mechanism for the entry of A. baumannii into the cytoplasm of host cells. PMID:22689572

  8. Stress, Coping, and Infectious Illness: Persistently Low Natural Killer Cell Activity as a Host Risk Factor.

    DTIC Science & Technology

    1988-03-14

    Persistently Low Natural Killer Cell Activity as a Host Risk Factor " 12 PERSONAL AUTHOR(S) Sandra M. Levy, Ph.D., Ronald B. Herberman, M.D., Theresa...killer (NK) cell activity. In this prevbus work, a subgroup of individuals characterized by persistently low NK activity, and self-reported depression and... depression and chronic anxiety. In a very preliminary fashion, we have also found a trend of association between this low NK activity pattern, and some

  9. Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA.

    PubMed

    Upadhyay, Alok; Dixit, Updesh; Manvar, Dinesh; Chaturvedi, Nootan; Pandey, Virendra N

    2013-06-01

    Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.

  10. Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells.

    PubMed

    Sachanonta, Navakanit; Medana, Isabelle M; Roberts, Rachel; Jones, Margaret; Day, Nicholas P J; White, Nicholas J; Ferguson, David J P; Turner, Gareth D H; Pongponratn, Emsri

    2008-03-01

    Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

  11. Host cell factors in HIV replication: meta-analysis of genome-wide studies.

    PubMed

    Bushman, Frederic D; Malani, Nirav; Fernandes, Jason; D'Orso, Iván; Cagney, Gerard; Diamond, Tracy L; Zhou, Honglin; Hazuda, Daria J; Espeseth, Amy S; König, Renate; Bandyopadhyay, Sourav; Ideker, Trey; Goff, Stephen P; Krogan, Nevan J; Frankel, Alan D; Young, John A T; Chanda, Sumit K

    2009-05-01

    We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an "encyclopedia" of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

  12. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  13. Analysis of functional domains of the host cell factor involved in VP16 complex formation.

    PubMed

    Hughes, T A; La Boissière, S; O'Hare, P

    1999-06-04

    We present biochemical analyses of the regions of the host cell factor (HCF) involved in VP16 complex formation and in the association between the N- and C-terminal domains of HCF itself. We show that the kelch repeat region of HCF (residues 1-380) is sufficient for VP16 complex formation, but that residues C-terminal to the repeats (positions 381-450) interfere with this activity. However, these latter residues are required for the interaction between the N- and C-terminal regions of HCF. The extreme C-terminal region of HCF, corresponding to an area of strong conservation with a Caenorhabditis elegans homologue, is sufficient for interaction with the N-terminal region. These results are discussed with respect to possible differences in the roles of HCF in VP16 activity versus its normal cellular function.

  14. Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

    PubMed

    Lenarcic, Erik M; Landry, Dori M; Greco, Todd M; Cristea, Ileana M; Thompson, Sunnie R

    2013-08-01

    Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.

  15. Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification

    PubMed Central

    Lenarcic, Erik M.; Landry, Dori M.; Greco, Todd M.; Cristea, Ileana M.

    2013-01-01

    Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses. PMID:23740976

  16. A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors.

    PubMed

    Kondorosi, Eva; Mergaert, Peter; Kereszt, Attila

    2013-01-01

    Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.

  17. Identification of a Transcription Factor That Regulates Host Cell Exit and Virulence of Mycobacterium tuberculosis

    PubMed Central

    Srinivasan, Lalitha; Gurses, Serdar A.; Hurley, Benjamin E.; Miller, Jessica L.; Karakousis, Petros C.; Briken, Volker

    2016-01-01

    The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb. PMID:27191591

  18. Human heme oxygenase 1 is a potential host cell factor against dengue virus replication

    PubMed Central

    Tseng, Chin-Kai; Lin, Chun-Kuang; Wu, Yu-Hsuan; Chen, Yen-Hsu; Chen, Wei-Chun; Young, Kung-Chia; Lee, Jin-Ching

    2016-01-01

    Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 μM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice’s brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection. PMID:27553177

  19. M062 is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host SAMD9 in human cells.

    PubMed

    Liu, Jia; Wennier, Sonia; Zhang, Leiliang; McFadden, Grant

    2011-04-01

    Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.

  20. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    PubMed Central

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

    2015-01-01

    ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and

  1. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

    PubMed Central

    Viktorovskaya, Olga V.; Greco, Todd M.; Cristea, Ileana M.; Thompson, Sunnie R.

    2016-01-01

    Background There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. Methodology/Principal Findings Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. Conclusions/Significance The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with

  2. Viral and Host Factors Required for Avian H5N1 Influenza A Virus Replication in Mammalian Cells

    PubMed Central

    Zhang, Hong; Hale, Benjamin G.; Xu, Ke; Sun, Bing

    2013-01-01

    Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions. PMID:23752648

  3. Host factors in childhood diarrhoea.

    PubMed

    Khan, M A; Yunus, M

    1990-06-01

    Host factors play a significant role in children under five who are the worst sufferers of this widespread and global scourge, both in regard to mortality and morbidity. This study deals with some of the host factors in relation to diarrhoea in a rural population. Age was found to have a definite and direct relationship to diarrhoea. Male children were effected more. Poor hygiene, malnutrition and the receipt of supplementary feeds, were found to have a significant association with childhood diarrhoeas.

  4. Dendritic Cell-Lymphocyte Cross Talk Downregulates Host Restriction Factor SAMHD1 and Stimulates HIV-1 Replication in Dendritic Cells

    PubMed Central

    Biedma, Marina Elizabeth; Lederle, Alexandre; Peressin, Maryse; Lambotin, Mélanie; Proust, Alizé; Decoville, Thomas; Schmidt, Sylvie; Laumond, Géraldine

    2014-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-α]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture. IMPORTANCE SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DCs and lymphocytes mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with downregulation of SAMHD1 expression and activation of innate immune sensing by DCs. We propose that DC-lymphocyte cross talk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing. PMID:24574390

  5. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  6. Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol▿

    PubMed Central

    Alonzo, Francis; Freitag, Nancy E.

    2010-01-01

    In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection. PMID:20823208

  7. Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection.

    PubMed

    Covarrubias, Charles; Cortez, Mauro; Ferreira, Daniele; Yoshida, Nobuko

    2007-12-01

    Outbreaks of severe acute Chagas' disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas' disease that has been reported in outbreaks of oral T. cruzi infection.

  8. Binding of Host Factors Influences Internalization and Intracellular Trafficking of Streptococcus uberis in Bovine Mammary Epithelial Cells

    PubMed Central

    Almeida, Raul A.; Dunlap, John R.; Oliver, Stephen P.

    2010-01-01

    We showed that internalization of Streptococcus uberis into bovine mammary epithelial cells occurred through receptor- (RME) and caveolae-mediated endocytosis (CME). We reported also that treatment of S. uberis with host proteins including lactoferrin (LF) enhanced its internalization into host cells. Since the underlying mechanism(s) involved in such enhancement was unknown we investigated if preincubation of S. uberis with host proteins drives internalization of this pathogen into host cells through CME. Thus, experiments involving coculture of collagen-, fibronectin-, and LF-pretreated S. uberis with bovine mammary epithelial cells treated with RME and CME inhibitors were conducted. Results showed that internalization of host proteins-pretreated S. uberis into mammary epithelial cells treated with RME inhibitors was higher than that of untreated controls. These results suggest that pretreatment with selected host proteins commits S. uberis to CME, thus avoiding intracellular bactericidal mechanisms and allowing its persistence into bovine mammary epithelial cells. PMID:20614000

  9. GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

    PubMed

    Nain, Minu; Mukherjee, Sriparna; Karmakar, Sonali Porey; Paton, Adrienne W; Paton, James C; Abdin, M Z; Basu, Anirban; Kalia, Manjula; Vrati, Sudhanshu

    2017-03-15

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric

  10. Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells

    SciTech Connect

    Wang, Bei; Zhang, XiaoYu; Zhao, Zhendong

    2013-08-02

    Highlights: •We introduced a new mutagenesis strategy named VBIM to the viral research. •This method can identify either host factors or host restriction factors. •Using VBIM system, we identified Nup214 as a host factor for EV71 replication in RD cells. -- Abstract: Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.

  11. Host factor PRPF31 is involved in cccDNA production in HBV-replicating cells.

    PubMed

    Kinoshita, Wataru; Ogura, Naoki; Watashi, Koichi; Wakita, Takaji

    2017-01-22

    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection and replication, and is an important factor for HBV surface antigen loss indicating the endpoint of HBV treatment. However, there is a known problem that current anti-HBV drugs, including interferons and nucleos(t)ide analogues, reduce HBV replication but have a little or no effect on reducing cccDNA. Therefore, the development of new therapeutic agents is necessary to eradicate cccDNA. In this study, we identified pre-mRNA processing factor 31 (PRPF31) by siRNA screening as a factor associated with cccDNA. PRPF31 knockdown by siRNA decreased cccDNA formation without serious cytotoxicity. In rescue experiments, expression of siRNA-resistant PRPF31 recovered cccDNA formation. PRPF31 knockdown did not affect HBV core protein and HBV core DNA levels in HBV-replicating cells. Chromatin immunoprecipitation and immunoprecipitation assays revealed an association between PRPF31 and cccDNA. Furthermore, co-overexpression of PRPF31 and HBx enhanced cccDNA formation in HepAD38 cells. Taken together, the present findings suggest that the interaction between PRPF31 and HBx may be a novel target for anti-HBV treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

    PubMed Central

    Tsang, Sabrina H.; Wang, Xin; Li, Jing; Buck, Christopher B.

    2014-01-01

    ABSTRACT Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. IMPORTANCE MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR

  13. The Host Cell Transcription Factor EGR1 Is Induced by Bacteria through the EGFR–ERK1/2 Pathway

    PubMed Central

    de Klerk, Nele; Saroj, Sunil D.; Wassing, Gabriela M.; Maudsdotter, Lisa; Jonsson, Ann-Beth

    2017-01-01

    The essential first step in bacterial colonization is adhesion to the host epithelial cells. The early host-responses post-bacterial adhesions are still poorly understood. Early growth response 1 (EGR1) is an early response transcriptional regulator that can be rapidly induced by various environmental stimuli. Several bacteria can induce EGR1 expression in host cells, but the involved bacterial characteristics and the underlying molecular mechanisms of this response are largely unknown. Here, we show that EGR1 can be induced in host epithelial cells by different species of bacteria independent of the adherence level, Gram-staining type and pathogenicity. However, bacterial viability and contact with host cells is necessary, indicating that an active interaction between bacteria and the host is important. Furthermore, the strongest response is observed in cells originating from the natural site of the infection, suggesting that the EGR1 induction is cell type specific. Finally, we show that EGFR–ERK1/2 and β1-integrin signaling are the main pathways used for bacteria-mediated EGR1 upregulation. In conclusion, the increase of EGR1 expression in epithelial cells is a common stress induced, cell type specific response upon host-bacteria interaction that is mediated by EGFR–ERK1/2 and β1-integrin signaling. PMID:28180113

  14. Nuclear Localization of the C1 Factor (Host Cell Factor) in Sensory Neurons Correlates with Reactivation of Herpes Simplex Virus from Latency

    NASA Astrophysics Data System (ADS)

    Kristie, Thomas M.; Vogel, Jodi L.; Sears, Amy E.

    1999-02-01

    After a primary infection, herpes simplex virus is maintained in a latent state in neurons of sensory ganglia until complex stimuli reactivate viral lytic replication. Although the mechanisms governing reactivation from the latent state remain unknown, the regulated expression of the viral immediate early genes represents a critical point in this process. These genes are controlled by transcription enhancer complexes whose assembly requires and is coordinated by the cellular C1 factor (host cell factor). In contrast to other tissues, the C1 factor is not detected in the nuclei of sensory neurons. Experimental conditions that induce the reactivation of herpes simplex virus in mouse model systems result in rapid nuclear localization of the protein, indicating that the C1 factor is sequestered in these cells until reactivation signals induce a redistribution of the protein. The regulated localization suggests that C1 is a critical switch determinant of the viral lytic-latent cycle.

  15. Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

    PubMed

    Realegeno, Susan; Puschnik, Andreas S; Kumar, Amrita; Goldsmith, Cynthia; Burgado, Jillybeth; Sambhara, Suryaprakash; Olson, Victoria A; Carroll, Darin; Damon, Inger; Hirata, Tetsuya; Kinoshita, Taroh; Carette, Jan E; Satheshkumar, Panayampalli Subbian

    2017-06-01

    Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe

  16. Minor ABO-mismatches are risk factors for acute graft-versus-host disease in hematopoietic stem cell transplant patients.

    PubMed

    Ludajic, Katarina; Balavarca, Yesilda; Bickeböller, Heike; Rosenmayr, Agathe; Fischer, Gottfried F; Faé, Ingrid; Kalhs, Peter; Pohlreich, David; Kouba, Michal; Dobrovolna, Marie; Greinix, Hildegard T

    2009-11-01

    We investigated the impact of ABO and Rhesus (Rh) blood group matching on the outcome of hematopoietic stem cell transplantation (HSCT) of 154 patients matched at 10/10 HLA loci with unrelated donors. ABO and Rh, as potential risk factors, were modeled with the clinical outcome--acute and chronic graft-versus-host disease (aGVHD, cGVHD), relapse, treatment-related mortality (TRM), and overall survival (OS)--by simple, multiple, and competing risk analyses. We found that minor ABO-mismatches represent a significant risk factor for aGVHD (II-IV) with an estimated risk increase of almost 3-fold (hazard ratio [HR]=2.92, 95% confidence interval [CI]: 1.43-5.95, P=.003), and even 4-fold for aGVHD (III-IV) (HR=4.24, 95% CI: 1.70-10.56, P=.002), but not for other transplant endpoints. No significant association of the Rh matching status with any of the HSCT endpoints was seen. These results suggest that ABO minor mismatches may play a role in aGvHD pathophysiology, possibly by providing the setting for T cell activation and antibody mediated damage. To decrease the risk of aGVHD, ABO matching should be considered in HSCT.

  17. POMC and TP53 genetic variability and risk of basal cell carcinoma of skin: Interaction between host and genetic factors.

    PubMed

    Rizzato, Cosmeri; Scherer, Dominique; Rudnai, Peter; Gurzau, Eugen; Koppova, Kvetoslava; Hemminki, Kari; Canzian, Federico; Kumar, Rajiv; Campa, Daniele

    2011-07-01

    Basal cell carcinoma (BCC) of the skin is the most common neoplasm among the Caucasian population of the western world. Ultraviolet (UV) radiation-induced p53 activation promotes cutaneous pigmentation by increasing transcriptional activity of pro-opiomelanocortin (POMC) in the skin. Induction of POMC/α-melanocyte-stimulating hormone (α-MSH) activates the melanocortin 1 receptor (MC1R), resulting in skin pigmentation. The tumor suppressor p53 is a key player in stress responses that preserve genomic stability, responding to a variety of insults including DNA damage, hypoxia, metabolic stress and oncogene activation. Malfunction of the p53 pathway is an almost universal hallmark of human tumors. Polymorphisms in the gene encoding p53 (TP53) alter its transcriptional activity, which in turn may influence the UV radiation-induced tanning response. The aim of the present work is to test association between POMC and TP53 genetic variability, the possible interplay with host factors and the risk of basal cell carcinoma of skin. We covered the variability of the two genes we used 17 tagging polymorphisms in 529 BCC cases and 532 healthy controls. We have also tested the possible interactions between the genetic variants and three known risk factors for BCC: skin complexion, sun effect and skin response to sun exposure. We did not observe any statistically significant association between SNPs in these two genes and BCC risk overall, nor interactions of SNPs with known BCC risk factors. However we found that, in the group of subjects with lower sun exposure, carriers of one copy of the C allele of the TP53 SNP rs12951053 had a decreased risk of BCC (OR=0.28, 95% CI 0.12-0.62, P=0.002). We have observed that the interplay of an environmental risk factor and one polymorphism in TP53 gene could modulate the risk of BCC. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  18. TUMOR AND HOST FACTORS THAT MAY LIMIT EFFICACY OF CHEMOTHERAPY IN NON-SMALL CELL AND SMALL CELL LUNG CANCER

    PubMed Central

    Stewart, David J.

    2010-01-01

    While chemotherapy provides useful palliation, advanced lung cancer remains incurable since those tumors that are initially sensitive to therapy rapidly develop acquired resistance. Resistance may arise from impaired drug delivery, extracellular factors, decreased drug uptake into tumor cells, increased drug efflux, drug inactivation by detoxifying factors, decreased drug activation or binding to target, altered target, increased damage repair, tolerance of damage, decreased proapoptotic factors, increased antiapoptotic factors, or altered cell cycling or transcription factors. Factors for which there is now substantial clinical evidence of a link to small cell lung cancer (SCLC) resistance to chemotherapy include MRP (for platinum-based combination chemotherapy) and MDR1/P-gp (for non-platinum agents). SPECT MIBI and Tc-TF scanning appears to predict chemotherapy benefit in SCLC. In non-small cell lung cancer (NSCLC), the strongest clinical evidence is for taxane resistance with elevated expression or mutation of class III β-tubulin (and possibly α tubulin), platinum resistance and expression of ERCC1 or BCRP, gemcitabine resistance and RRM1 expression, and resistance to several agents and COX-2 expression (although COX-2 inhibitors have had minimal impact on drug efficacy clinically). Tumors expressing high BRCA1 may have increased resistance to platinums but increased sensitivity to taxanes. Limited early clinical data suggest that chemotherapy resistance in NSCLC may also be increased with decreased expression of cyclin B1 or of Eg5, or with increased expression of ICAM, matrilysin, osteopontin, DDH, survivin, PCDGF, caveolin-1, p21WAF1/CIP1, or 14-3-3sigma, and that IGF-1R inhibitors may increase efficacy of chemotherapy, particularly in squamous cell carcinomas. Equivocal data (with some positive studies but other negative studies) suggest that NSCLC tumors with some EGFR mutations may have increased sensitivity to chemotherapy, while K-ras mutations and

  19. Binding of host-cell factors to DNA sequences in the long terminal repeat of human T-cell leukemia virus type I: implications for viral gene expression

    SciTech Connect

    Nyborg, J.K.; Dynan, W.S.; Chen, I.S.Y.; Wachsman, W.

    1988-03-01

    Efficient expression of human T-cell leukemia virus type I (HTLV-I) genes requires both host and viral proteins and is dependent on DNA sequences in the proviral long terminal repeats (LTRs). The authors have used DNase I-protection assays (footprinting) to construct a map of protein-DNA interactions over a 250-nucleotide region of the LTR upstream of the start site for viral RNA synthesis. They find that a host factor (host expression factor 1, or HEF-1) binds to the imperfect 21-nucleotide repeats that have previously been implicated in HTLV-I gene expression. HEF-1 binding activity is present in preparations from both lymphoid and nonlymphoid cells lines. However, the boundaries of the protected regions and the presence of a flanking DNase-hypersensitive site vary with cell type. Several regions of binding are detected in addition to the HEF-1 sites, including a complex group of sites 40-90 nucleotides upstream of the RNA start site. A comparison of HTLV-I transformed T lymphocytes that do and do not express the viral trans-activating protein p40/sup xI/ shows that none of the observed features of the DNase I footprint pattern correlate directly with the presence of this protein in the extract. These results suggest (i) that the primary recognition of promoter elements in the HTLV-I LTR involves specific interactions with host-cell proteins and (ii) that p40/sup xI/ influences the activity of one or more of these proteins, rather than interacting directly with the DNA.

  20. Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host

    SciTech Connect

    HayGlass, K.T.; Naides, S.J.; Benacerraf, B.; Sy, M.S.

    1985-04-01

    Suppressor T cell factor(s) (TsF/sub 1/) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF/sub 1/ is restricted by genes linked to Igh-1 allotypic markers. TsF/sub 1/ obtained from B6.Igh-1/sup n/ mice was unable to suppress the immune response in B6.Igh-1/sup b/ mice and vice versa. However, TsF/sub 1/ prepared from B6.Igh-1/sup n/ T cells parked in an Igh-congeneic B6.Igh-1/sup b/ environment displays an additional restriction specificity of the host. Thus, TsF/sub 1/ prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1/sup n/ (donor) and B6.Igh-1/sup b/ (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1/sup a/. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response.

  1. Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host.

    PubMed

    HayGlass, K T; Naides, S J; Benacerraf, B; Sy, M S

    1985-04-01

    Suppressor T-cell factor(s) (TsF1) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF1 is restricted by genes linked to Igh-1 allotypic markers. TsF1 obtained from B6.Igh-1n mice was unable to suppress the immune response in B6.Igh-1b mice and vice versa. However, TsF1 prepared from B6.Igh-1n T cells "parked" in an Igh-congeneic B6.Igh-1b environment displays an additional restriction specificity of the host. Thus, TsF1 prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1n (donor) and B6.Igh-1b (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1a. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response.

  2. Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host.

    PubMed Central

    HayGlass, K T; Naides, S J; Benacerraf, B; Sy, M S

    1985-01-01

    Suppressor T-cell factor(s) (TsF1) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF1 is restricted by genes linked to Igh-1 allotypic markers. TsF1 obtained from B6.Igh-1n mice was unable to suppress the immune response in B6.Igh-1b mice and vice versa. However, TsF1 prepared from B6.Igh-1n T cells "parked" in an Igh-congeneic B6.Igh-1b environment displays an additional restriction specificity of the host. Thus, TsF1 prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1n (donor) and B6.Igh-1b (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1a. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response. PMID:3920661

  3. Incidence, risk factors, and long-term outcomes of sclerotic graft-versus-host disease after allogeneic hematopoietic cell transplantation.

    PubMed

    Uhm, Jieun; Hamad, Nada; Shin, Elizabeth M; Michelis, Fotios V; Shanavas, Mohamed; Gupta, Vikas; Kuruvilla, John; Lipton, Jeffrey H; Messner, Hans A; Seftel, Matthew; Kim, Dennis Dong Hwan

    2014-11-01

    Sclerotic chronic graft-versus-host disease (sclGVHD) is associated with significant morbidity and a poor quality of life. We reviewed 502 patients diagnosed with chronic GVHD and analyzed the incidence and risk factors of sclGVHD and long-term outcomes and immunosuppressive therapy (IST) cessation in patients with sclGVHD. With a median onset at 18 months the cumulative incidence of sclGVHD was estimated at 22.6% at 5 years (95% confidence interval, 18.6% to 26.8%). Univariate and multivariate analysis identified 2 risk factors for sclGVHD: non-T cell depletion (hazard ratio [HR] 9.09, P < .001) and peripheral blood stem cell (HR 3.87, P < .001). Overall survival (OS) at 5 years was significantly better in the sclGVHD group (88.1%) compared with the non-sclGVHD group (62.7%; P < .001), as were nonrelapse mortality (7.3% versus 21.5% at 5 years) and relapse rates (9.1% versus 19.3% at 5 years). There was no difference in the rate of IST cessation at 5 years (44.8% versus 49.9%, P = .312), but there was a trend of longer IST duration in the sclGVHD group compared with the non-sclGVHD group (median 71.6 months versus 62.9 months). In conclusion, T cell depletion and graft source affect the risk of sclGVHD. SclGVHD did not adversely affect long-term outcomes or IST duration.

  4. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    SciTech Connect

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-05-25

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1{alpha}) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1{alpha} was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1{alpha}-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1{alpha} to occur. HIF-1{alpha} controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  5. Host cell factor-1 recruitment to E2F-bound and cell-cycle-control genes is mediated by THAP11 and ZNF143.

    PubMed

    Parker, J Brandon; Yin, Hanwei; Vinckevicius, Aurimas; Chakravarti, Debabrata

    2014-11-06

    Host cell factor-1 (HCF-1) is a metazoan transcriptional coregulator essential for cell-cycle progression and cell proliferation. Current models suggest a mechanism whereby HCF-1 functions as a direct coregulator of E2F proteins, facilitating the expression of genes necessary for cell proliferation. In this report, we show that HCF-1 recruitment to numerous E2F-bound promoters is mediated by the concerted action of zinc finger transcription factors THAP11 and ZNF143, rather than E2F proteins directly. THAP11, ZNF143, and HCF-1 form a mutually dependent complex on chromatin, which is independent of E2F occupancy. Disruption of the THAP11/ZNF143/HCF-1 complex results in altered expression of cell-cycle control genes and leads to reduced cell proliferation, cell-cycle progression, and cell viability. These data establish a model in which a THAP11/ZNF143/HCF-1 complex is a critical component of the transcriptional regulatory network governing cell proliferation.

  6. [Validation of the modified algorithm for predicting host susceptibility to viruses taking into account susceptibility parameters of primary target cell cultures and natural immunity factors].

    PubMed

    Zhukov, V A; Shishkina, L N; Safatov, A S; Sergeev, A A; P'iankov, O V; Petrishchenko, V A; Zaĭtsev, B N; Toporkov, V S; Sergeev, A N; Nesvizhskiĭ, Iu V; Vorob'ev, A A

    2010-01-01

    The paper presents results of testing a modified algorithm for predicting virus ID50 values in a host of interest by extrapolation from a model host taking into account immune neutralizing factors and thermal inactivation of the virus. The method was tested for A/Aichi/2/68 influenza virus in SPF Wistar rats, SPF CD-1 mice and conventional ICR mice. Each species was used as a host of interest while the other two served as model hosts. Primary lung and trachea cells and secretory factors of the rats' airway epithelium were used to measure parameters needed for the purpose of prediction. Predicted ID50 values were not significantly different (p = 0.05) from those experimentally measured in vivo. The study was supported by ISTC/DARPA Agreement 450p.

  7. Factors associated with bronchiolitis obliterans syndrome and chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation

    PubMed Central

    Gazourian, Lee; Rogers, Angela J.; Ibanga, Ruby; Weinhouse, Gerald L.; Pinto-Plata, Victor; Ritz, Jerome; Soiffer, Robert J.; Antin, Joseph H.; Washko, George R.; Baron, Rebecca M.; Ho, Vincent T.

    2015-01-01

    Bronchiolitis obliterans syndrome (BOS) is a form of chronic graft vs. host disease (cGVHD) and a highly morbid pulmonary complication after allogeneic hematopoietic stem cell transplantation (HSCT). We assessed the prevalence and risk factors for BOS and cGVHD in a cohort of HSCT recipients, including those who received reduced intensity conditioning (RIC) HSCT. Between January 1, 2000 and June 30, 2010, all patients who underwent allogeneic HSCT at our institution (n = 1854) were retrospectively screened for the development of BOS by PFT criteria. We matched the BOS cases with two groups of control patients: (1) patients who had concurrent cGVHD without BOS and (2) those who developed neither cGVHD nor BOS. Comparisons between BOS patients and controls were conducted using t-test or Fisher’s exact tests. Multivariate regression analysis was performed to examine factors associated with BOS diagnosis. All statistical analyses were performed using SAS 9.2. We identified 89 patients (4.8%) meeting diagnostic criteria for BOS at a median time of 491 days (range: 48–2067) after HSCT. Eighty-six (97%) of our BOS cohort had extra-pulmonary cGVHD. In multivariate analysis compared to patients without cGVHD, patients who received busulfan-based conditioning, had unrelated donors, and had female donors were significantly more likely to develop BOS, while ATG administration was associated with a lower risk of BOS. Our novel results suggest that busulfan conditioning, even in RIC transplantation, could be an important risk factor for BOS and cGVHD. PMID:24375545

  8. Circulating B-cell activating factor level predicts clinical response of chronic graft-versus-host disease to extracorporeal photopheresis.

    PubMed

    Whittle, Robert; Taylor, Peter C

    2011-12-08

    Extracorporeal photopheresis (ECP) is an important therapeutic option in steroid-refractory chronic graft-versus-host disease (cGVHD). Few biomarkers predicting response exist. We measured serum B-cell activating factor (BAFF) in 46 cGVHD patients receiving ECP before and during treatment course. BAFF level at 1 month of ECP predicted 3- and 6-month skin disease response, with BAFF less than 4 ng/mL associated with significant skin improvement and complete resolution in 11 of 20 patients. High BAFF at 1-month ECP associated with a worsening median 6-month skin score and resolution in 1 of 10 patients. BAFF level at 3 months also predicted the likelihood of maintaining skin disease improvement at 6 months. BAFF level was not correlated directly with extracutaneous cGVHD response, although full cutaneous responders exhibited improved extracutaneous organ response rates compared with skin nonresponders (65% vs 35%). This study suggests that early BAFF measurement during ECP for cGVHD represents a potentially useful biomarker in prediction of treatment outcome.

  9. Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells.

    PubMed

    Wang, Bei; Zhang, Xiaoyu; Zhao, Zhendong

    2013-08-02

    Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.

  10. The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

    PubMed Central

    Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

    2016-01-01

    Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species. PMID:26901159

  11. The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

    PubMed

    Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

    2016-01-01

    Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

  12. Selective recruitment of host factors by HSV-1 replication centers.

    PubMed

    Lang, Feng-Chao; Li, Xin; Vladmirova, Olga; Li, Zhuo-Ran; Chen, Gui-Jun; Xiao, Yu; Li, Li-Hong; Lu, Dan-Feng; Han, Hong-Bo; Zhou, Ju-Min

    2015-05-18

    Herpes simplex virus type 1 (HSV-1) enters productive infection after infecting epithelial cells, where it controls the host nucleus to make viral proteins, starts viral DNA synthesis and assembles infectious virions. In this process, replicating viral genomes are organized into replication centers to facilitate viral growth. HSV-1 is known to use host factors, including host chromatin and host transcription regulators, to transcribe its genes; however, the invading virus also encounters host defense and stress responses to inhibit viral growth. Recently, we found that HSV-1 replication centers recruit host factor CTCF but exclude γH2A.X. Thus, HSV-1 replication centers may selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively excluded modified histone H3, including heterochromatin mark H3K9me3, H3S10P and active chromatin mark H3K4me3, but not unmodified H3. We found a dynamic association between the viral replication centers and host RNA polymerase II. The centers also recruited components of the DNA damage response pathway, including 53BP1, BRCA1 and host antiviral protein SP100. Importantly, we found that ATM kinase was needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers took advantage of host signaling pathways to actively recruit or exclude host factors to benefit viral growth.

  13. Selective recruitment of host factors by HSV-1 replication centers

    PubMed Central

    LANG, Feng-Chao; LI, Xin; VLADMIROVA, Olga; LI, Zhuo-Ran; CHEN, Gui-Jun; XIAO, Yu; LI, Li-Hong; LU, Dan-Feng; HAN, Hong-Bo; ZHOU, Ju-Min

    2015-01-01

    Herpes simplex virus type 1 (HSV-1) enters productive infection after infecting epithelial cells, where it controls the host nucleus to make viral proteins, starts viral DNA synthesis and assembles infectious virions. In this process, replicating viral genomes are organized into replication centers to facilitate viral growth. HSV-1 is known to use host factors, including host chromatin and host transcription regulators, to transcribe its genes; however, the invading virus also encounters host defense and stress responses to inhibit viral growth. Recently, we found that HSV-1 replication centers recruit host factor CTCF but exclude βH2A.X. Thus, HSV-1 replication centers may selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively excluded modified histone H3, including heterochromatin mark H3K9me3, H3S10P and active chromatin mark H3K4me3, but not unmodified H3. We found a dynamic association between the viral replication centers and host RNA polymerase II. The centers also recruited components of the DNA damage response pathway, including 53BP1, BRCA1 and host antiviral protein SP100. Importantly, we found that ATM kinase was needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers took advantage of host signaling pathways to actively recruit or exclude host factors to benefit viral growth. PMID:26018857

  14. Host cells and cell banking.

    PubMed

    Stacey, Glyn N; Merten, Otto-Wilhelm

    2011-01-01

    Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus).

  15. Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion.

    PubMed

    van Wijk, Xander M; Döhrmann, Simon; Hallström, Björn M; Li, Shangzhong; Voldborg, Bjørn G; Meng, Brandon X; McKee, Karen K; van Kuppevelt, Toin H; Yurchenco, Peter D; Palsson, Bernhard O; Lewis, Nathan E; Nizet, Victor; Esko, Jeffrey D

    2017-01-10

    To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens. Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This

  16. Th17 cells and Mucosal Host Defense

    PubMed Central

    Aujla, Shean J.; Dubin, Patricia J.; Kolls, Jay K.

    2008-01-01

    Th17 cells are a new lineage of T-cells that are controlled by the transcription factor RORγt and develop independent of GATA-3, T-bet, Stat 4 and Stat 6. Novel effector molecules produced by these cells include IL-17A, IL-17F, IL-22, and IL-26. IL-17RA binds IL-17A and IL-17F and is critical for host defense against extracellular planktonic bacteria by regulating chemokine gradients for neutrophil emigration into infected tissue sites as well as host granulopoiesis. Moreover IL-17 and IL-22 regulate the production of antimicrobial proteins in mucosal epithelium. Although TGF-β1 and IL-6 have been shown to be critical for development of Th17 cells from naïve precursors, IL-23 is also important in regulating IL-17 release in mucosal tissues in response to infectious stimuli. Compared to Th1 cells, IL-23 and IL-17 show limited roles in controlling host defense against primary infections with intracellular bacteria such as Mycobacterium tuberculosis suggesting a predominate role of the Th17 lineage in host defense against extracellular pathogens. However in the setting of chronic biofilm infections, as that occurs with Cystic Fibrosis or bronchetctasis, Th17 cells may be key contributors of tissue injury. PMID:18054248

  17. The Poxvirus C7L Host Range Factor Superfamily

    PubMed Central

    Liu, Jia; Rothenburg, Stefan; McFadden, Grant

    2012-01-01

    Host range factors, expressed by the poxvirus family, determine the host tropism of species, tissue, and cell specificity. C7L family members exist in the genomes of most sequenced mammalian poxviruses, suggesting an evolutionarily conserved effort adapting to the hosts. In general, C7L orthologs influence the host tropism in mammalian cell culture, and for some poxviruses it is essential for the complete viral life cycle in vitro and in vivo. The C7L family members lack obvious sequence homology with any other known viral or cellular proteins. Here we review recent findings from an evolutionary perspective and summarize recent progress that broadens our view on the role of C7L family members in mediating poxvirus host range and antagonizing the host defense system. PMID:23103013

  18. Modified host cells with efflux pumps

    DOEpatents

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  19. Host resistance factors in human milk.

    PubMed

    Goldman, A S; Smith, C W

    1973-06-01

    This paper discusses the nature of host resistance factors in human milk and epidemiologic studies regarding infections and mortality rates in breastfed and nonbreastfed babies. The defense factors and their proposed modes of action are: 1) a growth enhancer of lactobacilli, which interferes with intestinal colonization of enteric pathogens; 2) antistaphylococcal factors, which inhibit staphylococci; 3) secretory IgA and other immunoglobulins, which protect the gut and respiratory tract; 4) C4 and C3 (complement components; C3 fragments have opsonic, chemotactic, and anaphylatoxic activities); 5) lysozome, lysis of bacterial cell wall; 6) lactoperoxidase, killing of streptococci; 7) lactoferrin, kills microorganism by chelating iron, and 8) macrophages and lymphocytes, phagocytosis and cell-mediated immunity. Although it can be postulated that the breastfed infant's resistance to infection would be superior on account of the greater presence of these factors in human milk compared to cow's milk, little is known about the effects of these defense factors on the infant. Epidemiologic studies have reported on the lower morbidity and mortality rates of breastfed infants as compared to bottlefed infants. Other studies have focused on the protective effects of human milk upon the infant, but these have been inconclusive. In countries with poor sanitation and high infection rates, the incidence of bacterial infections is lowest in breastfed infants. The advantages of human milk however are difficult to demonstrate in societies with high standards of sanitation and low infection rates. Infection and mortality rates in infants have in fact declined in developed countries as the practice of breastfeeding declined. Until it is established that immunity to common pathogens is transmitted to the infant by human milk, it will not be known whether human milk does have protective effects.

  20. Genome-Wide Screening Uncovers the Significance of N-sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection.

    PubMed

    Tanaka, Atsushi; Tumkosit, Uranan; Nakamura, Shota; Motooka, Daisuke; Kishishita, Natsuko; Priengprom, Thongkoon; Sa-Ngasang, Areerat; Kinoshita, Taroh; Takeda, Naokazu; Maeda, Yusuke

    2017-04-12

    The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3-E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of a clinically isolated CHIKV Thai #16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). Whereas glycosaminoglycans were commonly required for efficient infectivity of CHIKV, JEV and YFV as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at the later steps after the CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection, because it is involved in proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we

  1. Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy

    PubMed Central

    Spann, Kirsten M; Baturcam, Engin; Schagen, Johanna; Jones, Carmen; Straub, Claire P; Preston, F Maxine; Chen, Linping; Phipps, Simon; Sly, Peter D; Fantino, Emmanuelle

    2014-01-01

    Background Airway epithelial cells (AEC) from patients with asthma, appear to have an impaired interferon (IFN)-β and -λ response to infection with rhinovirus. Objectives To determine if impaired IFN responses can be identified in young children at risk of developing asthma due to atopy and/or early life wheeze, and if the site of infection or the infecting virus influence the antiviral response. Methods Nasal (N) and tracheal (T) epithelial cells (EC) were collected from children categorised with atopy and/or wheeze based on specific IgE to locally common aeroallergens and a questionnaire concerning respiratory health. Submerged primary cultures were infected with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), and IFN production, inflammatory cytokine expression and viral replication quantified. Results Nasal epithelial cells (NEC), but not tracheal epithelial cells (TEC), from children with wheeze and/or atopy produced less IFN-β, but not IFN-λ, in response to RSV infection; this was associated with higher viral shedding. However, IFN-regulated factors IRF-7, Mx-1 and CXCL-10, and inflammatory cytokines were not differentially regulated. NECs and TECs from children with wheeze and/or atopy demonstrated no impairment of the IFN response (β or λ) to hMPV infection. Despite this, more hMPV was shed from these cells. Conclusions AECs from children with wheeze and/or atopy do not have an intrinsic defect in the production of IFN-β or -λ, however, this response is influenced by the infecting virus. Higher viral load is associated with atopy and wheeze suggesting an impaired antiviral response to RSV and hMPV that is not influenced by production of IFNs. PMID:24811725

  2. Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy.

    PubMed

    Spann, Kirsten M; Baturcam, Engin; Schagen, Johanna; Jones, Carmen; Straub, Claire P; Preston, F Maxine; Chen, Linping; Phipps, Simon; Sly, Peter D; Fantino, Emmanuelle

    2014-10-01

    Airway epithelial cells (AEC) from patients with asthma, appear to have an impaired interferon (IFN)-β and -λ response to infection with rhinovirus. To determine if impaired IFN responses can be identified in young children at risk of developing asthma due to atopy and/or early life wheeze, and if the site of infection or the infecting virus influence the antiviral response. Nasal (N) and tracheal (T) epithelial cells (EC) were collected from children categorised with atopy and/or wheeze based on specific IgE to locally common aeroallergens and a questionnaire concerning respiratory health. Submerged primary cultures were infected with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), and IFN production, inflammatory cytokine expression and viral replication quantified. Nasal epithelial cells (NEC), but not tracheal epithelial cells (TEC), from children with wheeze and/or atopy produced less IFN-β, but not IFN-λ, in response to RSV infection; this was associated with higher viral shedding. However, IFN-regulated factors IRF-7, Mx-1 and CXCL-10, and inflammatory cytokines were not differentially regulated. NECs and TECs from children with wheeze and/or atopy demonstrated no impairment of the IFN response (β or λ) to hMPV infection. Despite this, more hMPV was shed from these cells. AECs from children with wheeze and/or atopy do not have an intrinsic defect in the production of IFN-β or -λ, however, this response is influenced by the infecting virus. Higher viral load is associated with atopy and wheeze suggesting an impaired antiviral response to RSV and hMPV that is not influenced by production of IFNs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  3. CD4+ CD25+ transforming growth factor-beta-producing T cells are present in the lung in murine tuberculosis and may regulate the host inflammatory response.

    PubMed

    Mason, C M; Porretta, E; Zhang, P; Nelson, S

    2007-06-01

    CD4(+) CD25(+) regulatory T cells produce the anti-inflammatory cytokines transforming growth factor (TGF)-beta or interleukin (IL)-10. Regulatory T cells have been recognized to suppress autoimmunity and promote self-tolerance. These cells may also facilitate pathogen persistence by down-regulating the host defence response during infection with Mycobacterium tuberculosis. We evaluated TGF-beta(+) and IL-10(+) lung CD4(+) CD25(+) T cells in a murine model of M. tuberculosis. BALB/c mice were infected with approximately 50 colony-forming units of M. tuberculosis H37Rv intratracheally. At serial times post-infection, lung cells were analysed for surface marker expression (CD3, CD4, CD25) and intracellular IL-10, TGF-beta, and interferon (IFN)-gamma production (following stimulation in vitro with anti-CD3 and anti-CD28 antibodies). CD4(+) lung lymphocytes were also selected positively after lung digestion, and stimulated in vitro for 48 h with anti-CD3 and anti-CD28 antibodies in the absence and presence of anti-TGF-beta antibody, anti-IL-10 antibody or rmTGF-beta soluble receptor II/human Fc chimera (TGFbetasrII). Supernatants were assayed for elicited IFN-gamma and IL-2. Fluorescence activated cell sorter analyses showed that TGF-beta- and IL-10-producing CD4(+) CD25(+) T cells are present in the lungs of infected mice. Neutralization of TGF-beta and IL-10 each resulted in increases in elicited IFN-gamma, with the greatest effect seen when TGFbetasrII was used. Elicited IL-2 was not affected significantly by TGF-beta neutralization. These results confirm the presence of CD4(+) CD25(+) TGF-beta(+) T cells in murine pulmonary tuberculosis, and support the possibility that TGF-beta may contribute to down-regulation of the host response.

  4. Host cell invasion by medically important fungi.

    PubMed

    Sheppard, Donald C; Filler, Scott G

    2014-11-03

    To infect the host and cause disease, many medically important fungi invade normally nonphagocytic host cells, such as endothelial cells and epithelial cells. Host cell invasion is a two-step process consisting of adherence followed by invasion. There are two general mechanisms of host cell invasion, induced endocytosis and active penetration. Furthermore, fungi can traverse epithelial or endothelial cell barriers either by proteolytic degradation of intercellular tight junctions or via a Trojan horse mechanism in which they are transported by leukocytes. Although these mechanisms of host cell invasion have been best studied using Candida albicans and Cryptococcus neoformans, it is probable that other invasive fungi also use one or more of these mechanisms to invade host cells. Identification of these invasion mechanisms holds promise to facilitate the development of new approaches to inhibit fungal invasion and thereby prevent disease. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  5. Host Cell Invasion by Medically Important Fungi

    PubMed Central

    Sheppard, Donald C.; Filler, Scott G.

    2015-01-01

    To infect the host and cause disease, many medically important fungi invade normally nonphagocytic host cells, such as endothelial cells and epithelial cells. Host cell invasion is a two-step process consisting of adherence followed by invasion. There are two general mechanisms of host cell invasion, induced endocytosis and active penetration. Furthermore, fungi can traverse epithelial or endothelial cell barriers either by proteolytic degradation of intercellular tight junctions or via a Trojan horse mechanism in which they are transported by leukocytes. Although these mechanisms of host cell invasion have been best studied using Candida albicans and Cryptococcus neoformans, it is probable that other invasive fungi also use one or more of these mechanisms to invade host cells. Identification of these invasion mechanisms holds promise to facilitate the development of new approaches to inhibit fungal invasion and thereby prevent disease. PMID:25367974

  6. Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *

    PubMed Central

    Matsubayashi, Makoto; Teramoto-Kimata, Isao; Uni, Shigehiko; Lillehoj, Hyun S.; Matsuda, Haruo; Furuya, Masaru; Tani, Hiroyuki; Sasai, Kazumi

    2013-01-01

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis. PMID:24085304

  7. Discovery of Insect and Human Dengue Virus Host Factors

    PubMed Central

    Sessions, October M.; Barrows, Nicholas J.; Souza-Neto, Jayme A.; Robinson, Timothy J.; Hershey, Christine L.; Rodgers, Mary A.; Ramirez, Jose L.; Dimopoulos, George; Yang, Priscilla L.; Pearson, James L.; Garcia-Blanco, Mariano A.

    2012-01-01

    Dengue fever (DF) is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection1. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make DF a global public health threat1, 2. Given their compact genomes, dengue viruses (DENV 1-4) and other flaviviruses likely require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors have been identified3-10. To discover insect host factors required for DENV-2 propagation, we carried out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 dsRNA library. This screen identified 116 candidate dengue virus host factors (DVHFs) (Supplementary Fig. 1). While some were previously associated with flaviviruses (e.g., V-ATPases and alpha-glucosidases)3-5, 7, 9, 10, most DVHFs were newly implicated in DENV propagation. The dipteran DVHFs had eighty-two readily recognizable human homologues and, using a targeted siRNA screen, we showed that forty-two of these are human DVHFs. This indicates remarkable conservation of required factors between dipteran and human hosts. This work suggests novel approaches to control infection in the insect vector and the mammalian host. PMID:19396146

  8. KSHV miRNAs decrease expression of lytic genes in latently infected PEL and endothelial cells by targeting host transcription factors.

    PubMed

    Plaisance-Bonstaff, Karlie; Choi, Hong Seok; Beals, Tyler; Krueger, Brian J; Boss, Isaac W; Gay, Lauren A; Haecker, Irina; Hu, Jianhong; Renne, Rolf

    2014-10-23

    Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3'UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3'UTR downstream from the luciferase reporter. Knockdown of miR‑K12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down-regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors.

  9. The network of microRNAs, transcription factors, target genes and host genes in human renal cell carcinoma

    PubMed Central

    SONG, CHENGLU; XU, ZHIWEN; JIN, YUE; ZHU, MINGHUI; WANG, KUNHAO; WANG, NING

    2015-01-01

    At present, scientists have performed numerous studies investigating the morbidity of renal cell carcinoma (RCC) in the genetic and microRNA (miRNA) fields, obtaining a substantial amount of knowledge. However, the experimentally validated data of genes, miRNA and transcription factors (TFs) cannot be found in a unified form, which makes it challenging to decipher the regulatory mechanisms. In the present study, the genes, miRNAs and TFs involved in RCC are regarded as elements in the regulatory network, and the present study therefore focuses on the association between each entity. Three regulatory networks were constructed hierarchically to indicate the regulatory association between the genes, miRNAs and TFs clearly, including the differentially expressed, associated and global networks. All the elements were macroscopically investigated in these networks, instead of only investigating one or several of them. The present study not only compared and analyzed the similarities and the differences between the three networks, but also systematically expounded the pathogenesis of RCC and supplied theoretical foundations for future gene therapy investigations. Following the construction of the three networks, certain important pathways were highlighted. The upstream and downstream element table of differentially expressed genes and miRNAs was listed, in which self-adaption associations and circle-regulations were identified. In future studies, the identified genes and miRNAs should be granted more attention. PMID:25436016

  10. Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication.

    PubMed

    Flores, O; Lee, G; Kessler, J; Miller, M; Schlief, W; Tomassini, J; Hazuda, D

    1999-06-22

    HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.

  11. Salmonella - at home in the host cell.

    PubMed

    Malik-Kale, Preeti; Jolly, Carrie E; Lathrop, Stephanie; Winfree, Seth; Luterbach, Courtney; Steele-Mortimer, Olivia

    2011-01-01

    The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic "trigger"-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.

  12. Interaction of chlamydiae and host cells in vitro.

    PubMed Central

    Moulder, J W

    1991-01-01

    The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models. PMID:2030670

  13. Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus

    PubMed Central

    de Medeiros, Ricardo B.; Figueiredo, Juliana; Resende, Renato de O.; De Avila, Antonio C.

    2005-01-01

    Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses. PMID:15657123

  14. Recipient CTLA-4*CT60-AA genotype is a prognostic factor for acute graft-versus-host disease in hematopoietic stem cell transplantation for thalassemia.

    PubMed

    Orrù, Sandro; Orrù, Nicola; Manolakos, Emmanouil; Littera, Roberto; Caocci, Giovanni; Giorgiani, Giovanna; Bertaina, Alice; Pagliara, Daria; Giardini, Claudio; Nesci, Sonia; Locatelli, Franco; Carcassi, Carlo; La Nasa, Giorgio

    2012-03-01

    Polymorphisms of the cytotoxic T-lymphocyte antigen-4 gene (CTLA-4) have been associated with autoimmune diseases and it has recently been reported that donor genotypes correlate with the outcome of allogeneic hematopoietic stem cell transplantation in leukemia patients. With the aim of confirming this finding in thalassemia patients, we investigated the influence of genotype distribution of 3 CTLA-4 gene polymorphisms in 72 thalassemia patients and their unrelated donors. A significant association was observed for recipient CT60-AA genotype and onset of grade II-IV (63.2% vs 24.5%; p = 0.001) and grade III-IV (36.4% vs 7.6%; p = 0.005) acute graft-versus-host disease (aGVHD). The same association was observed for the 88-base-pair allele of the CTLA-4 (AT)n polymorphism, which was determined to be in complete linkage disequilibrium with the CT60 A allele. Multinomial Cox regression demonstrated that this association was independent of CT60 donor genotypes or other risk factors (p = 0.016; hazard ratio = 2.8). Our data confirm that the genetic variability in CTLA-4 is an important prognostic factor for aGVHD and suggest that some of the risk factors for this complication are generated by recipient cells that persist after the myeloablative conditioning regimen. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  15. The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles.

    PubMed

    Trautz, Birthe; Wiedemann, Hannah; Lüchtenborg, Christian; Pierini, Virginia; Kranich, Jan; Glass, Bärbel; Kräusslich, Hans-Georg; Brocker, Thomas; Pizzato, Massimo; Ruggieri, Alessia; Brügger, Britta; Fackler, Oliver T

    2017-08-18

    The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Host Range Factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) Is an Essential Viral Factor Required for Productive Infection of NPVs in IPLB-Ld652Y Cells Derived from L. dispar

    PubMed Central

    Ishikawa, Hiroki; Ikeda, Motoko; Felipe Alves, Cristiano A.; Thiem, Suzanne M.; Kobayashi, Michihiro

    2004-01-01

    Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells. PMID:15507661

  17. Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

    PubMed Central

    van Wijk, Xander M.; Döhrmann, Simon; Hallström, Björn M.; Li, Shangzhong; Voldborg, Bjørn G.; Meng, Brandon X.; McKee, Karen K.; van Kuppevelt, Toin H.; Yurchenco, Peter D.; Palsson, Bernhard O.; Lewis, Nathan E.; Nizet, Victor

    2017-01-01

    ABSTRACT To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens. PMID:28074024

  18. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

    PubMed Central

    Oberli, Alexander; Slater, Leanne M.; Cutts, Erin; Brand, Françoise; Mundwiler-Pachlatko, Esther; Rusch, Sebastian; Masik, Martin F. G.; Erat, Michèle C.; Beck, Hans-Peter; Vakonakis, Ioannis

    2014-01-01

    Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.—Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. PMID:24983468

  19. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    PubMed

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

  20. Host and Viral Factors in HIV-Mediated Bystander Apoptosis

    PubMed Central

    Garg, Himanshu; Joshi, Anjali

    2017-01-01

    Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

  1. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    PubMed Central

    Shrestha, Archana; Hendricks, Matthew R.; Bomberger, Jennifer M.

    2016-01-01

    ABSTRACT Clostridium perfringens enterotoxin (CPE) binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s) from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s) in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease. PMID:27965452

  2. Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

    PubMed Central

    Boehm, Manja; Krause-Gruszczynska, Malgorzata; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Oyarzabal, Omar A.; Backert, Steffen

    2011-01-01

    Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin−/−, integrin beta1−/−, and focal adhesion kinase (FAK)−/− deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin−/−, integrin beta1−/−, and FAK−/− knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells. PMID:22919583

  3. Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

    PubMed Central

    Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M. Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

    2017-01-01

    Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

  4. Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

    PubMed

    Bielaszewska, Martina; Rüter, Christian; Bauwens, Andreas; Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

    2017-02-01

    Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

  5. Contrasting Lifestyles Within the Host Cell

    PubMed Central

    Case, Elizabeth Di Russo; Samuel, James E.

    2015-01-01

    CHAPTER SUMMARY Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host, and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without its hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH, and contains degradative enzymes, and reactive oxygen species resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, like Shigella, Listeria, Francisella, and Rickettsia escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  6. Elongation Factor-1a is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum*

    USDA-ARS?s Scientific Manuscript database

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess a unique complex of organelles at the anterior end, referred to as the apical complex, which is involved in host cell invasion. Previously, we generated the chicken m...

  7. The chromosome localization and the HCF repeats of the human host cell factor gene (HCFC1) are conserved in the mouse homologue

    SciTech Connect

    Frattini, A.; Faranda, S.; Sacco, M.G.; Villa, A.; Vezzoni, P.

    1996-03-01

    The gene encoding the human host cell factor (HCFC1) has recently been cloned and mapped to Xq28. HCFC1 codes for a family of related polypeptides that apparently arise from posttranslational processing. Six extremely conserved 19-amino-acid (aa)-long motifs, unique to HCFC1 and located in the middle of the protein, could play a role in this processing or could be instrumental to the physiological role of the protein. Alternatively, these repeats could have arisen from recent duplications and may not have any specific function. To resolve this issue, we cloned the homologous region from the mouse Hcfc1 gene and demonstrated that the 19-aa motifs are extremely conserved in sequence, number, and genomic organization, while the {open_quotes}linker{close_quotes} region between the third and fourth repeat is not. This suggests an important function for these repeats. In addition, by RT-PCR analysis of human RNA and comparison to the human genomic sequence, an alternative transcript including a 44-aa in-frame insertion, driving from the 3{prime} nd of intron 18, was found. The significance of this alternative transcript is unknown, since it was not detectable in the mouse. The mouse Hcfc1 gene maps to a region syntenic to Xq28, and, as in human, is in close proximity to the Renin-binding protein gene, in a 100-kb region also including the L1cam and Vasopressin receptor type 2 genes. 8 refs., 2 figs.

  8. Bartonella entry mechanisms into mammalian host cells.

    PubMed

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment.

  9. Inhibition of host cell apoptosis by Eimeria bovis sporozoites.

    PubMed

    Lang, Mirjam; Kann, Michael; Zahner, Horst; Taubert, Anja; Hermosilla, Carlos

    2009-03-09

    Sophisticated evasion strategies of obligate intracellular parasites, in particular prevention of host cell apoptosis, are necessary to ensure successful replication. To study the ability of Eimeria bovis in this regard, in vitro experiments were performed applying bovine foetal gastrointestinal cells (BFGC), bovine umbilical vein endothelial cells (BUVEC) and African green monkey kidney cells (VERO) as host cells. BUVEC and BFGC allow maturation of sporozoites to macromeronts, in VERO cells sporozoites survive for weeks without showing further development. In highly infected BUVEC monolayers, infected cells survived until merozoite release whereas uninfected cells underwent apoptosis. Light microscopy and TUNEL assays performed 3-10 days p.i. showed that, within infected BFGC and VERO cell monolayers, uninfected cells underwent programmed cell death after application of various inducers of apoptosis, whereas infected cells survived. Incidentally, the anti-apoptotic efficacies in infected cells were independent of the drugs and the host cell type. We could not demonstrate significant differences between infected and uninfected cells after colchicin treatment in terms of translation of phosphatidylserines to the host cell surface, caspase 3 activity and cytochrome c release, probably since obtainable infection rates were too low. However, we could show by laser scanning confocal microscopy on single cell levels that the expression of the anti-apoptotic factors cellular Flice inhibitory protein (c-FLIP) and cellular inhibition of apoptosis protein 1 (c-IAP1) were enhanced in E. bovis infected cells after application of colchicin, in the latter case also in non-infected cells directly neighbouring infected ones. Our data show that E. bovis protects its host cell from apoptosis by increasing expression of c-IAP1 and c-FLIP.

  10. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  11. CONTRIBUTION OF HOST-DERIVED TISSUE FACTOR TO TUMOR NEOVASCULARIZATION

    PubMed Central

    Yu, Joanne; May, Linda; Milsom, Chloe; Anderson, G. Mark; Weitz, Jeffrey I.; Luyendyk, James P.; Broze, George; Mackman, Nigel; Rak, Janusz

    2010-01-01

    Objective The role of host-derived tissue factor (TF) in tumor growth, angiogenesis and metastasis has hitherto been unclear, and was investigated in this study. Methods We compared tumor growth, vascularity and responses to cyclophosphamide (CTX) of tumors in wild type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Results Global growth rate of three different types of transplantable tumors (LLC, B16F1 and ES teratoma), or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (i) reduced tumor blood vessel size in B16F1 tumors; (ii) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (iii) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF-/-) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels, and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. Conclusions Our study points to an important, but context-dependent role of host TF in tumor formation, angiogenesis and therapy. PMID:18772494

  12. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells.

    PubMed

    Popa, Crina M; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding.

  13. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  14. Alterations of host cell ubiquitination machinery by pathogenic bacteria.

    PubMed

    Alomairi, Jaafar; Bonacci, Thomas; Ghigo, Eric; Soubeyran, Philippe

    2015-01-01

    Response of immune and non-immune cells to pathogens infections is a very dynamic process. It involves the activation/modulation of many pathways leading to actin remodeling, membrane engulfing, phagocytosis, vesicle trafficking, phagolysosome formation, aiming at the destruction of the intruder. These sophisticated and rapid mechanisms rely on post-translational modifications (PTMs) of key host cells' factors, and bacteria have developed various strategies to manipulate them to favor their survival. Among these important PTMs, ubiquitination has emerged as a major mediator/modulator/regulator of host cells response to infections that pathogens have also learned to use for their own benefit. In this mini-review, we summarize our current knowledge about the normal functions of ubiquitination during host cell infection, and we detail its hijacking by model pathogens to escape clearance and to proliferate.

  15. Interactions between Bdellovibrio and its host cell.

    PubMed

    Stolp, H

    1979-04-11

    The bdellovibrios are extremely small bacteria with the unique property of being parasites of other (gram-negative) bacteria. In the presence of viable and susceptible bacteria a Bdellovibrio cell physically 'attacks' an individual host cell, attaches to its surface, penetrates the cell wall, and multiples within the periplasmic (intramural) space of its prey. The invading Bdellovibrio and its progeny degrade and consume the cellular constituents of the invaded host bacterium. This process finally results in complete lysis of the host cell and release of the Bdellovibrio progeny. From a population of parasitic bdellovibrios, derivatives can be selected that grow on complex nutrient media. Currently, none of the different nutritional types can be propagated in a fully defined synthetic medium. By degradation of the cellular constituents of the host the Bdellovibrio cell in its periplasmic space has available all the monomeric subunits needed to synthesis of the macromolecules. Peculiarities of Bdellovibrio metabolism with respect to uptake of preformed molecules and energy efficiency are discussed.

  16. Somatic Host Cell Alterations in HPV Carcinogenesis

    PubMed Central

    Litwin, Tamara R.; Clarke, Megan A.; Dean, Michael; Wentzensen, Nicolas

    2017-01-01

    High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions. PMID:28771191

  17. Neuroendocrine Factors Alter Host Defense by Modulating Immune Function

    PubMed Central

    Butts, Cherie L.; Sternberg, Estner M.

    2008-01-01

    An increasing body of evidence demonstrates that there is bidirectional communication between the neuroendocrine and immune systems. Interactions between these systems results in a variety of outcomes, including the well documented “sickness behavior” elicited by cytokines of the immune system that can enter the brain or activate second messengers that modify neuronal activity. Crosstalk between the neuroendocrine and immune systems can also result in production of factors by the nervous and endocrine systems that alter immune cell function and subsequent modulation of immune responses against infectious agents and other pathogens. Continued exposure to molecules produced by the neuroendocrine system has also been shown to increase susceptibility and/or severity of disease. Furthermore, neuroendocrine factors are thought to play a major role in the gender-specific difference in development of certain disorders, including autoimmune/inflammatory diseases that have a 2- to 10-fold higher incidence in females compared to males. Neuroendocrine factors can affect immune cells at the level of gene transcription but have also been shown to modify immune cell activity by interacting with intracellular signal transduction molecules, resulting in modified ability of these cells to mount a potent immune response. In this review, we will consider the various effects of the neuroendocrine system and its proteins on specific populations of immune cells and associated responses in host immunity against pathogens. We will further discuss how this modification of immune cell activity by the neuroendocrine system can contribute to susceptibility/severity of development of diseases. PMID:18329009

  18. Adverse Host Factors Exacerbate Occult HIV-Associated Nephropathy

    PubMed Central

    Kumar, Dileep; Salhan, Divya; Magoon, Sandeep; Torri, Deepti D.; Sayeneni, Swapna; Sagar, Ankita; Bandhlish, Anshu; Malhotra, Ashwani; Chander, Praveen N.; Singhal, Pravin C.

    2011-01-01

    In the present study, we hypothesized that HIV-1–induced occult HIV-associated nephropathy (HIVAN) would become apparent in the presence of adverse host factors. To test our hypothesis, Vpr mice (which display doxycycline-dependent Vpr expression in podocytes) with two, three, and four copies of the angiotensinogen (Agt) gene (Vpr-Agt-2, Vpr-Agt-3, and Vpr-Agt-4) were administered doxycycline for 3 weeks (to develop clinically occult HIVAN) followed by doxycycline-free water during the next 3 weeks. Subsequently, renal biomarkers were measured, and kidneys were harvested for renal histology. Vpr-Agt-2 developed neither proteinuria nor elevated blood pressure, and displayed minimal glomerular and tubular lesions only, without any microcyst formation. Vpr-Agt-3 showed mild glomerular and tubular lesions and microcyst formation, whereas Vpr-Agt-4 showed moderate proteinuria, hypertension, glomerular sclerosis, tubular dilation, microcysts, and expression of epithelial mesenchymal transition markers. Vpr-Agt-4 not only displayed enhanced renal tissue expression of Agt, renin, and angiotensin-converting enzyme, but also had higher renal tissue concentrations of angiotensin II. Moreover, renal cells in Vpr-Agt-4 showed enhanced expression of transforming growth factor-β, connective tissue growth factor, and vascular endothelial growth factor. These findings indicate that adverse host factors, such as the activation of the renin-angiotensin system, promote the progression of occult HIVAN to apparent HIVAN. PMID:21871425

  19. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis.

    PubMed

    Martín-Hernández, Raquel; Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host's cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite's survival within the cell.

  20. Acanthamoeba induces cell-cycle arrest in host cells.

    PubMed

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

    2004-08-01

    Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

  1. Graft monocytic myeloid-derived suppressor cell content predicts the risk of acute graft-versus-host disease after allogeneic transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.

    PubMed

    Vendramin, Antonio; Gimondi, Silvia; Bermema, Anisa; Longoni, Paolo; Rizzitano, Sara; Corradini, Paolo; Carniti, Cristiana

    2014-12-01

    Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Lin(low/neg)HLA-DR(-)CD11b(+)CD33(+)CD14(+)) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  2. Complement factor H in host defense and immune evasion.

    PubMed

    Parente, Raffaella; Clark, Simon J; Inforzato, Antonio; Day, Anthony J

    2017-05-01

    Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

  3. Nuclear factor-κ B inducing kinase is required for graft-versus-host disease

    PubMed Central

    Sánchez-Valdepeñas, Carmen; Casanova, Lucía; Colmenero, Isabel; Arriero, Mar; González, África; Lozano, Nieves; González-Vicent, Marta; Díaz, Miguel A.; Madero, Luís; Fresno, Manuel; Ramírez, Manuel

    2010-01-01

    Background Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease. Design and Methods We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control). Results We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells. Conclusions Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes. PMID:20823135

  4. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    PubMed Central

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  5. High levels of B cell activating factor during the peritransplantation period are associated with a reduced incidence of acute graft-versus-host disease following myeloablative allogeneic stem cell transplantation.

    PubMed

    Cho, Byung-Sik; Min, Chang-Ki; Kim, Hee-Je; Lee, Seok; Kim, Yoo-Jin; Lim, Ji-Young; Jeong, Dae-Chul; Cho, Bin; Kim, Hack-Ki; Eom, Ki-Seong; Cho, Seok-Goo; Kim, Dong-Wook; Lee, Jong-Wook; Min, Woo-Sung; Kim, Chun-Choo; Chung, Nak-Gyun

    2010-05-01

    B cell activating factor (BAFF), also known as B cell survival and activation factor, is associated with autoimmune disease and chronic graft-versus-host disease (cGVHD). T cells are known to be modulated by soluble BAFF (sBAFF). Considering the possible association of sBAFF with T cell as well as B cell function, sBAFF during the peritransplantation period may affect the development of acute GVHD (aGVHD). To test this hypothesis, we evaluated 45 patients who had undergone myeloablative (MA) allogeneic stem cell transplantation (SCT) for hematologic malignancy. Serum sBAFF levels were measured before conditioning and on day 0, day +7, and day +14. Thirty-three of the 45 patients (cumulative incidence, 73%) developed aGVHD between 16 days and 98 days posttransplantation. Repeated-measures analysis of variance revealed significantly lower sBAFF levels during the peritransplantation period in patients with aGVHD than in those without aGVHD (P=.001). Receiver operating characteristic curve analysis revealed that sBAFF levels at every time point were available for the prediction of aGVHD development, and that patients with a sBAFF level >43 pg/mL at each time point (which could ensure 75% sensitivity and 73%-82% specificity for the prediction of aGVHD at every time point) had a significantly lower cumulative incidence of aGVHD. This study is the first to demonstrate that sBAFF level during the peritransplantation period not only may be predictive of aGVHD, but also may have a protective effect against aGVHD in humans. Further investigation is needed to confirm our findings. Copyright 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  6. Host cell targets for African swine fever virus.

    PubMed

    Muñoz-Moreno, Raquel; Galindo, Inmaculada; Cuesta-Geijo, Miguel Ángel; Barrado-Gil, Lucía; Alonso, Covadonga

    2015-11-02

    Viruses are strict intracellular pathogens that require the cellular environment to complete a successful infection. Among them, African swine fever virus (ASFV) is an evolutionary ancient DNA virus, endemic in Africa, which is nowadays causing an emergent disease in Europe with a potential high economic impact in the pig industry. It is well known that host-cell components are critical crossroads mapping the virus path for a productive infection, some of them at the endocytic pathway. Considering that ASFV infectious cycle strongly relies in several factors from the host cell, the study of virus-host interactions remains crucial as they will reveal the obstacles, routes and tracks, hints and the target waypoint in the virus journey to destination. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

    PubMed Central

    Nielsen, Judith N.; Charlier, Caroline; Baltes, Nicholas J.; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, Kirsten

    2010-01-01

    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection. PMID:20585559

  8. The cell cycle of symbiotic Chlorella. I. The relationship between host feeding and algal cell growth and division.

    PubMed

    McAuley, P J

    1985-08-01

    When green hydra were starved, cell division of the symbiotic algae within their digestive cells was inhibited, but algal cell growth, measured as increase in either mean volume or protein content per cell, was not. Therefore, control of algal division by the host digestive cells must be effected by direct inhibition of algal mitosis rather than by controlling algal cell growth. The number of algae per digestive cell increased slightly during starvation, eventually reaching a new stable level. A number of experiments demonstrated that although there was a relationship between host cell and algal mitosis, this was not causal: the apparent entrainment of algal mitosis to that of the host cells could be disrupted. Thus, there was a delay in algal but not host cell mitosis when hydra were fed after prolonged starvation, and algae repopulated starved hydra with lower than normal numbers of algae (reinfected aposymbionts or hydra transferred to light after growth in continuous darkness). Two experiments demonstrated a direct stimulation of algal cell division by host feeding. Relationships of algal and host cell mitosis to numbers of Artemia digested per hydra were different, and in hydra fed extracted Artemia algal, but not host cell, mitosis was reduced in comparison to that in control hydra fed live shrimp. It is proposed that algal division may be dependent on a division factor, derived from host digestion of prey, whose supply is controlled by the host cells. Numbers of algae per cell would be regulated by competition for division factor, except at host cell mitosis, when the algae may have temporarily uncontrolled access to host pools of division factor. The identity of the division factor is not known, but presumably is a metabolite needed by both host cells and algae.

  9. Concepts of papillomavirus entry into host cells.

    PubMed

    Day, Patricia M; Schelhaas, Mario

    2014-02-01

    Papillomaviruses enter basal cells of stratified epithelia. Assembly of new virions occurs in infected cells during terminal differentiation. This unique biology is reflected in the mechanism of entry. Extracellularly, the interaction of nonenveloped capsids with several host cell proteins, after binding, results in discrete conformational changes. Asynchronous internalization occurs over several hours by an endocytic mechanism related to, but distinct from macropinocytosis. Intracellular trafficking leads virions through the endosomal system, and from late endosomes to the trans-Golgi-network, before nuclear delivery. Here, we discuss the existing data with the aim to synthesize an integrated model of the stepwise process of entry, thereby highlighting key open questions. Additionally, we relate data from experiments with cultured cells to in vivo results.

  10. The Host Specificities of Baculovirus per os Infectivity Factors

    PubMed Central

    Song, Jingjiao; Wang, Xi; Hou, Dianhai; Huang, Huachao; Liu, Xijia; Deng, Fei; Wang, Hualin; Arif, Basil M.; Hu, Zhihong; Wang, Manli

    2016-01-01

    Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host’s midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses. PMID:27454435

  11. Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

    PubMed

    Torriani, Giulia; Galan-Navarro, Clara; Kunz, Stefan

    2017-02-15

    Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  12. Novel HIV-1 Therapeutics through Targeting Altered Host Cell Pathways

    PubMed Central

    Coley, William; Kehn-Hall, Kylene; Van Duyne, Rachel; Kashanchi, Fatah

    2009-01-01

    The emergence of drug-resistant human immunodeficiency virus type I (HIV-1) strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse-transcriptase, viral protease, or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we will discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host’s proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be performed to better design therapeutics against HIV-1. PMID:19732026

  13. Ongoing graft-versus-host disease is a risk factor for azoospermia after allogeneic hematopoietic stem cell transplantation: a survey of the Late Effects Working Party of the European Group for Blood and Marrow Transplantation.

    PubMed

    Rovó, Alicia; Aljurf, Mahmoud; Chiodi, Sandra; Spinelli, Simonetta; Salooja, Nina; Sucak, Gülsan; Hunter, Ann; Kim, Tan Swee; Socié, Gérard; van Lint, Maria Teresa; Passweg, Jakob R; Arat, Mutlu; Badoglio, Manuela; Tichelli, André

    2013-03-01

    The aim of this study was to assess the degree of spermatogenesis defects in sperm analysis in long-term male survivors after allogeneic hematopoietic stem cell transplantation in order to identify the risk factors related to potential infertility after hematopoietic stem cell transplantation and to provide data on longitudinal sperm recovery after hematopoietic stem cell transplantation. Here, the Late Effects Working Party of the European Group for Blood and Marrow Transplantation reports data of sperm analysis from 224 males who underwent hematopoietic stem cell transplantation. Median time between transplantation and sperm analysis was 63 months (8-275 months). At last sperm analysis, presence of any degree of spermatozoa was reported in 70 (31%) and complete azoospermia in 154 (69%) patients. In multivariate analysis, being conditioned with total body irradiation (RR 7.1; 95% CI: 3.4-14.8) and age over 25 years at transplantation (RR 2.4; 95% CI: 1.09-5.2) were significantly associated with higher risk for azoospermia. In patients not conditioned with total body irradiation, ongoing chronic graft-versus-host disease is the main adverse factor for sperm recovery (RR of 3.11; 95% CI: 1.02-9.47; P=0.045). Already established risk factors, such as total body irradiation and age older than 25 years at hematopoietic stem cell transplantation, were seen to be the most relevant adverse risk factor for sperm production after hematopoietic stem cell transplantation. Furthermore, for the first time, ongoing graft-versus-host disease has been shown to be the most relevant adverse factor for sperm recovery, particularly in patients conditioned without total body irradiation. We also introduce a useful scoring system to predict the probability of male long-term survivors' azoospermia.

  14. Ongoing graft-versus-host disease is a risk factor for azoospermia after allogeneic hematopoietic stem cell transplantation: a survey of the Late Effects Working Party of the European Group for Blood and Marrow Transplantation

    PubMed Central

    Rovó, Alicia; Aljurf, Mahmoud; Chiodi, Sandra; Spinelli, Simonetta; Salooja, Nina; Sucak, Gülsan; Hunter, Ann; Kim, Tan Swee; Socié, Gérard; van Lint, Maria Teresa; Passweg, Jakob R.; Arat, Mutlu; Badoglio, Manuela; Tichelli, André

    2013-01-01

    The aim of this study was to assess the degree of spermatogenesis defects in sperm analysis in long-term male survivors after allogeneic hematopoietic stem cell transplantation in order to identify the risk factors related to potential infertility after hematopoietic stem cell transplantation and to provide data on longitudinal sperm recovery after hematopoietic stem cell transplantation. Here, the Late Effects Working Party of the European Group for Blood and Marrow Transplantation reports data of sperm analysis from 224 males who underwent hematopoietic stem cell transplantation. Median time between transplantation and sperm analysis was 63 months (8–275 months). At last sperm analysis, presence of any degree of spermatozoa was reported in 70 (31%) and complete azoospermia in 154 (69%) patients. In multivariate analysis, being conditioned with total body irradiation (RR 7.1; 95% CI: 3.4–14.8) and age over 25 years at transplantation (RR 2.4; 95% CI: 1.09–5.2) were significantly associated with higher risk for azoospermia. In patients not conditioned with total body irradiation, ongoing chronic graft-versus-host disease is the main adverse factor for sperm recovery (RR of 3.11; 95% CI: 1.02–9.47; P=0.045). Already established risk factors, such as total body irradiation and age older than 25 years at hematopoietic stem cell transplantation, were seen to be the most relevant adverse risk factor for sperm production after hematopoietic stem cell transplantation. Furthermore, for the first time, ongoing graft-versus-host disease has been shown to be the most relevant adverse factor for sperm recovery, particularly in patients conditioned without total body irradiation. We also introduce a useful scoring system to predict the probability of male long-term survivors’ azoospermia. PMID:22929982

  15. Plant cell proliferation inside an inorganic host.

    PubMed

    Perullini, Mercedes; Rivero, María Mercedes; Jobbágy, Matías; Mentaberry, Alejandro; Bilmes, Sara A

    2007-01-10

    In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

  16. Yersinia virulence factors - a sophisticated arsenal for combating host defences

    PubMed Central

    Atkinson, Steve; Williams, Paul

    2016-01-01

    The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six ‘effector’ proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen. PMID:27347390

  17. Yersinia virulence factors - a sophisticated arsenal for combating host defences.

    PubMed

    Atkinson, Steve; Williams, Paul

    2016-01-01

    The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six 'effector' proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen.

  18. The roles of host factors in tombusvirus RNA recombination.

    PubMed

    Nagy, Peter D

    2011-01-01

    RNA viruses are the champions of evolution due to high frequency mutations and genetic recombination occurring during virus replication. These genetic events are due to the error-prone nature of viral RNA-dependent RNA polymerases (RdRp). Recently emerging models on viral RNA recombination, however, also include key roles for host and environmental factors. Accordingly, genome-wide screens and global proteomics approaches with Tomato bushy stunt virus (TBSV) and yeast (Saccharomyces cerevisiae) as a model host have identified 38 host proteins affecting viral RNA recombination. Follow-up studies have identified key host proteins and cellular pathways involved in TBSV RNA recombination. In addition, environmental factors, such as salt stress, have been shown to affect TBSV recombination via influencing key host or viral factors involved in the recombination process. These advances will help build more accurate models on viral recombination, evolution, and adaptation.

  19. Breast cancer cells induce osteoclast formation by stimulating host IL-11 production and downregulating granulocyte/macrophage colony-stimulating factor.

    PubMed

    Morgan, Hayley; Tumber, Anthony; Hill, Peter A

    2004-05-01

    Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by

  20. Transforming growth factor-beta expression by host cells is elicited locally by the filarial nematode Onchocerca volvulus in hyporeactive patients independently from Wolbachia.

    PubMed

    Korten, Simone; Kaifi, Jussuf T; Büttner, Dietrich W; Hoerauf, Achim

    2010-07-01

    Transforming growth factor-beta (TGF-beta) is a key cytokine in immune regulation, cell differentiation, development, wound healing, and tissue remodelling. It mediates immunosuppression in filarial infections facilitating parasite persistence, while attenuating immunopathology, which is induced by migrating microfilariae. Immunosuppression rises with parasite burden, but it remains unknown whether filariae elicit local release of immunosuppressive cytokines. Therefore, using immunohistology, we investigated the expression of stable, released latent TGF-beta1 in subcutaneous nodules from highly infected, hyporeactive onchocerciasis patients, harbouring adult Onchocerca volvulus. Since many cell types produce TGF-beta, we elucidated the cellular source, distribution and dependency on the worms' sex, productivity and vitality. We found TGF-beta1 to be abundantly expressed by T cells, plasma/B cells, macrophages, mast cells, fibrocytes, and vascular endothelial cells, particularly in onchocercomas with productive or previously productive females, damaged, dead and resorbed adult worms or microfilariae. We conclude TGF-beta to be antigen induced by the filariae since expression was scarce around subcutaneous arthropods or cholesterol crystals in onchocercomas. Enhanced expression after ivermectin or endobacteria-depleting doxycycline treatment indicates induction to depend on filariae and not on Wolbachia endobacteria. TGF-beta(+) cells were reduced in HIV co-infection. This finding of local and sustained TGF-beta induction by vital and dead filariae, untreated and after treatment, adds new aspects to immunomodulation by helminths.

  1. Intracellular Voyeurism: Examining the Modulation of Host Cell Activities bySalmonella enterica Serovar Typhimurium.

    PubMed

    Szeto, Jason; Brumell, John H

    2005-11-01

    Salmonella spp. can infect host cells by gaining entry through phagocytosis or by inducing host cell membrane ruffling that facilitates bacterial uptake. With its wide host range, Salmonella enterica serovar Typhimurium has proven to be an important model organism for studying intracellular bacterial pathogenesis. Upon entry into host cells, serovar Typhimurium typically resides within a membrane-bound compartment termed the Salmonella-containing vacuole (SCV). From the SCV, serovar Typhimurium can inject several effector proteins that subvert many normal host cell systems, including endocytic trafficking, cytoskeletal rearrangements, lipid signaling and distribution, and innate and adaptive host defenses. The study of these intracellular events has been made possible through the use of various imaging techniques, ranging from classic methods of transmission electron microscopy to advanced livecell fluorescence confocal microscopy. In addition, DNA microarrays have now been used to provide a "snapshot" of global gene expression in serovar Typhimurium residing within the infected host cell. This review describes key aspects of Salmonella-induced subversion of host cell activities, providing examples of imaging that have been used to elucidate these events. Serovar Typhimurium engages specific host cell machinery from initial contact with the host cell to replication within the SCV. This continuous interaction with the host cell has likely contributed to the extensive arsenal that serovar Typhimurium now possesses, including two type III secretion systems, a range of ammunition in the form of TTSS effectors, and a complex genetic regulatory network that coordinates the expression of hundreds of virulence factors.

  2. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

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

  3. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

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

  4. Neurons Are Host Cells for Mycobacterium tuberculosis

    PubMed Central

    Randall, Philippa J.; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M.; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston

    2014-01-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system. PMID:24566619

  5. Neurons are host cells for Mycobacterium tuberculosis.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

    2014-05-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

  6. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  7. Interaction of human tumor viruses with host cell surface receptors and cell entry.

    PubMed

    Schäfer, Georgia; Blumenthal, Melissa J; Katz, Arieh A

    2015-05-22

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  8. Neuroendocrine host factors and inflammatory disease susceptibility.

    PubMed Central

    Ligier, S; Sternberg, E M

    1999-01-01

    The etiology of autoimmune diseases is multifactorial, resulting from a combination of genetically predetermined host characteristics and environmental exposures. As the term autoimmune implies, immune dysfunction and dysregulated self-tolerance are key elements in the pathophysiology of all these diseases. The neuroendocrine and sympathetic nervous systems are increasingly recognized as modulators of the immune response at the levels of both early inflammation and specific immunity. As such, alterations in their response represent a potential mechanism by which pathologic autoimmunity may develop. Animal models of autoimmune diseases show pre-existing changes in neuroendocrine responses to a variety of stimuli, and both animal and human studies have shown altered stress responses in the setting of active immune activation. The potential role of the neuroendocrine system in linking environmental exposures and autoimmune diseases is 2-fold. First, it may represent a direct target for toxic compounds. Second, its inadequate function may result in the inappropriate response of the immune system to an environmental agent with immunogenic properties. This article reviews the relationship between autoimmune diseases and the neuroendocrine system and discusses the difficulties and pitfalls of investigating a physiologic response that is sensitive to such a multiplicity of environmental exposures. PMID:10502534

  9. Host cell contact induces expression of virulence factors and VieA, a cyclic di-GMP phosphodiesterase, in Vibrio cholerae.

    PubMed

    Dey, Amit K; Bhagat, Abha; Chowdhury, Rukhsana

    2013-05-01

    Vibrio cholerae, a noninvasive bacterium, colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes the severe fluid loss characteristic of the disease cholera. In this study, we demonstrate that adherence of V. cholerae to the intestinal epithelial cell line INT 407 strongly induces the expression of the major virulence genes ctxAB and tcpA and the virulence regulatory gene toxT. No induction of toxR and tcpP, which encode transcriptional activators of toxT, was observed in adhered bacteria, and the adherence-dependent upregulation of toxT expression was independent of ToxR and TcpP. A sharp increase in the expression of the vieA gene, which encodes a cyclic di-GMP (c-di-GMP) phosphodiesterase, was observed in INT 407-adhered V. cholerae immediately after infection. Induction of toxT, ctxAB, and tcpA in INT 407-adhered vieA mutant strain O395 ΔvieA was consistently lower than in the parent strain, although no effect was observed in unadhered bacteria, suggesting that VieA has a role in the upregulation of toxT expression specifically in host cell-adhered V. cholerae. Furthermore, though VieA has both a DNA binding helix-turn-helix domain and an EAL domain conferring c-di-GMP phosphodiesterase activity, the c-di-GMP phosphodiesterase activity of VieA is necessary and sufficient for the upregulation of toxT expression.

  10. Bacterial effectors target the plant cell nucleus to subvert host transcription

    PubMed Central

    Canonne, Joanne; Rivas, Susana

    2012-01-01

    In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells. PMID:22353865

  11. Host restriction factors in retroviral infection: promises in virus-host interaction

    PubMed Central

    2012-01-01

    Retroviruses have an intricate life cycle. There is much to be learned from studying retrovirus-host interactions. Among retroviruses, the primate lentiviruses have one of the more complex genome structures with three categories of viral genes: structural, regulatory, and accessory genes. Over time, we have gained increasing understanding of the lentivirus life cycle from studying host factors that support virus replication. Similarly, studies on host restriction factors that inhibit viral replication have also made significant contributions to our knowledge. Here, we review recent progress on the rapidly growing field of restriction factors, focusing on the antiretroviral activities of APOBEC3G, TRIM5, tetherin, SAMHD1, MOV10, and cellular microRNAs (miRNAs), and the counter-activities of Vif, Vpu, Vpr, Vpx, and Nef. PMID:23254112

  12. Host cell kinases and the hepatitis C virus life cycle.

    PubMed

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Induction of virulence factors in Giardia duodenalis independent of host attachment

    PubMed Central

    Emery, Samantha J.; Mirzaei, Mehdi; Vuong, Daniel; Pascovici, Dana; Chick, Joel M.; Lacey, Ernest; Haynes, Paul A.

    2016-01-01

    Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response. PMID:26867958

  14. Eimeria bovis: an update on parasite-host cell interactions.

    PubMed

    Hermosilla, Carlos; Ruiz, Antonio; Taubert, Anja

    2012-10-01

    Apicomplexan parasites are obligate intracellular protozoans and are well recognized modulators of the host cell machinery on varying levels such as host cell metabolism, MHC expression, cell cycle, or apoptosis in order to guarantee their intracellular development and survival. One of the most thoroughly examined apicomplexan pathogens demonstrating a potent manipulative capacity with respect to various host cell functions is Toxoplasma gondii, a protozoon exhibiting rapid intracellular development with small meronts in any nucleated cell, almost irrespective of the cell type or host origin. In contrast, Eimeria bovis merogony I is host- and cell type-restricted and occurs exclusively in bovine endothelial host cells. Furthermore, as a peculiarity, intracellular E. bovis meront I development is a long-lasting process (up to 3 weeks), leading to the formation of huge macromeronts of up to 300 μm in size, containing up to 120,000 merozoites I as offspring. In consequence, the necessity for intense host cell modulation to support this particular development appears even more pressing than in other apicomplexan parasite cases. Here we review the data currently available on E. bovis-host cell interactions, indicating the intriguing capacity of this protozoan to exploit and utilize its host cell for its own benefit.

  15. Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells.

    PubMed

    Devadas, Krishnakumar; Biswas, Santanu; Haleyurgirisetty, Mohan; Wood, Owen; Ragupathy, Viswanath; Lee, Sherwin; Hewlett, Indira

    2016-01-01

    HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2.

  16. Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells

    PubMed Central

    Devadas, Krishnakumar; Biswas, Santanu; Haleyurgirisetty, Mohan; Wood, Owen; Ragupathy, Viswanath; Lee, Sherwin; Hewlett, Indira

    2016-01-01

    HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2. PMID:26821323

  17. Pathogen Cell-to-cell Variability Drives Heterogeneity In Host Immune Responses

    PubMed Central

    Avraham, Roi; Haseley, Nathan; Brown, Douglas; Penaranda, Cristina; Jijon, Humberto B; Trombetta, John J; Satija, Rahul; Shalek, Alex K; Xavier, Ramnik; Regev, Aviv; Hung, Deborah T

    2015-01-01

    Summary Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize gene expression variation that underlies distinct infection outcomes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers, monitoring infection phenotypes. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host Type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo. PMID:26343579

  18. Analysis of an in vivo model to study the interaction of host factors with Candida albicans.

    PubMed Central

    Poor, A H; Cutler, J E

    1981-01-01

    Conditions were established under which membrane diffusion chambers surgically implanted into mice could be used to study interactions between host defense factors and Candida albicans within the chambers. Depending on the size of membrane pores, soluble host substances and phagocytic cells entered the chambers during the first 24 h after chamber implantation. By 7 days in vivo, the membranes of chambers appeared impermeable to these host factors. Mouse phagocytic cells were found to be functional within the in vivo chambers whether the cells emigrated to the chambers on their own accord or were placed there before chamber implantation. Opsonic factors such as antibody and complement appeared to be biologically functional within the in vivo chambers. Conditions suitable for harvesting C. albicans from the chambers also were determined. PMID:7014455

  19. Virulence Factors and Strategies of Leptopilina spp.: Selective Responses in Drosophila Hosts

    PubMed Central

    Lee, Mark J.; Kalamarz, Marta E.; Paddibhatla, Indira; Small, Chiyedza; Rajwani, Roma; Govind, Shubha

    2010-01-01

    To ensure survival, parasitic wasps of Drosophila have evolved strategies to optimize host development to their advantage. They also produce virulence factors that allow them to overcome or evade host defense. Wasp infection provokes cellular and humoral defense reactions, resulting in alteration in gene expression of the host. The activation of these reactions is controlled by conserved mechanisms shared by other invertebrate and vertebrate animals. Application of genomics and bioinformatics approaches is beginning to reveal comparative host gene expression changes after infection by different parasitic wasps. We analyze this comparison in the context of host physiology and immune cells, as well as the biology of the venom factors that wasps introduce into their hosts during oviposition. We compare virulence strategies of Leptopilina boulardi and L. heterotoma, in relation to genome-wide changes in gene expression in the fly hosts after infection. This analysis highlights fundamental differences in the changes that the host undergoes in its immune and general physiology in response to the two parasitic wasps. Such a comparative approach has the potential of revealing mechanisms governing the evolution of pathogenicity and how it impacts host range. PMID:19773069

  20. Early Vertebrate Evolution of the Host Restriction Factor Tetherin

    PubMed Central

    Heusinger, Elena; Kluge, Silvia F.; Kirchhoff, Frank

    2015-01-01

    release of newly formed progeny virions from infected cells. Although tetherin targets a broad range of enveloped viruses, including retro-, filo-, herpes-, and arenaviruses, the evolutionary origin of this restriction factor and its antiviral activity remained obscure. Here, we examined diverse vertebrate genomes for genes encoding cellular proteins that share with tetherin the highly unusual combination of an N-terminal transmembrane domain and a C-terminal glycosylphosphatidylinositol anchor. We show that tetherin orthologs are found in fish, reptiles, and birds and demonstrate that alligator tetherin efficiently inhibits the release of retroviral particles. Our findings identify tetherin as an evolutionarily ancient restriction factor and provide new important insights into the continuous arms race between viruses and their hosts. PMID:26401043

  1. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  2. Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

    PubMed Central

    Tawaratsumida, Kazuki; Phan, Van; Hrincius, Eike R.; High, Anthony A.; Webby, Richard; Redecke, Vanessa

    2014-01-01

    ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host

  3. Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

    PubMed Central

    Padi, Megha; Korkhin, Anna; James, Robert L.; Adelmant, Guillaume; Yoon, Rosa; Guo, Luxuan; Berrios, Christian; Zhang, Ying; Calderwood, Michael A.; Velmurgan, Soundarapandian; Cheng, Jingwei; Marto, Jarrod A.; Hill, David E.; Cusick, Michael E.; Vidal, Marc; Florens, Laurence; Washburn, Michael P.; Litovchick, Larisa; DeCaprio, James A.

    2012-01-01

    The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. PMID:23093934

  4. Variation in RNA Virus Mutation Rates across Host Cells

    PubMed Central

    Combe, Marine; Sanjuán, Rafael

    2014-01-01

    It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10−6 to 10−4 substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10−5 s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature. PMID:24465205

  5. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors.

    PubMed

    Park, Ryan J; Wang, Tim; Koundakjian, Dylan; Hultquist, Judd F; Lamothe-Molina, Pedro; Monel, Blandine; Schumann, Kathrin; Yu, Haiyan; Krupzcak, Kevin M; Garcia-Beltran, Wilfredo; Piechocka-Trocha, Alicja; Krogan, Nevan J; Marson, Alexander; Sabatini, David M; Lander, Eric S; Hacohen, Nir; Walker, Bruce D

    2017-02-01

    Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.

  6. Interactions between host factors and the skin microbiome

    PubMed Central

    SanMiguel, Adam; Grice, Elizabeth A.

    2015-01-01

    The skin is colonized by an assemblage of microorganisms which, for the most part, peacefully coexist with their hosts. In some cases, these communities also provide vital functions to cutaneous health through the modulation of host factors. Recent studies have illuminated the role of anatomical skin site, gender, age, and the immune system in shaping the cutaneous ecosystem. Alterations to microbial communities have also been associated with, and likely contribute to, a number of cutaneous disorders. This review focuses on the host factors that shape and maintain skin microbial communities, and the reciprocal role of microbes in modulating skin immunity. A greater understanding of these interactions is critical to elucidating the forces that shape cutaneous populations and their contributions to skin homeostasis. This knowledge can also inform the tendency of perturbations to predispose and/or bring about certain skin disorders. PMID:25548803

  7. Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

    PubMed

    Fischer, Annette; Rudel, Thomas

    2016-05-13

    Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

  8. Methods for production of proteins in host cells

    DOEpatents

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  9. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  10. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  11. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens.

    PubMed

    Colonne, Punsiri M; Winchell, Caylin G; Voth, Daniel E

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  12. Host Factors in the Infection Cycle of Bamboo mosaic virus

    PubMed Central

    Huang, Ying-Ping; Chen, I-Hsuan; Tsai, Ching-Hsiu

    2017-01-01

    To complete the infection cycle efficiently, the virus must hijack the host systems in order to benefit for all the steps and has to face all the defense mechanisms from the host. This review involves a discussion of how these positive and negative factors regulate the viral RNA accumulation identified for the Bamboo mosaic virus (BaMV), a single-stranded RNA virus. The genome of BaMV is approximately 6.4 kb in length, encoding five functional polypeptides. To reveal the host factors involved in the infection cycle of BaMV, a few different approaches were taken to screen the candidates. One of the approaches is isolating the viral replicase-associated proteins by co-immunoprecipitation with the transiently expressed tagged viral replicase in plants. Another approach is using the cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes derived from N. benthamiana plants after infection. The candidates are examined by knocking down the expression in plants using the Tobacco rattle virus-based virus-induced gene silencing technique following BaMV inoculation. The positive or negative regulators could be described as reducing or enhancing the accumulation of BaMV in plants when the expression levels of these proteins are knocked down. The possible roles of these host factors acting on the accumulation of BaMV will be discussed. PMID:28360904

  13. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface.

    PubMed

    Oberli, Alexander; Slater, Leanne M; Cutts, Erin; Brand, Françoise; Mundwiler-Pachlatko, Esther; Rusch, Sebastian; Masik, Martin F G; Erat, Michèle C; Beck, Hans-Peter; Vakonakis, Ioannis

    2014-10-01

    Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome. © FASEB.

  14. Early Bunyavirus-Host Cell Interactions.

    PubMed

    Albornoz, Amelina; Hoffmann, Anja B; Lozach, Pierre-Yves; Tischler, Nicole D

    2016-05-24

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion.

  15. Early Bunyavirus-Host Cell Interactions

    PubMed Central

    Albornoz, Amelina; Hoffmann, Anja B.; Lozach, Pierre-Yves; Tischler, Nicole D.

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  16. Inhibition of Plasmodium sporozoites infection by targeting the host cell

    PubMed Central

    Leitao, Ricardo; Rodriguez, Ana

    2010-01-01

    There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and P. yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion. PMID:20493847

  17. Factors affecting host range in a generalist seed pathogen of semi-arid shrublands

    Treesearch

    Julie Beckstead; Susan E. Meyer; Kurt O. Reinhart; Kellene M. Bergen; Sandra R. Holden; Heather F. Boekweg

    2014-01-01

    Generalist pathogens can exhibit differential success on different hosts, resulting in complex host range patterns. Several factors operate to reduce realized host range relative to potential host range, particularly under field conditions. We explored factors influencing host range of the naturally occurring generalist ascomycete grass seed pathogen Pyrenophora...

  18. In situ regeneration of skeletal muscle tissue through host cell recruitment.

    PubMed

    Ju, Young Min; Atala, Anthony; Yoo, James J; Lee, Sang Jin

    2014-10-01

    Standard reconstructive procedures for restoring normal function after skeletal muscle defects involve the use of existing host tissues such as muscular flaps. In many instances, this approach is not feasible and delays the rehabilitation process and restoration of tissue function. Currently, cell-based tissue engineering strategies have been used for reconstruction; however, donor tissue biopsy and ex vivo cell manipulation are required prior to implantation. The present study aimed to overcome these limitations by demonstrating mobilization of muscle cells into a target-specific site for in situ muscle regeneration. First, we investigated whether host muscle cells could be mobilized into an implanted scaffold. Poly(l-lactic acid) (PLLA) scaffolds were implanted in the tibialis anterior (TA) muscle of rats, and the retrieved scaffolds were characterized by examining host cell infiltration in the scaffolds. The host cell infiltrates, including Pax7+ cells, gradually increased with time. Second, we demonstrated that host muscle cells could be enriched by a myogenic factor released from the scaffolds. Gelatin-based scaffolds containing a myogenic factor were implanted in the TA muscle of rats, and the Pax7+ cell infiltration and newly formed muscle fibers were examined. By the second week after implantation, the Pax7+ cell infiltrates and muscle formation were significantly accelerated within the scaffolds containing insulin-like growth factor 1 (IGF-1). Our data suggest an ability of host stem cells to be recruited into the scaffolds with the capability of differentiating to muscle cells. In addition, the myogenic factor effectively promoted host cell recruitment, which resulted in accelerating muscle regeneration in situ.

  19. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host.

  20. Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors.

    PubMed

    Anacker, Brian L; Rank, Nathan E; Hüberli, Daniel; Garbelotto, Matteo; Gordon, Sarah; Harnik, Tami; Whitkus, Richard; Meentemeyer, Ross

    2008-01-01

    Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.

  1. Increased interferon-gamma, interleukin-12p40 and IL-8 production in Propionibacterium acnes-treated peripheral blood mononuclear cells from patient with acne vulgaris: host response but not bacterial species is the determinant factor of the disease.

    PubMed

    Sugisaki, Hitomi; Yamanaka, Keiichi; Kakeda, Masato; Kitagawa, Hiroshi; Tanaka, Kaori; Watanabe, Kunitomo; Gabazza, Esteban C; Kurokawa, Ichiro; Mizutani, Hitoshi

    2009-07-01

    Acne vulgaris is a multifactorial inflammatory disease of the sebaceous follicles of the face and torso that frequently occurs in adolescence. Initially, acne starts as a non-inflammatory comedo. Subsequently, inflammatory reactions evolve to pustules, granulomas and cystic lesions. Many pathogenic mechanisms have been proposed including sebum excretion, obstruction of hair follicles, impaired keratinization of hair epithelium, bacterial overgrowth and immunological mechanisms; the role of Propionibacterium acnes (P. acnes) is particularly important. Facultative anaerobic gram-positive rods have been implicated in acne pathogenesis. However, the host immune response to P. acnes has not been as yet elucidated. The aim of the present study is to evaluate the importance of the immune response to P. acnes and the bacteriological factor in the pathogenesis of acne. P. acnes isolated from acne lesions and healthy volunteers skin were cultured. The peripheral blood mononuclear cells (PBMC) from acne patients or healthy volunteers were stimulated with viable P. acnes, and cytokine production was evaluated using RT-PCR and ELISA. IFN-gamma, IL-12p40, and IL-8 mRNA and protein production were significantly increased in PBMC from acne patients compared to that from normal donors. However, different P. acnes species isolated from acne lesions or normal subjects showed no difference in cytokines production from acne patients and normal subjects PBMC. The inflammatory response of acne appears to be attributable to P. acnes-induced host immune response rather than P. acnes strains from normal skin or acne lesions.

  2. Exploring the role of host cell chaperones/PPIases during cellular up-take of bacterial ADP-ribosylating toxins as basis for novel pharmacological strategies to protect mammalian cells against these virulence factors.

    PubMed

    Barth, Holger

    2011-03-01

    Bacterial exotoxins exploit protein transport pathways of their mammalian target cells to deliver their enzymatic active moieties into the cytosol. There, they modify their specific substrate molecules resulting in cell damage and the clinical symptoms characteristic for each individual toxin. We have investigated the cellular uptake of the binary actin ADP-ribosylating C2 toxin from Clostridium botulinum and the binary lethal toxin from Bacillus anthracis, a metalloprotease. Both toxins are composed of a binding/translocation component and a separate enzyme component. During cellular uptake, the binding/translocation components form pores in membranes of acidified endosomes, and the enzyme components translocate as unfolded proteins through the pores into the cytosol. We found by using specific pharmacological inhibitors that the host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase cyclophilin A are crucial for membrane translocation of the enzyme component of the C2 toxin but not of the lethal toxin, although the structures of the binding/translocation components and the overall uptake mechanisms of both toxins are widely comparable. In conclusion, the new findings imply that Hsp90 and cyclophilin function selectively in promoting translocation of certain bacterial toxins depending on the enzyme domains of the individual toxins. The targeted pharmacological inhibition of individual host cell chaperones/PPIases prevents uptake of certain bacterial exotoxins into the cytosol of mammalian cells and thus protects cells from intoxication. Such substances could represent attractive lead substances for development of novel therapeutics to prevent toxic effects during infection with toxin-producing bacteria.

  3. Host and Environmental Factors Influencing Individual Human Cytokine Responses.

    PubMed

    Ter Horst, Rob; Jaeger, Martin; Smeekens, Sanne P; Oosting, Marije; Swertz, Morris A; Li, Yang; Kumar, Vinod; Diavatopoulos, Dimitri A; Jansen, Anne F M; Lemmers, Heidi; Toenhake-Dijkstra, Helga; van Herwaarden, Antonius E; Janssen, Matthijs; van der Molen, Renate G; Joosten, Irma; Sweep, Fred C G J; Smit, Johannes W; Netea-Maier, Romana T; Koenders, Mieke M J F; Xavier, Ramnik J; van der Meer, Jos W M; Dinarello, Charles A; Pavelka, Norman; Wijmenga, Cisca; Notebaart, Richard A; Joosten, Leo A B; Netea, Mihai G

    2016-11-03

    Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    PubMed

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  5. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

    PubMed Central

    Wu, Kan Xing; Phuektes, Patchara; Kumar, Pankaj; Goh, Germaine Yen Lin; Moreau, Dimitri; Chow, Vincent Tak Kwong; Bard, Frederic; Chu, Justin Jang Hann

    2016-01-01

    Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets. PMID:27748395

  6. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle.

    PubMed

    Catta-Preta, Carolina M C; Brum, Felipe L; da Silva, Camila C; Zuma, Aline A; Elias, Maria C; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number.

  7. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

    PubMed Central

    Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

  8. A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes

    PubMed Central

    Sulaiman, Azad A.; Zolnierczyk, Katarzyna; Japa, Ornampai; Owen, Jonathan P.; Maddison, Ben C.; Hodgkinson, Jane E.; Gough, Kevin C.

    2016-01-01

    The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced

  9. Innate lymphoid cells in graft-versus-host disease.

    PubMed

    Konya, V; Mjösberg, J

    2015-11-01

    Innate lymphoid cells (ILC) are lymphocytes lacking rearranged antigen receptors such as those expressed by T and B cells. ILC are important effector and regulatory cells of the innate immune system, controlling lymphoid organogenesis, tissue inflammation, and homeostasis. The family of ILC consists of cytotoxic NK cells and the more recently described noncytotoxic group 1, 2, and 3 ILC. The classification of noncytotoxic ILC-in many aspects-mirrors that of T helper cells, which is based on the expression of master transcription factors and signature cytokines specific for each subset. The IL-22 producing RORγt(+) ILC3 subset was recently found to be critical in the prevention of intestinal graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (HCT) via strengthening the intestinal mucosal barrier. In this review, we summarize the current view of the immunological functions of human noncytotoxic ILC subsets and discuss the potentially beneficial features of IL-22 producing ILC3 in improving allo-HCT efficacy by attenuating susceptibility to GVHD. In addition, we explore the possibility of other ILC subsets playing a role in GVHD. © 2015 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of American Society of Transplant Surgeons.

  10. Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice.

    PubMed

    Vercauteren, Koen; Brown, Richard J P; Mesalam, Ahmed Atef; Doerrbecker, Juliane; Bhuju, Sabin; Geffers, Robert; Van Den Eede, Naomi; McClure, C Patrick; Troise, Fulvia; Verhoye, Lieven; Baumert, Thomas; Farhoudi, Ali; Cortese, Riccardo; Ball, Jonathan K; Leroux-Roels, Geert; Pietschmann, Thomas; Nicosia, Alfredo; Meuleman, Philip

    2016-12-01

    Direct-acting antivirals (DAAs) inhibit hepatitis C virus (HCV) infection by targeting viral proteins that play essential roles in the replication process. However, selection of resistance-associated variants (RAVs) during DAA therapy has been a cause of therapeutic failure. In this study, we wished to address whether such RAVs could be controlled by the co-administration of host-targeting entry inhibitors that prevent intrahepatic viral spread. We investigated the effect of adding an entry inhibitor (the anti-scavenger receptor class B type I mAb1671) to a DAA monotherapy (the protease inhibitor ciluprevir) in human-liver mice chronically infected with HCV of genotype 1b. Clinically relevant non-laboratory strains were used to achieve viraemia consisting of a cloud of related viral variants (quasispecies) and the emergence of RAVs was monitored at high resolution using next-generation sequencing. HCV-infected human-liver mice receiving DAA monotherapy rapidly experienced on-therapy viral breakthrough. Deep sequencing of the HCV protease domain confirmed the manifestation of drug-resistant mutants upon viral rebound. In contrast, none of the mice treated with a combination of the DAA and the entry inhibitor experienced on-therapy viral breakthrough, despite detection of RAV emergence in some animals. This study provides preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses. Our approach is an excellent strategy to prevent therapeutic failure caused by on-therapy rebound of DAA-RAVs. Inclusion of an entry inhibitor to the newest DAA combination therapies may further increase response rates, especially in difficult-to-treat patient populations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  11. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  12. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  13. Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

    PubMed

    Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

    2016-03-01

    Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research.

  14. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

    PubMed

    Alderete, J F; Garza, G E

    1985-12-01

    The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface

  15. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

    PubMed Central

    Alderete, J F; Garza, G E

    1985-01-01

    The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface

  16. Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

    PubMed

    Mehlitz, Adrian; Eylert, Eva; Huber, Claudia; Lindner, Buko; Vollmuth, Nadine; Karunakaran, Karthika; Goebel, Werner; Eisenreich, Wolfgang; Rudel, Thomas

    2017-03-01

    Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with (13) C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous (13) C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous (13) C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

  17. Chew on this: Amoebic trogocytosis and host cell killing by Entamoeba histolytica

    PubMed Central

    Ralston, Katherine S.

    2015-01-01

    Entamoeba histolytica was named “histolytica” (histo-: tissue; lytic-: dissolving) for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. Here we review this process, termed “amoebic trogocytosis” (trogo-: nibble), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. “Nibbling” processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange. PMID:26070402

  18. Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment.

    PubMed

    Alvarez, Maite; Sun, Kai; Murphy, William J

    2016-03-03

    Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as "licensed" and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively.

  19. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis

    PubMed Central

    Barott, Katie L.; Venn, Alexander A.; Perez, Sidney O.; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-01

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H+-ATPase (VHA), which acidifies the symbiosome space down to pH ∼4. Inhibition of VHA results in a significant decrease in average H+ activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts. PMID:25548188

  20. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

    PubMed

    Barott, Katie L; Venn, Alexander A; Perez, Sidney O; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-13

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

  1. Capacitive immunosensor for the detection of host cell proteins.

    PubMed

    Teeparuksapun, Kosin; Hedström, Martin; Kanatharana, Proespichaya; Thavarungkul, Panote; Mattiasson, Bo

    2012-01-01

    A new analysis for monitoring host cell proteins in preparations of transgenically produced protein pharmaceuticals is described. A capacitive biosensor with a very high sensitivity is used to monitor trace amounts of host cell proteins. The sensor consists of a gold electrode, the surface of which is well insulated and on which a preparation of a population of polyclonal antibodies raised against the complete protein set-up of the host cell are immobilized. Host cell proteins are present at very low concentrations during the production of a transgenic protein. The system studied here is a model system with an enzyme expressed in Escherichia coli (E. coli). Due to the high sensitivity, it may even be possible to dilute the samples to be analyzed, thereby reducing a negative influence from non-specific binding to the sensor surface.

  2. CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.

    PubMed

    Li, Da-Jiang; Verma, Dinesh; Mosbruger, Tim; Swaminathan, Sankar

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as

  3. Fungal Invasion of Normally Non-Phagocytic Host Cells

    PubMed Central

    Filler, Scott G; Sheppard, Donald C

    2006-01-01

    Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research. PMID:17196036

  4. Fungal invasion of normally non-phagocytic host cells.

    PubMed

    Filler, Scott G; Sheppard, Donald C

    2006-12-01

    Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

  5. Host cells and methods for producing isoprenyl alkanoates

    SciTech Connect

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  6. Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

    PubMed Central

    de Macedo Dossin, Fernando; Choi, Seo Yeon; Kim, Nam Youl; Kim, Hi Chul; Jung, Sung Yong; Schenkman, Sergio; Almeida, Igor C.; Emans, Neil; Freitas-Junior, Lucio H.

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. PMID:21625474

  7. Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

    PubMed Central

    Leitão, Elsa; Costa, Ana Catarina; Brito, Cláudia; Costa, Lionel; Pombinho, Rita; Cabanes, Didier; Sousa, Sandra

    2014-01-01

    Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation. PMID:24552813

  8. Select host restriction factors are associated with HIV persistence during antiretroviral therapy.

    PubMed

    Abdel-Mohsen, Mohamed; Wang, Charlene; Strain, Matthew C; Lada, Steven M; Deng, Xutao; Cockerham, Leslie R; Pilcher, Christopher D; Hecht, Frederick M; Liegler, Teri; Richman, Douglas D; Deeks, Steven G; Pillai, Satish K

    2015-02-20

    The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-long terminal repeat (2-LTR) circle HIV-1 DNA and immunophenotypes of CD4 T cells in 72 HIV-1-infected individuals on suppressive ART (23 individuals initiated ART less than 1 year post-infection, and 49 individuals initiated ART greater than 1 year post-infection). Correlations were analysed using nonparametric tests. The enhanced expression of a few select host restriction factors, p21, schlafen 11 and PAF1, was strongly associated with reduced CD4 T-cell associated HIV RNA during ART (P < 0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4 T-cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in individuals who initiated ART during early versus chronic infection and may contribute to the reduced reservoir size observed in these individuals. Intrinsic immune responses modulate HIV persistence during suppressive ART and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo.

  9. Select Host Restriction Factors Are Associated with HIV Persistence During Antiretroviral Therapy

    PubMed Central

    ABDEL-MOHSEN, Mohamed; WANG, Charlene; STRAIN, Matthew C.; LADA, Steven M.; DENG, Xutao; COCKERHAM, Leslie R.; PILCHER, Christopher D.; HECHT, Frederick M.; LIEGLER, Teri; RICHMAN, Douglas D.; DEEKS, Steven G.; PILLAI, Satish K.

    2015-01-01

    Objective The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. Design We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. Methods We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-LTR circle HIV-1 DNA, and immunophenotypes of CD4+ T cells in 72 HIV-1-infected subjects on suppressive ART (23 subjects initiated ART <1 year post-infection, and 49 subjects initiated ART >1 year post-infection). Correlations were analyzed using non-parametric tests. Results The enhanced expression of a few select host restriction factors, p21, schlafen 11, and PAF1, was strongly associated with reduced CD4+ T cell-associated HIV RNA during ART (p<0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4+ T cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in subjects who initiated ART during early versus chronic infection, and may contribute to the reduced reservoir size observed in these individuals. Conclusions Intrinsic immune responses modulate HIV persistence during suppressive ART, and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo. PMID:25602681

  10. Similarity in viral and host promoters couples viral reactivation with host cell migration

    PubMed Central

    Bohn-Wippert, Kathrin; Tevonian, Erin N.; Megaridis, Melina R.; Dar, Roy D.

    2017-01-01

    Viral–host interactomes map the complex architecture of an evolved arms race during host cell invasion. mRNA and protein interactomes reveal elaborate targeting schemes, yet evidence is lacking for genetic coupling that results in the co-regulation of promoters. Here we compare viral and human promoter sequences and expression to test whether genetic coupling exists and investigate its phenotypic consequences. We show that viral–host co-evolution is imprinted within promoter gene sequences before transcript or protein interactions. Co-regulation of human immunodeficiency virus (HIV) and human C-X-C chemokine receptor-4 (CXCR4) facilitates migration of infected cells. Upon infection, HIV can actively replicate or remain dormant. Migrating infected cells reactivate from dormancy more than non-migrating cells and exhibit differential migration–reactivation responses to drugs. Cells producing virus pose a risk for reinitiating infection within niches inaccessible to drugs, and tuning viral control of migration and reactivation improves strategies to eliminate latent HIV. Viral–host genetic coupling establishes a mechanism for synchronizing transcription and guiding potential therapies. PMID:28462923

  11. The Golgi associated ERI3 is a Flavivirus host factor

    PubMed Central

    Ward, Alex Michael; Calvert, Meredith E. K.; Read, Leah R.; Kang, Seokyoung; Levitt, Brandt E.; Dimopoulos, George; Bradrick, Shelton S.; Gunaratne, Jayantha; Garcia-Blanco, Mariano A.

    2016-01-01

    Dengue virus (DENV) is a mosquito-borne Flavivirus classified into four serotypes (DENV-1-4) that causes Dengue fever (DF), Dengue hemorrhagic Fever (DHF) or Dengue shock syndrome (DSS). An estimated 390 million people are at risk for infection with DENV and there are no effective vaccines or therapeutics. We utilized RNA chromatography coupled with quantitative mass spectrometry (qMS) to identify host RNA binding proteins (RBPs) that interact with DENV-2 RNA. We identified ERI3 (also PRNPIP and PINT1), a putative 3′–5′ RNA exonuclease, which preferentially associates with DENV-2 genomic RNA via interactions with dumbbell structures in the 3′ UTR. ERI3 is required for accumulation of DENV-2 genomic RNA and production of infectious particles. Furthermore, the mosquito homologue of ERI3 is required for DENV-2 replication in adult Aedes aegypti mosquitos implying that the requirement for ERI3 is conserved in both DENV hosts. In human cells ERI3 localizes to the Golgi in uninfected cells, but relocalizes near sites of DENV-2 replication in infected cells. ERI3 is not required for maintaining DENV-2 RNA stability or translation of the viral polyprotein, but is required for viral RNA synthesis. Our results define a specific role for ERI3 and highlight the importance of Golgi proteins in DENV-2 replication. PMID:27682269

  12. Host-derived, pore-forming toxin–like protein and trefoil factor complex protects the host against microbial infection

    PubMed Central

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng’an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-01-01

    Aerolysins are virulence factors belonging to the bacterial β-pore–forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens. PMID:24733922

  13. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    PubMed

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  14. Identification of Essential Filovirion-associated Host Factors by Serial Proteomic Analysis and RNAi Screen*

    PubMed Central

    Spurgers, Kevin B.; Alefantis, Tim; Peyser, Brian D.; Ruthel, Gordon T.; Bergeron, Alison A.; Costantino, Julie A.; Enterlein, Sven; Kota, Krishna P.; Boltz, R. C. Dutch; Aman, M. Javad; DelVecchio, Vito G.; Bavari, Sina

    2010-01-01

    An assessment of the total protein composition of filovirus (ebolavirus and marburgvirus) virions is currently lacking. In this study, liquid chromatography-linked tandem mass spectrometry of purified ebola and marburg virions was performed to identify associated cellular proteins. Host proteins involved in cell adhesion, cytoskeleton, cell signaling, intracellular trafficking, membrane organization, and chaperones were identified. Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types. However, the great majority of proteins identified here have not been previously described to be incorporated within filovirus particles. Host proteins identified by liquid chromatography-linked tandem mass spectrometry could lack biological relevance because they represent protein contaminants in the virus preparation, or because they are incorporated within virions by chance. These issues were addressed using siRNA library-mediated gene knockdown (targeting each identified virion-associated host protein), followed by filovirus infection. Knockdown of several host proteins (e.g. HSPA5 and RPL18) significantly interfered with ebolavirus and marburgvirus infection, suggesting specific and relevant virion incorporation. Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses. The proteomic analysis presented here contributes to a greater understanding of filovirus biology and potentially identifies host factors that can be targeted for antiviral drug development. PMID:20702783

  15. Dengue virus life cycle: viral and host factors modulating infectivity.

    PubMed

    Rodenhuis-Zybert, Izabela A; Wilschut, Jan; Smit, Jolanda M

    2010-08-01

    Dengue virus (DENV 1-4) represents a major emerging arthropod-borne pathogen. All four DENV serotypes are prevalent in the (sub) tropical regions of the world and infect 50-100 million individuals annually. Whereas the majority of DENV infections proceed asymptomatically or result in self-limited dengue fever, an increasing number of patients present more severe manifestations, such as dengue hemorrhagic fever and dengue shock syndrome. In this review we will give an overview of the infectious life cycle of DENV and will discuss the viral and host factors that are important in controlling DENV infection.

  16. Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

    PubMed

    Hegde, Shrilakshmi; Gabriel, Cordula; Kragl, Martin; Chopra-Dewasthaly, Rohini

    2015-10-01

    Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

  17. Hookworm virulence factors: making the most of the host.

    PubMed

    Periago, Maria V; Bethony, Jeffrey M

    2012-12-01

    Hookworm disease from Necator americanus and Ancylostoma duodenale affects approximately 700 million people, with N. americanus being the predominant species. Unlike other pathogens (e.g., bacterial infections), where "virulence" is described in regards to acute pathogenesis and case-fatality, hookworms are well-evolved, multicellular parasites that establish long-term infections in their human hosts with a subtle and chronic, but insidious, pathogenesis, usually in the form of iron deficiency anemia from parasite blood feeding that, over time, has devastating effects on the human host especially when it involves children or women of child bearing years. As such, many of the typical terms for "virulence factors" used in other reviews in this special edition cannot be applied to hookworm (e.g., "colonization", "invasion", "or "toxicity"); rather the virulence of hookworm infection comes in terms of their ability to maintain a chronic blood-feeding infection in the lumen of relatively healthy human hosts, an infection that is usually measured in years but can sometimes be measured in decades. In the current manuscript, we describe the routes of invasion hookworms take into their human hosts and the means by which they modulate the human immune system to maintain this long-term parasitism. Little data on hookworm infection comes from actual human infections; instead, much of the data is derived from observations of laboratory animal models, in which hookworms fail to establish this distinctive "chronic infection," either due to physiological or immunological responses of these animal models. Hence, the mode and effects of chronic immunity must be extrapolated from this very different sort of infection to humans. Herein, we aim to synthesize immunological information from both types of models in the context of immune regulation and protection in order to identify future research focuses for the development of new treatment alternatives (i.e. drugs and vaccines).

  18. HTLV-1 subgroups associated with the risk of HAM/TSP are related to viral and host gene expression in peripheral blood mononuclear cells, independent of the transactivation functions of the viral factors.

    PubMed

    Yasuma, Keiko; Matsuzaki, Toshio; Yamano, Yoshihisa; Takashima, Hiroshi; Matsuoka, Masao; Saito, Mineki

    2016-08-01

    Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, the risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) across lifetime differs between ethnic groups. There is an association between HTLV-1 tax gene subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. In this study, we investigated the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples. The functional differences in the viral transcriptional regulators Tax and HTLV-1 bZIP factor (HBZ) between each subgroup and the relationships between subgroups and the clinical and laboratory characteristics of HAM/TSP patients were evaluated. The results of these analyses indicated the following: (1) distinct nucleotide substitutions corresponding to each subgroup were associated with nucleotide substitutions in viral structural, regulatory, and accessory genes; (2) the HBZ messenger RNA (mRNA) expression in HTLV-1-infected cells was significantly higher in HAM/TSP patients with subgroup-B than in those with subgroup-A; (3) a positive correlation was observed between the expression of HBZ mRNA and its target Foxp3 mRNA in HAM/TSP patients with subgroup-B, but not in patients with subgroup-A; (4) no clear differences were noted in clinical and laboratory characteristics between HAM/TSP patients with subgroup-A and subgroup-B; and (5) no functional differences were observed in Tax and HBZ between each subgroup based on reporter gene assays. Our results indicate that although different HTLV-1 subgroups are characterized by different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients.

  19. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host

    PubMed Central

    Popoff, Michel R

    2014-01-01

    Actin cytoskeleton is a main target of many bacterial pathogens. Among the multiple regulation steps of the actin cytoskeleton, bacterial factors interact preferentially with RhoGTPases. Pathogens secrete either toxins which diffuse in the surrounding environment, or directly inject virulence factors into target cells. Bacterial toxins, which interfere with RhoGTPases, and to some extent with RasGTPases, catalyze a covalent modification (ADPribosylation, glucosylation, deamidation, adenylation, proteolysis) blocking these molecules in their active or inactive state, resulting in alteration of epithelial and/or endothelial barriers, which contributes to dissemination of bacteria in the host. Injected bacterial virulence factors preferentially manipulate the RhoGTPase signaling cascade by mimicry of eukaryotic regulatory proteins leading to local actin cytoskeleton rearrangement, which mediates bacterial entry into host cells or in contrast escape to phagocytosis and immune defense. Invasive bacteria can also manipulate RhoGTPase signaling through recognition and stimulation of cell surface receptor(s). Changes in RhoGTPase activation state is sensed by the innate immunity pathways and allows the host cell to adapt an appropriate defense response. PMID:25203748

  20. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Voth, Daniel E.

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions. PMID:27713866

  1. Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions*

    PubMed Central

    Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V.; Kwon, Keehwan; Townsend, Katherine; Yu, Chenggang; Yu, Xueping; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

    2013-01-01

    Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin

  2. Transcription factor ATF3 links host adaptive response to breast cancer metastasis

    PubMed Central

    Wolford, Chris C.; McConoughey, Stephen J.; Jalgaonkar, Swati P.; Leon, Marino; Merchant, Anand S.; Dominick, Johnna L.; Yin, Xin; Chang, Yiseok; Zmuda, Erik J.; O’Toole, Sandra A.; Millar, Ewan K.A.; Roller, Stephanie L.; Shapiro, Charles L.; Ostrowski, Michael C.; Sutherland, Robert L.; Hai, Tsonwin

    2013-01-01

    Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell–selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes. PMID:23921126

  3. p53 Is a Host Cell Regulator during Herpes Simplex Encephalitis

    PubMed Central

    Maruzuru, Yuhei; Koyanagi, Naoto; Takemura, Naoki; Uematsu, Satoshi; Matsubara, Daisuke; Suzuki, Yutaka; Arii, Jun; Kato, Akihisa

    2016-01-01

    ABSTRACT p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knockout reduced viral replication in the brains of mice and led to significantly reduced rates of mortality due to herpes simplex encephalitis. These results suggest that p53 is an important host cell regulator of HSV-1 replication and pathogenesis in the central nervous system (CNS). IMPORTANCE HSV-1 causes sporadic cases of encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism, which may suggest novel clinical treatment options for herpes simplex encephalitis. PMID:27170756

  4. p53 Is a Host Cell Regulator during Herpes Simplex Encephalitis.

    PubMed

    Maruzuru, Yuhei; Koyanagi, Naoto; Takemura, Naoki; Uematsu, Satoshi; Matsubara, Daisuke; Suzuki, Yutaka; Arii, Jun; Kato, Akihisa; Kawaguchi, Yasushi

    2016-08-01

    p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knockout reduced viral replication in the brains of mice and led to significantly reduced rates of mortality due to herpes simplex encephalitis. These results suggest that p53 is an important host cell regulator of HSV-1 replication and pathogenesis in the central nervous system (CNS). HSV-1 causes sporadic cases of encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism, which may suggest novel clinical treatment options for herpes simplex encephalitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Cif type III effector protein: a smart hijacker of the host cell cycle.

    PubMed

    Samba-Louaka, Ascel; Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2009-09-01

    During coevolution with their hosts, bacteria have developed functions that allow them to interfere with the mechanisms controlling the proliferation of eukaryotic cells. Cycle inhibiting factor (Cif) is one of these cyclomodulins, the family of bacterial effectors that interfere with the host cell cycle. Acquired early during evolution by bacteria isolated from vertebrates and invertebrates, Cif is an effector protein of type III secretion machineries. Cif blocks the host cell cycle in G1 and G2 by inducing the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). The x-ray crystal structure of Cif reveals it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases. This review summarizes and discusses what we know about Cif, from the bacterial gene to the host target.

  6. Viroporins Customize Host Cells for Efficient Viral Propagation

    PubMed Central

    Giorda, Kristina M.

    2013-01-01

    Viruses are intracellular parasites that must access the host cell machinery to propagate. Viruses hijack the host cell machinery to help with entry, replication, packaging, and release of progeny to infect new cells. To carry out these diverse functions, viruses often transform the cellular environment using viroporins, a growing class of viral-encoded membrane proteins that promote viral proliferation. Viroporins modify the integrity of host membranes, thereby stimulating the maturation of viral infection, and are critical for virus production and dissemination. Significant advances in molecular and cell biological approaches have helped to uncover some of the roles that viroporins serve in the various stages of the viral life cycle. In this study, the ability of viroporins to modify the cellular environment will be discussed, with particular emphasis on their role in the stepwise progression of the viral life cycle. PMID:23945006

  7. Host epithelial geometry regulates breast cancer cell invasiveness.

    PubMed

    Boghaert, Eline; Gleghorn, Jason P; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C; Nelson, Celeste M

    2012-11-27

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.

  8. Bacterial Colonization of Host Cells in the Absence of Cholesterol

    PubMed Central

    Gilk, Stacey D.; Cockrell, Diane C.; Luterbach, Courtney; Hansen, Bryan; Knodler, Leigh A.; Ibarra, J. Antonio; Steele-Mortimer, Olivia; Heinzen, Robert A.

    2013-01-01

    Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24−/− mouse embryonic fibroblasts (MEFs). DHCR24−/− MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24−/− MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24−/− MEFs. In contrast, C. burnetii entry was significantly decreased in −cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated αVβ3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24−/− MEFs lacked the CD63-postive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. PMID:23358892

  9. Aquatic viruses induce host cell death pathways and its application.

    PubMed

    Reshi, Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-01-04

    Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell's death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Molecular mechanisms of host cell traversal by malaria sporozoites.

    PubMed

    Yang, Annie S P; Boddey, Justin A

    2017-02-01

    Malaria is a pernicious infectious disease caused by apicomplexan parasites of the genus Plasmodium. Each year, malaria afflicts over 200million people, causing considerable morbidity, loss to gross domestic product of endemic countries, and more than 420,000 deaths. A central feature of the virulence of malaria parasites is the ability of sporozoite forms injected by a mosquito to navigate from the inoculation site in the skin through host tissues to infect the liver. The ability for sporozoites to traverse through different host cell types is very important for the successful development of parasites within the mammalian host. Over the past decade, our understanding of the role of host cell traversal has become clearer through important studies with rodent models of malaria. However, we still do not understand the stepwise process of host cell entry and exit or know the molecular mechanisms governing each step. We know even less about cell traversal by malaria parasite species that infect humans. Here, we review current knowledge regarding the role and molecular mechanisms of sporozoite cell traversal and highlight recent advances that prompt new ways of thinking about this important process. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  11. Invasion of Host Cells and Tissues by Uropathogenic Bacteria

    PubMed Central

    Lewis, Adam J.; Richards, Amanda C.; Mulvey, Matthew A.

    2016-01-01

    Within the mammalian urinary tract uropathogenic bacteria face many challenges, including the shearing flow of urine, numerous antibacterial molecules, the bactericidal effects of phagocytes, and a scarcity of nutrients. These problems may be circumvented in part by the ability of uropathogenic Escherichia coli (UPEC) and several other uropathogens to invade the epithelial cells that line the urinary tract. By entering host cells, uropathogens can gain access to additional nutrients and protection from both host defenses and antibiotic treatments. Translocation through host cells can facilitate bacterial dissemination within the urinary tract, while the establishment of stable intracellular bacterial populations may create reservoirs for relapsing and chronic urinary tract infections (UTIs). Here we review the mechanisms and consequences of host cell invasion by uropathogenic bacteria, with consideration of the defenses that are brought to bear against facultative intracellular pathogens within the urinary tract. The relevance of host cell invasion to the pathogenesis of UTIs in human patients is also assessed, along with some of the emerging treatment options that build upon our growing understanding of the infectious life cycle of UPEC and other uropathogenic bacteria. PMID:28087946

  12. Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells

    PubMed Central

    Deiser, Katrin; Stoycheva, Diana; Bank, Ute; Blankenstein, Thomas; Schüler, Thomas

    2016-01-01

    The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols. PMID:27447484

  13. Host Cell Factor and an Uncharacterized SANT Domain Protein Are Stable Components of ATAC, a Novel dAda2A/dGcn5-Containing Histone Acetyltransferase Complex in Drosophila

    PubMed Central

    Guelman, Sebastián; Suganuma, Tamaki; Florens, Laurence; Swanson, Selene K.; Kiesecker, Cheri L.; Kusch, Thomas; Anderson, Scott; Yates, John R.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2006-01-01

    Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells. PMID:16428443

  14. Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.

    PubMed

    Capalbo, Gianni; Mueller-Kuller, Thea; Markovic, Sandra; Klein, Stefan A; Dietrich, Ursula; Hoelzer, Dieter; Ottmann, Oliver G; Scheuring, Urban J

    2011-12-01

    Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

  15. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

    PubMed

    Gerold, Gisa; Meissner, Felix; Bruening, Janina; Welsch, Kathrin; Perin, Paula M; Baumert, Thomas F; Vondran, Florian W; Kaderali, Lars; Marcotrigiano, Joseph; Khan, Abdul G; Mann, Matthias; Rice, Charles M; Pietschmann, Thomas

    2015-08-04

    Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Host cell proteins in biotechnology-derived products: A risk assessment framework.

    PubMed

    de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

    2015-11-01

    To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

  17. Role of the host cell in bacteriophage T4 development. II. Characterization of host mutants that have pleiotropic effects on T4 growth.

    PubMed Central

    Stitt, B L; Revel, H R; Lielausis, I; Wood, W B

    1980-01-01

    Mutant host-defective Escherichi coli that fail to propagate bacteriophage T4 and have a pleiotropic effect on T4 development have been isolated and characterized. In phage-infected mutant cells, specific early phage proteins are absent or reduced in amount, phage DNA synthesis is depressed by about 50%, specific structural phage proteins, including some tail and collar components, are deficient or missing, and host-cell lysis is delayed and slow. Almost all phage that can overcome the host block carry mutantions that map in functionally undefined 'nonessential' regions of the T4 genome, most near gene 39. The mutant host strains are temperature sensitive for growth and show simultaneous reversion of the ts phenotype and the inability to propagate T4+. The host mutations are cotransduced with ilv (83 min) and may lie in the gene for transcription termination factor rho. Images PMID:6999171

  18. CRISPR Immunological Memory Requires a Host Factor for Specificity.

    PubMed

    Nuñez, James K; Bai, Lawrence; Harrington, Lucas B; Hinder, Tracey L; Doudna, Jennifer A

    2016-06-16

    Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.

  19. Expression profile of host restriction factors in HIV-1 elite controllers.

    PubMed

    Abdel-Mohsen, Mohamed; Raposo, Rui André Saraiva; Deng, Xutao; Li, Manqing; Liegler, Teri; Sinclair, Elizabeth; Salama, Mohamed S; Ghanem, Hussam El-Din A; Hoh, Rebecca; Wong, Joseph K; David, Michael; Nixon, Douglas F; Deeks, Steven G; Pillai, Satish K

    2013-10-16

    Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals. Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r² = 0.597, p < 0.0001), viral load (r² = 0.34, p = 0.015), and expression of ISG15 (r² = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers. Host restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.

  20. Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation.

    PubMed

    Pillai, Asha B; George, Tracy I; Dutt, Suparna; Teo, Pearline; Strober, Samuel

    2007-05-15

    Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.

  1. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  2. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2015-12-28

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

  3. Bacterial toxins can inhibit host cell autophagy through cAMP generation.

    PubMed

    Shahnazari, Shahab; Namolovan, Anton; Mogridge, Jeremy; Kim, Peter K; Brumell, John H

    2011-09-01

    Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of 'camouflage' proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.

  4. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells.

    PubMed

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-28

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.

  5. The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes

    PubMed Central

    Gamradt, Pia; Xu, Yun; Gratz, Nina; Duncan, Kellyanne; Kobzik, Lester; Högler, Sandra; Decker, Thomas

    2016-01-01

    Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen. PMID:27973535

  6. Host cell infiltration into PDT-treated tumor

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Krosl, Gorazd; Dougherty, Graeme J.; Chaplin, David J.

    1992-06-01

    C3H mice bearing SCCVII squamous cell carcinoma were treated with photodynamic therapy (PDT) 24 hours after receiving Photofrin (25 mg/kg, i.v.). Single cell suspensions obtained by the enzymatic digestion of tumors excised either 30 minutes or 4 hours after PDT were analyzed for the content of host immune cells and colony forming ability of malignant cells. The results were compared to the data obtained with non-treated tumors. It is shown that there is a marked increase in the content of cells expressing Mac-1 (monocytes/macrophages or granulocytes) in the tumor 30 minutes post PDT, while a high level of other leucocytes are found within the tumors by 4 hours after PDT. As elaborated in Discussion, the infiltration rate of host immune cells, dying of malignant tumor cells, and yet unknown death rate of host cells originally present in PDT treated tumor occurring concomitantly during this time period complicates this analysis. The results of this study suggest a massive infiltration of macrophages and other leucocytes in PDT treated SCCVII tumor, supporting the suggestion that a potent immune reaction is one of the main characteristics of PDT action in solid tumors. It remains to be determined to what extent is the activity of tumor infiltrating immune cells responsible for its eradication by PDT.

  7. Identification of Entry Factors Involved in Hepatitis C Virus Infection Based on Host-Mimicking Short Linear Motifs

    PubMed Central

    2017-01-01

    Host factors that facilitate viral entry into cells can, in principle, be identified from a virus-host protein interaction network, but for most viruses information for such a network is limited. To help fill this void, we developed a bioinformatics approach and applied it to hepatitis C virus (HCV) infection, which is a current concern for global health. Using this approach, we identified short linear sequence motifs, conserved in the envelope proteins of HCV (E1/E2), that potentially can bind human proteins present on the surface of hepatocytes so as to construct an HCV (envelope)-host protein interaction network. Gene Ontology functional and KEGG pathway analyses showed that the identified host proteins are enriched in cell entry and carcinogenesis functionalities. The validity of our results is supported by much published experimental data. Our general approach should be useful when developing antiviral agents, particularly those that target virus-host interactions. PMID:28129350

  8. Mechanisms of outer membrane vesicle entry into host cells.

    PubMed

    O'Donoghue, Eloise J; Krachler, Anne Marie

    2016-11-01

    Bacterial outer membrane vesicles (OMVs) are nano-sized compartments consisting of a lipid bilayer that encapsulates periplasm-derived, luminal content. OMVs, which pinch off of Gram-negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV-host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

  9. Mechanisms of outer membrane vesicle entry into host cells

    PubMed Central

    O'Donoghue, Eloise J.

    2016-01-01

    Abstract Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies. PMID:27529760

  10. Toxoplasma Co-opts Host Cells It Does Not Invade

    PubMed Central

    Koshy, Anita A.; Dietrich, Hans K.; Christian, David A.; Melehani, Jason H.; Shastri, Anjali J.; Hunter, Christopher A.; Boothroyd, John C.

    2012-01-01

    Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large. PMID:22910631

  11. The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol.

    PubMed

    Bonassi, Stefano; Coskun, Erdem; Ceppi, Marcello; Lando, Cecilia; Bolognesi, Claudia; Burgaz, Sema; Holland, Nina; Kirsh-Volders, Micheline; Knasmueller, Siegfried; Zeiger, Errol; Carnesoltas, Deyanira; Cavallo, Delia; da Silva, Juliana; de Andrade, Vanessa M; Demircigil, Gonca Cakmak; Domínguez Odio, Aníbal; Donmez-Altuntas, Hamiyet; Gattas, Gilka; Giri, Ashok; Giri, Sarbani; Gómez-Meda, Belinda; Gómez-Arroyo, Sandra; Hadjidekova, Valeria; Haveric, Anja; Kamboj, Mala; Kurteshi, Kemajl; Martino-Roth, Maria Grazia; Montero Montoya, Regina; Nersesyan, Armen; Pastor-Benito, Susana; Favero Salvadori, Daisy Maria; Shaposhnikova, Alina; Stopper, Helga; Thomas, Philip; Torres-Bugarín, Olivia; Yadav, Abhay Singh; Zúñiga González, Guillermo; Fenech, Michael

    2011-01-01

    The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Host cell modulation by human, animal and plant pathogens.

    PubMed

    Andersson, Siv G E; Kempf, Volkhard A J

    2004-04-01

    Members of the alpha-proteobacteria display a broad range of interactions with higher eukaryotes. Some are pathogens of humans, such as Rickettsia and Bartonella that are associated with diseases like epidemic typhus, trench fever, cat scratch disease and bacillary angiomatosis. Others like the Brucella cause abortions in pregnant animals. Yet other species have evolved elaborate interactions with plants; in this group we find both plant symbionts and parasites. Despite radically different host preferences, extreme genome size variations and the absence of toxin genes, similarities in survival strategies and host cell interactions can be recognized among members of the alpha-proteobacteria. Here, we review some of these similarities, with a focus on strategies for modulation of the host target cell.

  13. Functional genomics approach for the identification of human host factors supporting dengue viral propagation.

    PubMed

    Barrows, Nicholas J; Jamison, Sharon F; Bradrick, Shelton S; Le Sommer, Caroline; Kim, So Young; Pearson, James; Garcia-Blanco, Mariano A

    2014-01-01

    Dengue virus (DENV) is endemic throughout tropical regions of the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high-throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle.

  14. Functional genomics approach for the identification of human host factors supporting dengue viral propagation

    PubMed Central

    Barrows, Nicholas J.; Jamison, Sharon F.; Bradrick, Shelton S.; Le Sommer, Caroline; Kim, So Young; Pearson, James; Garcia-Blanco, Mariano A.

    2014-01-01

    Dengue virus (DENV) is endemic throughout tropical regions around the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle. PMID:24696344

  15. Host genetic variation is a contributable factor for imperfectly-immunizing vaccination

    USDA-ARS?s Scientific Manuscript database

    Vaccine protective efficacy is determined by multiple factors including host genetics, vaccine type, vaccine dosage, challenge virus virulence, challenge virus dose, and interval between vaccination and exposure to challenge viruses. About two decades ago, studies conducted to evaluate host genetic ...

  16. A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

    PubMed Central

    Durrani, Zeeshan; Pillai, Sreerekha S.; Baird, Margaret; Shiels, Brian R.

    2013-01-01

    Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner. PMID:23840536

  17. A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression.

    PubMed

    Kinnaird, Jane H; Weir, William; Durrani, Zeeshan; Pillai, Sreerekha S; Baird, Margaret; Shiels, Brian R

    2013-01-01

    Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.

  18. Salidroside exhibits anti-dengue virus activity by upregulating host innate immune factors.

    PubMed

    Sharma, Navita; Mishra, K P; Ganju, Lilly

    2016-12-01

    Dengue is an arboviral disease with no effective therapy available. Therefore, there is an urgent need to find a potent antiviral agent against dengue virus (DENV). In the present study, salidroside, a main bioactive compound of Rhodiola rosea, was evaluated for its antiviral potential against DENV serotype-2 infection and its effect on host innate immune factors. Antiviral effects of salidroside were examined in DENV-infected cells by western blotting, flow cytometry and real-time PCR. Its underlying mechanism involved in antiviral action was determined by evaluating expression of host innate immune factors including RIG-I, IRF-3, IRF-7, PKR, P-eIF2α and NF-κB. Salidroside potently inhibited DENV infection by decreasing DENV envelope protein expression more than tenfold. Salidroside exerts its antiviral activity by increasing expression of RNA helicases such as RIG-I, thereby initiating a downstream signaling cascade that induces upregulation of IRF-3 and IRF-7. It prevents viral protein synthesis by increasing the expression of PKR and P-eIF2α while decreasing NF-κB expression. It was also found to induce the expression of IFN-α. In addition, the number of NK cells and CD8(+) T cells were also found to be increased by salidroside treatment in human PBMCs, which are important in limiting DENV replication during early stages of infection. The findings presented here suggest that salidroside exhibits antiviral activity against DENV by inhibiting viral protein synthesis and boosting host immunity by increasing the expression of host innate immune factors and hence could be considered for the development of an effective therapeutic agent against DENV infection.

  19. Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

    PubMed

    Licona-Limón, Paula; Henao-Mejia, Jorge; Temann, Angela U; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De'broski R; Flavell, Richard A

    2013-10-17

    Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.

  20. Th9 cells drive host immunity against gastrointestinal worm infection

    PubMed Central

    Licona-Limón, Paula; Henao-Mejía, Jorge; Temann, Angela U.; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De’Broski R.; Flavell, Richard A.

    2013-01-01

    Type 2 inflammatory cytokines including interleukin-4 (IL-4), -5, -9, and -13 drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial and the importance of IL-9 vs. IL-4 producing CD4+ effector T cells in Type 2 immunity is incompletely defined. Herein, we generated IL-9 deficient and IL-9 fluorescent reporter mice that demonstrated an essential role for this cytokine in the early Type 2 immunity against Nippostrongylus brasiliensis. Whereas Th9 cells and Type 2 Innate Lymphoid Cells (ILC2) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells caused rapid worm expulsion, marked basophilia and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and non-redundant role for Th9 cells and IL-9 in host protective Type 2 immunity against parasitic worm infection. PMID:24138883

  1. Impact of sex on prognostic host factors in surgical patients with lung cancer.

    PubMed

    Wainer, Zoe; Wright, Gavin M; Gough, Karla; Daniels, Marissa G; Choong, Peter; Conron, Matthew; Russell, Prudence A; Alam, Naveed Z; Ball, David; Solomon, Benjamin

    2016-09-14

    Lung cancer has markedly poorer survival in men. Recognized important prognostic factors are divided into host, tumour and environmental factors. Traditional staging systems that use only tumour factors to predict prognosis are of limited accuracy. By examining sex-based patterns of disease-specific survival in non-small cell lung cancer patients, we determined the effect of sex on the prognostic value of additional host factors. Two cohorts of patients treated surgically with curative intent between 2000 and 2009 were utilized. The primary cohort was from Melbourne, Australia, with an independent validation set from the American Surveillance, Epidemiology and End Results (SEER) database. Univariate and multivariate analyses of validated host-related prognostic factors were performed in both cohorts to investigate the differences in survival between men and women. The Melbourne cohort had 605 patients (61% men) and SEER cohort comprised 55 681 patients (51% men). Disease-specific 5-year survival showed men had statistically significant poorer survival in both cohorts (P < 0.001); Melbourne men at 53.2% compared with women at 68.3%, and SEER 53.3% men and 62.0% women were alive at 5 years. Being male was independently prognostic for disease-specific mortality in the Melbourne cohort after adjustment for ethnicity, smoking history, performance status, age, pathological stage and histology (hazard ratio = 1.54, 95% confidence interval: 1.10-2.16, P = 0.012). Sex differences in non-small cell lung cancer are important irrespective of age, ethnicity, smoking, performance status and tumour, node and metastasis stage. Epidemiological findings such as these should be translated into research and clinical paradigms to determine the factors that influence the survival disadvantage experienced by men. © 2016 Royal Australasian College of Surgeons.

  2. Host Factors That Affect Ty3 Retrotransposition in Saccharomyces cerevisiae

    PubMed Central

    Aye, Michael; Irwin, Becky; Beliakova-Bethell, Nadejda; Chen, Eric; Garrus, Jennifer; Sandmeyer, Suzanne

    2004-01-01

    The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNAVal(AAC) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets. PMID:15579677

  3. Characterization of the host factors required for hepadnavirus covalently closed circular (ccc) DNA formation.

    PubMed

    Guo, Haitao; Xu, Chunxiao; Zhou, Tianlun; Block, Timothy M; Guo, Ju-Tao

    2012-01-01

    Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.

  4. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression. © 2013 John Wiley & Sons Ltd.

  5. siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication.

    PubMed

    Kok, Kin-Hang; Lei, Ting; Jin, Dong-Yan

    2009-08-27

    A recent RNAi screen used a genome-wide shRNA library to search for cellular factors required for HIV-1 replication. This work complements three other siRNA-based screening studies and potentially opens the door to the discovery of factors that are important for HIV-1 replication in physiological host cells such as T lymphocytes. shRNA screens can be further improved, and they could promise to unravel new pathways and new facets of virus-cell interactions.

  6. Fundamental factors determining the nature of parasite aggregation in hosts.

    PubMed

    Gourbière, Sébastien; Morand, Serge; Waxman, David

    2015-01-01

    The distribution of parasites in hosts is typically aggregated: a few hosts harbour many parasites, while the remainder of hosts are virtually parasite free. The origin of this almost universal pattern is central to our understanding of host-parasite interactions; it affects many facets of their ecology and evolution. Despite this, the standard statistical framework used to characterize parasite aggregation does not describe the processes generating such a pattern. In this work, we have developed a mathematical framework for the distribution of parasites in hosts, starting from a simple statistical description in terms of two fundamental processes: the exposure of hosts to parasites and the infection success of parasites. This description allows the level of aggregation of parasites in hosts to be related to the random variation in these two processes and to true host heterogeneity. We show that random variation can generate an aggregated distribution and that the common view, that encounters and success are two equivalent filters, applies to the average parasite burden under neutral assumptions but it does not apply to the variance of the parasite burden, and it is not true when heterogeneity between hosts is incorporated in the model. We find that aggregation decreases linearly with the number of encounters, but it depends non-linearly on parasite success. We also find additional terms in the variance of the parasite burden which contribute to the actual level of aggregation in specific biological systems. We have derived the formal expressions of these contributions, and these provide new opportunities to analyse empirical data and tackle the complexity of the origin of aggregation in various host-parasite associations.

  7. Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

    PubMed Central

    de SOUZA, João Antonio Chaves; ROSSA JUNIOR, Carlos; GARLET, Gustavo Pompermaier; NOGUEIRA, Andressa Vilas Boas; CIRELLI, Joni Augusto

    2012-01-01

    Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment. PMID:22666826

  8. Macaque-tropic human immunodeficiency virus type 1: breaking out of the host restriction factors

    PubMed Central

    Saito, Akatsuki; Akari, Hirofumi

    2013-01-01

    Macaque monkeys serve as important animal models for understanding the pathogenesis of lentiviral infections. Since human immunodeficiency virus type 1 (HIV-1) hardly replicates in macaque cells, simian immunodeficiency virus (SIV) or chimeric viruses between HIV-1 and SIV (SHIV) have been used as challenge viruses in this research field. These viruses, however, are genetically distant from HIV-1. Therefore, in order to evaluate the efficacy of anti-HIV-1 drugs and vaccines in macaques, the development of a macaque-tropic HIV-1 (HIV-1mt) having the ability to replicate efficiently in macaques has long been desired. Recent studies have demonstrated that host restriction factors, such as APOBEC3 family and TRIM5, impose a strong barrier against HIV-1 replication in macaque cells. By evading these restriction factors, others and we have succeeded in developing an HIV-1mt that is able to replicate in macaques. In this review, we have attempted to shed light on the role of host factors that affect the susceptibility of macaques to HIV-1mt infection, especially by focusing on TRIM5-related factors. PMID:23847610

  9. Deviance partitioning of host factors affecting parasitization in the European brown hare ( Lepus europaeus)

    NASA Astrophysics Data System (ADS)

    Alzaga, Vanesa; Tizzani, Paolo; Acevedo, Pelayo; Ruiz-Fons, Francisco; Vicente, Joaquín; Gortázar, Christian

    2009-10-01

    Deviance partitioning can provide new insights into the ecology of host-parasite interactions. We studied the host-related factors influencing parasite prevalence, abundance, and species richness in European brown hares ( Lepus europaeus) from northern Spain. We defined three groups of explanatory variables: host environment, host population, and individual factors. We hypothesised that parasite infection rates and species richness were determined by different host-related factors depending on the nature of the parasite (endo- or ectoparasite, direct or indirect life cycle). To assess the relative importance of these components, we used deviance partitioning, an innovative approach. The explained deviance (ED) was higher for parasite abundance models, followed by those of prevalence and then by species richness, suggesting that parasite abundance models may best describe the host factors influencing parasitization. Models for parasites with a direct life cycle yielded higher ED values than those for indirect life cycle ones. As a general trend, host individual factors explained the largest proportion of the ED, followed by host environmental factors and, finally, the interaction between host environmental and individual factors. Similar hierarchies were found for parasite prevalence, abundance, and species richness. Individual factors comprised the most relevant group of explanatory variables for both types of parasites. However, host environmental factors were also relevant in models for indirect life-cycle parasites. These findings are consistent with the idea of the host as the main habitat of the parasite; whereas, for indirect life-cycle parasites, transmission would be also modulated by environmental conditions. We suggest that parasitization can be used not only as an indicator of individual fitness but also as an indicator of environmental quality for the host. This research underlines the importance of monitoring parasite rates together with environmental

  10. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    PubMed Central

    Markwell, M A; Paulson, J C

    1980-01-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells. Images PMID:6255459

  11. Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

    PubMed Central

    Chin, Elizabeth; Kirker, Kelly; Zuck, Meghan; James, Garth; Hybiske, Kevin

    2012-01-01

    The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism. PMID

  12. Recombinant host cells and media for ethanol production

    SciTech Connect

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  13. Extracellular vesicles from Trypanosoma brucei mediate virulence factor transfer and cause host anemia

    PubMed Central

    Szempruch, Anthony J.; Sykes, Steven E.; Kieft, Rudo; Denison, Lauren; Becker, Allison C.; Gartrell, Anzio; Martin, William J.; Nakayasu, Ernesto S.; Almeida, Igor C.; Hajduk, Stephen L.; Harrington, John M.

    2015-01-01

    Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling inducing anemia. PMID:26771494

  14. Cancer Cell Discrimination Using Host-Guest "Doubled" Arrays.

    PubMed

    Le, Ngoc D B; Yesilbag Tonga, Gulen; Mout, Rubul; Kim, Sung-Tae; Wille, Marcos E; Rana, Subinoy; Dunphy, Karen A; Jerry, D Joseph; Yazdani, Mahdieh; Ramanathan, Rajesh; Rotello, Caren M; Rotello, Vincent M

    2017-06-14

    We report a nanosensor that uses cell lysates to rapidly profile the tumorigenicity of cancer cells. This sensing platform uses host-guest interactions between cucurbit[7]uril and the cationic headgroup of a gold nanoparticle to non-covalently modify the binding of three fluorescent proteins of a multi-channel sensor in situ. This approach doubles the number of output channels to six, providing single-well identification of cell lysates with 100% accuracy. Significantly, this classification could be extended beyond the training set, determining the invasiveness of novel cell lines. The unique fingerprint of these cell lysates required minimal sample quantity (200 ng, ∼1000 cells), making the methodology compatible with microbiopsy technology.

  15. Host Factors and Cancer Progression: Biobehavioral Signaling Pathways and Interventions

    PubMed Central

    Lutgendorf, Susan K.; Sood, Anil K.; Antoni, Michael H.

    2010-01-01

    Whereas evidence for the role of psychosocial factors in cancer initiation has been equivocal, support continues to grow for links between psychological factors such as stress, depression, and social isolation and progression of cancer. In vitro, in vivo, and clinical studies show that stress- related processes can impact pathways implicated in cancer progression, including immuno-regulation, angiogenesis, and invasion. Contributions of systemic factors, such as stress hormones to the crosstalk between tumor and stromal cells, appear to be critical in modulating downstream signaling pathways with important implications for disease progression. Inflammatory pathways may also be implicated in fatigue and other factors related to quality of life. Although substantial evidence supports a positive effect of psychosocial interventions on quality of life in cancer, the clinical evidence for efficacy of stress-modulating psychosocial interventions in slowing cancer progression remains inconclusive, and the biobehavioral mechanisms that might explain such effects are still being established. This article reviews research findings to date and outlines future avenues of research in this area. PMID:20644093

  16. RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

    PubMed Central

    Esau, Katherine; Cronshaw, James

    1967-01-01

    The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts. PMID:6036529

  17. Adhesins and Host Serum Factors Drive Yop Translocation by Yersinia into Professional Phagocytes during Animal Infection

    PubMed Central

    Maldonado-Arocho, Francisco J.; Green, Carlos; Fisher, Michael L.; Paczosa, Michelle K.; Mecsas, Joan

    2013-01-01

    Yersinia delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process requiring several adhesins and host complement. When WT Yptb or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by ΔailΔinvΔyadA but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that Yersinia adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, Yersinia uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes. PMID:23818844

  18. Adhesins and host serum factors drive Yop translocation by yersinia into professional phagocytes during animal infection.

    PubMed

    Maldonado-Arocho, Francisco J; Green, Carlos; Fisher, Michael L; Paczosa, Michelle K; Mecsas, Joan

    2013-01-01

    Yersinia delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process requiring several adhesins and host complement. When WT Yptb or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by ΔailΔinvΔyadA but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that Yersinia adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, Yersinia uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.

  19. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses

    PubMed Central

    Di Genova, Bruno M.; Tonelli, Renata R.

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite–host interaction and in the mechanisms implicated in the diseases’ pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  20. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  1. Brucella T4SS: the VIP pass inside host cells.

    PubMed

    Lacerda, Thais Lourdes Santos; Salcedo, Suzana Pinto; Gorvel, Jean-Pierre

    2013-02-01

    For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

  2. Host cell autophagy in immune response to zoonotic infections.

    PubMed

    Skendros, Panagiotis; Mitroulis, Ioannis

    2012-01-01

    Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered within double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. Accumulating evidence supports the pivotal role of autophagy in host defense against intracellular pathogens implicating both innate and adaptive immunity. Many of these pathogens cause common zoonotic infections worldwide. The induction of the autophagic machinery by innate immune receptors signaling, such as TLRs, NOD1/2, and p62/SQSTM1 in antigen-presenting cells results in inhibition of survival and elimination of invading pathogens. Furthermore, Th1 cytokines induce the autophagic process, whereas autophagy also contributes to antigen processing and MHC class II presentation, linking innate to adaptive immunity. However, several pathogens have developed strategies to avoid autophagy or exploit autophagic machinery to their advantage. This paper focuses on the role of host cell autophagy in the regulation of immune response against intracellular pathogens, emphasizing on selected bacterial and protozoan zoonoses.

  3. Secretomic Analysis of Host-Pathogen Interactions Reveals That Elongation Factor-Tu Is a Potential Adherence Factor of Helicobacter pylori during Pathogenesis.

    PubMed

    Chiu, Kuo-Hsun; Wang, Ling-Hui; Tsai, Tsung-Ting; Lei, Huan-Yao; Liao, Pao-Chi

    2017-01-06

    The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

  4. In Vivo Imaging of Differences in Early Donor Cell Proliferation in Graft-Versus-Host Disease Hosts with Different Pre-Conditioning Doses

    PubMed Central

    Song, Myung Geun; Kang, Bora; Jeon, Ji Yeong; Chang, Jun; Lee, Seungbok; Min, Chang-Ki; Youn, Hyewon; Choi, Eun Young

    2012-01-01

    Graft-versus-host disease (GVHD) results from immune-mediated attacks on recipient tissues by donor-originated cells through the recognition of incompatible antigens expressed on host cells. The pre-conditioning irradiation dose is a risk factor influencing GVHD severity. In this study, using newly generated luciferase transgenic mice on a B6 background (B6.LucTg) as bone marrow and splenocyte donors, we explored the effects of irradiation doses on donor cell dynamics in major histocompatibility complex (MHC)-matched allogeneic GVHD hosts via bioluminescence imaging (BLI). Results from BLI of GVHD hosts showed higher emission intensities of luminescence signals from hosts irradiated with 900 cGy as compared with those irradiated with 400 cGy. In particular, BLI signals from target organs, such as the spleen, liver, and lung, and several different lymph nodes fluctuated with similar time kinetics soon after transplantation, reflecting the synchronous proliferation of donor cells in the different organs in hosts irradiated with 900 cGy. The kinetic curves of the BLI signals were not synchronized between the target organs and the secondary organs in hosts irradiated with 400 cGy. These results demonstrate that pre-conditioning doses influence the kinetics and degree of proliferation in the target organs soon after transplantation. The results from this study are the first describing donor cell dynamics in MHC-matched allogeneic GVHD hosts and the influence of irradiation doses on proliferation dynamics, and will provide spatiotemporal information to help understand GVHD pathophysiology. PMID:22228184

  5. Centrality of host cell death in plant-microbe interactions.

    PubMed

    Dickman, Martin B; Fluhr, Robert

    2013-01-01

    Programmed cell death (PCD) is essential for proper growth, development, and cellular homeostasis in all eukaryotes. The regulation of PCD is of central importance in plant-microbe interactions; notably, PCD and features associated with PCD are observed in many host resistance responses. Conversely, pathogen induction of inappropriate cell death in the host results in a susceptible phenotype and disease. Thus, the party in control of PCD has a distinct advantage in these battles. PCD processes appear to be of ancient origin, as indicated by the fact that many features of cell death strategy are conserved between animals and plants; however, some of the details of death execution differ. Mammalian core PCD genes, such as caspases, are not present in plant genomes. Similarly, pro- and antiapoptotic mammalian regulatory elements are absent in plants, but, remarkably, when expressed in plants, successfully impact plant PCD. Thus, subtle structural similarities independent of sequence homology appear to sustain operational equivalence. The vacuole is emerging as a key organelle in the modulation of plant PCD. Under different signals for cell death, the vacuole either fuses with the plasmalemma membrane or disintegrates. Moreover, the vacuole appears to play a key role in autophagy; evidence suggests a prosurvival function for autophagy, but other studies propose a prodeath phenotype. Here, we describe and discuss what we know and what we do not know about various PCD pathways and how the host integrates signals to activate salicylic acid and reactive oxygen pathways that orchestrate cell death. We suggest that it is not cell death as such but rather the processes leading to cell death that contribute to the outcome of a given plant-pathogen interaction.

  6. Toxoplasma Actin Is Required for Efficient Host Cell Invasion

    PubMed Central

    Drewry, Lisa L.

    2015-01-01

    ABSTRACT Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of Δact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible Δact1 parasites as long as 5 days after ACT1 deletion. We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in Δact1 parasites being responsible for their invasive ability. Furthermore, invasion by the Δact1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by Δact1 parasites. However, Δact1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion. PMID:26081631

  7. A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

    PubMed Central

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E.; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D.; Mörgelin, Matthias; Karpman, Diana

    2015-01-01

    Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system. PMID:25719452

  8. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    PubMed

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D; Mörgelin, Matthias; Karpman, Diana

    2015-02-01

    Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  9. Comparative Analysis of Host Cell Entry of Ebola Virus From Sierra Leone, 2014, and Zaire, 1976.

    PubMed

    Hofmann-Winkler, Heike; Gnirß, Kerstin; Wrensch, Florian; Pöhlmann, Stefan

    2015-10-01

    The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors.

  10. Comparative Analysis of Host Cell Entry of Ebola Virus From Sierra Leone, 2014, and Zaire, 1976

    PubMed Central

    Hofmann-Winkler, Heike; Gnirß, Kerstin; Wrensch, Florian; Pöhlmann, Stefan

    2015-01-01

    The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors. PMID:25840443

  11. Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria.

    PubMed

    Basu, Rajatava; O'Quinn, Darrell B; Silberger, Daniel J; Schoeb, Trenton R; Fouser, Lynette; Ouyang, Wenjun; Hatton, Robin D; Weaver, Casey T

    2012-12-14

    Interleukin-22 (IL-22) is central to host protection against bacterial infections at barrier sites. Both innate lymphoid cells (ILCs) and T cells produce IL-22. However, the specific contributions of CD4(+) T cells and their developmental origins are unclear. We found that the enteric pathogen Citrobacter rodentium induced sequential waves of IL-22-producing ILCs and CD4(+) T cells that were each critical to host defense during a primary infection. Whereas IL-22 production by ILCs was strictly IL-23 dependent, development of IL-22-producing CD4(+) T cells occurred via an IL-6-dependent mechanism that was augmented by, but not dependent on, IL-23 and was dependent on both transcription factors T-bet and AhR. Transfer of CD4(+) T cells differentiated with IL-6 in the absence of TGF-β ("Th22" cells) conferred complete protection of infected IL-22-deficient mice whereas transferred Th17 cells did not. These findings establish Th22 cells as an important component of mucosal antimicrobial host defense. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Th22 Cells are an Important Source of IL-22 for Host Protection against Enteropathogenic Bacteria

    PubMed Central

    Basu, Rajatava; O’Quinn, Darrell B.; Silberger, Daniel J.; Schoeb, Trenton R.; Fouser, Lynette; Ouyang, Wenjun; Hatton, Robin D.; Weaver, Casey T.

    2013-01-01

    SUMMARY Interleukin-22 (IL-22) is central to host protection against bacterial infections at barrier sites. Both innate lymphoid cells (ILCs) and T cells produce IL-22. However, the specific contributions of CD4+ T cells and their developmental origins are unclear. We found that the enteric pathogen Citrobacter rodentium induced sequential waves of IL-22 producing ILCs and CD4+ T cells that were each critical to host defense during a primary infection. Whereas IL-22 production by ILCs was strictly IL-23–dependent, development of IL-22 producing CD4+ T cells occurred via an IL-6–dependent mechanism that was augmented by, but not dependent on, IL-23, and was dependent on both transcription factors T-bet and AhR. Transfer of CD4+ T cells differentiated with IL-6 in the absence of TGF-β (“Th22” cells) conferred protection of infected IL-22-deficient mice whereas transferred Th17 cells did not. These findings establish Th22 cells as an important component of mucosal anti-microbial host defense. PMID:23200827

  13. Hijacking of host cell IKK signalosomes by the transforming parasite Theileria.

    PubMed

    Heussler, Volker T; Rottenberg, Sven; Schwab, Rebekka; Küenzi, Peter; Fernandez, Paula C; McKellar, Susan; Shiels, Brian; Chen, Zhijian J; Orth, Kim; Wallach, David; Dobbelaere, Dirk A E

    2002-11-01

    Parasites have evolved a plethora of mechanisms to ensure their propagation and evade antagonistic host responses. The intracellular protozoan parasite Theileria is the only eukaryote known to induce uncontrolled host cell proliferation. Survival of Theileria-transformed leukocytes depends strictly on constitutive nuclear factor kappa B (NF-kappaB) activity. We found that this was mediated by recruitment of the multisubunit IkappaB kinase (IKK) into large, activated foci on the parasite surface. IKK signalosome assembly was specific for the transforming schizont stage of the parasite and was down-regulated upon differentiation into the nontransforming merozoite stage. Our findings provide insights into IKK activation and how pathogens subvert host-cell signaling pathways.

  14. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    USDA-ARS?s Scientific Manuscript database

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  15. Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance.

    PubMed

    Dodds, Peter N; Rafiqi, Maryam; Gan, Pamela H P; Hardham, Adrienne R; Jones, David A; Ellis, Jeffrey G

    2009-01-01

    Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

  16. Virus and Host Factors Affecting the Clinical Outcome of Bluetongue Virus Infection

    PubMed Central

    Caporale, Marco; Di Gialleonorado, Luigina; Janowicz, Anna; Wilkie, Gavin; Shaw, Andrew; Savini, Giovanni; Van Rijn, Piet A.; Mertens, Peter; Di Ventura, Mauro

    2014-01-01

    ABSTRACT Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence. IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected

  17. Expression profile of host restriction factors in HIV-1 elite controllers

    PubMed Central

    2013-01-01

    Background Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals. Results Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r2 = 0.597, p < 0.0001), viral load (r2 = 0.34, p = 0.015), and expression of ISG15 (r2 = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers. Conclusions Host restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control. PMID:24131498

  18. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell.

    PubMed

    Rolando, Monica; Buchrieser, Carmen

    2014-12-01

    Intracellular bacterial pathogens modulate the host response to persist and replicate inside a eukaryotic cell and cause disease. Legionella pneumophila, the causative agent of Legionnaires' disease, is present in freshwater environments and represents one of these pathogens. During coevolution with protozoan cells, L. pneumophila has acquired highly sophisticated and diverse strategies to hijack host cell processes. It secretes hundreds of effectors into the host cell, and these manipulate host signaling pathways and key cellular processes. Recently it has been shown that L. pneumophila is also able to alter the transcription and translation machinery of the host and to exploit epigenetic mechanisms in the cells it resides in to counteract host responses.

  19. Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis.

    PubMed

    Viboud, Gloria I; Bliska, James B

    2005-01-01

    A type III secretion system (TTSS) is encoded on a virulence plasmid that is common to three pathogenic Yersinia species: Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. Pathogenic Yersinia species require this TTSS to survive and replicate within lymphoid tissues of their animal or human hosts. A set of pathogenicity factors, including those known as Yersinia outer proteins (Yops), is exported by this system upon bacterial infection of host cells. Two translocator Yops (YopB and YopD) insert into the host plasma membrane and function to transport six effector Yops (YopO, YopH, YopM, YopT, YopJ, and YopE) into the cytosol of the host cell. Effector Yops function to counteract multiple signaling responses in the infected host cell. The signaling responses counteracted by Yops are initiated by phagocytic receptors, Toll-like receptors, translocator Yops, and additional mechanisms. Innate and adaptive immune responses are thwarted as a consequence of Yop activities. A biochemical function for each effector Yop has been established, and the importance of these proteins for the pathogenesis process is being elucidated. This review focuses on the biochemical functions of Yops, the signaling pathways they modulate, and the role of these proteins in Yersinia virulence.

  20. IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

    PubMed Central

    Copenhaver, Alan M.; Casson, Cierra N.; Nguyen, Hieu T.; Duda, Matthew M.; Shin, Sunny

    2015-01-01

    The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response. PMID:26034289

  1. IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis.

    PubMed

    Copenhaver, Alan M; Casson, Cierra N; Nguyen, Hieu T; Duda, Matthew M; Shin, Sunny

    2015-06-16

    The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response.

  2. Bifidobacteria-Host Interactions—An Update on Colonisation Factors

    PubMed Central

    Grimm, Verena; Westermann, Christina; Riedel, Christian U.

    2014-01-01

    Bifidobacteria are one of the predominant bacterial groups of the human intestinal microbiota and have important functional properties making them interesting for the food and dairy industries. Numerous in vitro and preclinical studies have shown beneficial effects of particular bifidobacterial strains or strain combinations on various health parameters of their hosts. This indicates the potential of bifidobacteria in alternative or supplementary therapeutic approaches in a number of diseased states. Based on these observations, bifidobacteria have attracted considerable interest by the food, dairy, and pharmaceutical industries and they are widely used as so-called probiotics. As a consequence of the rapidly increasing number of available bifidobacterial genome sequences and their analysis, there has been substantial progress in the identification of bifidobacterial structures involved in colonisation of and interaction with the host. With the present review, we aim to provide an update on the current knowledge on the mechanisms by which bifidobacteria colonise their hosts and exert health promoting effects. PMID:25295282

  3. Host co-factors of the retrovirus-like transposon Ty1

    PubMed Central

    2012-01-01

    Background Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. Results To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. Conclusion Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are

  4. Dynamic Quantification of Host Schwann Cell Migration into Peripheral Nerve Allografts

    PubMed Central

    Whitlock, Elizabeth L.; Myckatyn, Terence M.; Tong, Alice Y.; Yee, Andrew; Yan, Ying; Magill, Christina K.; Johnson, Philip J.; Mackinnon, Susan E.

    2010-01-01

    Host Schwann cell (SC) migration into nerve allografts is the limiting factor in the duration of immunosuppression following peripheral nerve allotransplantation, and may be affected by different immunosuppressive regimens. Our objective was to compare SC migration patterns between clinical and experimental immunosuppression regimens both over time and at the harvest endpoint. Eighty mice that express GFP under the control of the Schwann cell specific S100 promoter were engrafted with allogeneic, nonfluorescent sciatic nerve grafts. Mice received immunosuppression with either tacrolimus (FK506), or experimental T-cell triple costimulation blockade (CSB), consisting of CTLA4-immunoglobulin fusion protein, anti-CD40 monoclonal antibody, and anti-inducible costimulator monoclonal antibody. Migration of GFP-expressing host SCs into wild-type allografts was assessed in vivo every 3 weeks until 15 weeks postoperatively, and explanted allografts were evaluated for immunohistochemical staining patterns to differentiate graft from host SCs. Immunosuppression with tacrolimus exhibited a plateau of SC migration, characterized by significant early migration (< 3 weeks) followed by a constant level of host SCs in the graft (15 weeks). At the endpoint, graft fluorescence was decreased relative to surrounding host nerve, and donor SCs persisted within the graft. CSB-treated mice displayed gradually increasing migration of host SCs into the graft, without the plateau noted in tacrolimus-treated mice, and also maintained a population of donor SCs at the 15-week endpoint. SC migration patterns are affected by immunosuppressant choice, particularly in the immediate postoperative period, and the use of a single treatment of CSB may allow for gradual population of nerve allografts with host SCs. PMID:20633557

  5. Phylogeographic origin of Helicobacter pylori determines host-adaptive responses upon coculture with gastric epithelial cells.

    PubMed

    Sheh, Alexander; Chaturvedi, Rupesh; Merrell, D Scott; Correa, Pelayo; Wilson, Keith T; Fox, James G

    2013-07-01

    While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA, vacA, and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.

  6. Host Cell Invasion in Mucormycosis: Role of Iron

    PubMed Central

    Ibrahim, Ashraf S.

    2011-01-01

    Clinical hallmarks of mucormycosis infections include the unique susceptibility of patients with increased available serum iron, the propensity of the organism to invade blood vessels, and defective phagocytic function. These hallmarks underscore the critical roles of iron metabolism, phagocyte function, and interactions with endothelial cells lining blood vessels, in the organism’s virulence strategy. In an attempt to understand how Mucorales invade the host, we will review the current knowledge about interactions between Mucorales and the host while evading phagocyte-mediated killing. Additionally, since iron is an important determinant of the disease, we will focus on the role of iron on these interactions. Ultimately, a superior understanding of the pathogenesis of mucormycosis will enable development of novel therapies for this disease. PMID:21807554

  7. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens

    PubMed Central

    Marceau, Caleb D.; Puschnik, Andreas S.; Majzoub, Karim; Ooi, Yaw Shin; Brewer, Susan M.; Fuchs, Gabriele; Swaminathan, Kavya; Mata, Miguel A.; Elias, Joshua E.; Sarnow, Peter; Carette, Jan E.

    2016-01-01

    Summary The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide1. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates2. Also hepatitis C virus (HCV) remains a major medical problem with 160 million chronically infected patients and only expensive treatment on the market3. Despite distinct differences in pathogenesis and mode of transmission, the two viruses share common replication strategies4. A detailed understanding of the host functions that determine viral infection is lacking. Here, we utilized a pooled CRISPR genetic screening strategy5,6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified ER-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER associated degradation. Dengue virus replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as critical step requiring the OST complex. Moreover, we showed that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects7. In contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA binding proteins and enzymes involved in metabolism. We discovered an unexpected link between intracellular FAD levels and HCV replication. This study shows remarkable divergence in host dependency factors between DENV and HCV and illuminates novel host targets for antiviral therapy. PMID:27383987

  8. Modulation of activation-associated host cell gene expression by the apicomplexan parasite Theileria annulata

    PubMed Central

    Durrani, Zeeshan; Weir, William; Pillai, Sreerekha; Kinnaird, Jane; Shiels, Brian

    2012-01-01

    Summary Infection of bovine leucocytes by Theileria annulata results in establishment of transformed, infected cells. Infection of the host cell is known to promote constitutive activation of pro-inflammatory transcription factors that have the potential to be beneficial or detrimental. In this study we have compared the effect of LPS activation on uninfected bovine leucocytes (BL20 cells) and their Theileria-infected counterpart (TBL20). Gene expression profiles representing activated uninfected BL20 relative to TBL20 cells were also compared. The results show that while prolonged stimulation with LPS induces cell death and activation of NF-κB in BL20 cells, the viability of Theileria-infected cells was unaffected. Analysis of gene expression networks provided evidence that the parasite establishes tight control over pathways associated with cellular activation by modulating reception of extrinsic stimuli and by significantly altering the expression outcome of genes targeted by infection-activated transcription factors. Pathway analysis of the data set identified novel candidate genes involved in manipulation of cellular functions associated with the infected transformed cell. The data indicate that the T. annulata parasite can irreversibly reconfigure host cell gene expression networks associated with development of inflammatory disease and cancer to generate an outcome thatis beneficial to survival and propagation of the infected leucocyte. PMID:22533473

  9. HIV-Induced Epigenetic Alterations in Host Cells.

    PubMed

    Abdel-Hameed, Enass A; Ji, Hong; Shata, Mohamed Tarek

    2016-01-01

    Human immunodeficiency virus (HIV), a member of the Retroviridae family, is a positive-sense, enveloped RNA virus. HIV, the causative agent of acquired immunodeficiency syndrome (AIDS) has two major types, HIV-1 and HIV-2 In HIV-infected cells the single stranded viral RNA genome is reverse transcribed and the double-stranded viral DNA integrates into the cellular DNA, forming a provirus. The proviral HIV genome is controlled by the host epigenetic regulatory machinery. Cellular epigenetic regulators control HIV latency and reactivation by affecting the chromatin state in the vicinity of the viral promoter located to the 5' long terminal repeat (LTR) sequence. In turn, distinct HIV proteins affect the epigenotype and gene expression pattern of the host cells. HIV-1 infection of CD4(+) T cells in vitro upregulated DNMT activity and induced hypermethylation of distinct cellular promoters. In contrast, in the colon mucosa and peripheral blood mononuclear cells from HIV-infected patients demethylation of the FOXP3 promoter was observed, possibly due to the downregulation of DNA methyltransferase 1. For a curative therapy of HIV infected individuals and AIDS patients, a combination of antiretroviral drugs with epigenetic modifying compounds have been suggested for the reactivation of latent HIV-1 genomes. These epigenetic drugs include histone deacetylase inhibitors (HDACI), histone methyltransferase inhibitors (HMTI), histone demethylase inhibitors, and DNA methyltransferase inhibitors (DNMTI).

  10. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  11. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  12. Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.

    PubMed

    Taylor, Martin S; LaCava, John; Mita, Paolo; Molloy, Kelly R; Huang, Cheng Ran Lisa; Li, Donghui; Adney, Emily M; Jiang, Hua; Burns, Kathleen H; Chait, Brian T; Rout, Michael P; Boeke, Jef D; Dai, Lixin

    2013-11-21

    LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

  13. A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by Toxoplasma gondii

    PubMed Central

    Gaji, Rajshekhar Y.; Huynh, My-Hang; Carruthers, Vern B.

    2013-01-01

    Toxoplasma gondii critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that Toxoplasma entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in Toxoplasma entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for Toxoplasma invasion along with identifying several novel host factors that potentially participate in parasite entry. PMID:23741372

  14. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

    PubMed Central

    Lupberger, Joachim; Zeisel, Mirjam B.; Xiao, Fei; Thumann, Christine; Fofana, Isabel; Zona, Laetitia; Davis, Christopher; Mee, Christopher J.; Turek, Marine; Gorke, Sebastian; Royer, Cathy; Fischer, Benoit; Zahid, Muhammad N.; Lavillette, Dimitri; Fresquet, Judith; Cosset, François-Loïc; Rothenberg, S Michael; Pietschmann, Thomas; Patel, Arvind H.; Pessaux, Patrick; Doffoël, Michel; Raffelsberger, Wolfgang; Poch, Olivier; Mckeating, Jane A.; Brino, Laurent; Baumert, Thomas F.

    2011-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease. Therapeutic options are limited and preventive strategies are absent. Entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen we identified epidermal growth factor receptor and ephrin receptor A2 as host co-factors for HCV entry. Blocking of kinase function by approved inhibitors broadly inhibited HCV infection of all major HCV genotypes and viral escape variants in cell culture and an animal model in vivo. Receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and membrane fusion. These results identify RTKs as novel HCV entry co-factors and uncover that kinase inhibitors have significant antiviral activity. Inhibition of RTK function may constitute a novel approach for prevention and treatment of HCV infection. PMID:21516087

  15. Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion

    PubMed Central

    Chopra, Martin; Brandl, Andreas; Amich, Jorge; Mottok, Anja; Jordán-Garrote, Ana-Laura; Bäuerlein, Carina A.; Brede, Christian; Ribechini, Eliana; Fick, Andrea; Polz, Johannes; Nishikii, Hidekazu; Mattenheimer, Katharina; Schwinn, Stefanie; Winter, Thorsten; Krappmann, Sven; Einsele, Hermann; Reddehase, Matthias J.; Lutz, Manfred B.

    2016-01-01

    Donor CD4+Foxp3+ regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell–dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo. PMID:27526711

  16. Helicobacter pylori: A Paradigm Pathogen for Subverting Host Cell Signal Transmission.

    PubMed

    Naumann, Michael; Sokolova, Olga; Tegtmeyer, Nicole; Backert, Steffen

    2017-04-01

    Helicobacter pylori colonizes the gastric mucosa in the human stomach and represents a major risk factor for peptic ulcer disease and gastric cancer. Here, we summarize our current knowledge of the complex impact of H. pylori on manipulating host signalling networks, that is, by the cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). We show that H. pylori infections reflect a paradigm for interspecies contact-dependent molecular communication, which includes the disruption of cell-cell junctions and cytoskeletal rearrangements, as well as proinflammatory, cell cycle-related, proliferative, antiapoptotic, and DNA damage responses. The contribution of these altered signalling cascades to disease outcome is discussed.

  17. Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

    PubMed Central

    Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric

    2017-01-01

    ABSTRACT   Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. PMID:28119472

  18. Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis.

    PubMed

    Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric; Attrée, Ina

    2017-01-24

    Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication.

  19. Inkjet printing of silk nest arrays for cell hosting.

    PubMed

    Suntivich, Rattanon; Drachuk, Irina; Calabrese, Rossella; Kaplan, David L; Tsukruk, Vladimir V

    2014-04-14

    An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 μm and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure. These "locked-in" silk nests remained anchored to the substrate during incubation in cell growth media to provide a biotemplated platform for printing-in, immobilization, encapsulation and growth of cells. The process of inkjet-assisted printing is versatile and can be applied on any type of substrate, including rigid and flexible, with scalability and facile formation.

  20. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

    PubMed Central

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-01

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

  1. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells

    PubMed Central

    Baldridge, Gerald D.; Carroll, Elissa M.; Kurtz, Cassandra M.

    2015-01-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7–10 mosquito cells decreases in riboflavin-depleted culture medium. A well studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7–10 cells with an LC50 of approximately 12 µg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell. PMID:24789726

  2. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

    PubMed

    Fallon, Ann M; Baldridge, Gerald D; Carroll, Elissa M; Kurtz, Cassandra M

    2014-09-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

  3. SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

    PubMed Central

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin; Mund, Andreas; Wimmer, Peter; Schubert, Tobias; Groitl, Peter; Will, Hans; Dobner, Thomas

    2013-01-01

    Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming

  4. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes.

    PubMed

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-04-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.

  5. The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis.

    PubMed

    Chavez, Carolina Varela; Jubelin, Grégory; Courties, Gabriel; Gomard, Aurélie; Ginibre, Nadège; Pages, Sylvie; Taïeb, Frédéric; Girard, Pierre-Alain; Oswald, Eric; Givaudan, Alain; Zumbihl, Robert; Escoubas, Jean-Michel

    2010-12-01

    Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G(2)/M phase transition in human cell lines. We report here the first direct functional analysis of Cif(Pl), from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif(Pl) gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif(Pl) was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif(Pl) inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G(2)/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif(Pl) catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  6. Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells.

    PubMed

    Pizarro-Cerdá, Javier; Sousa, Sandra; Cossart, Pascale

    2004-02-01

    Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

  7. Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells.

    PubMed

    Pizarro-Cerdá, Javier; Sousa, Sandra; Cossart, Pascale

    2004-06-01

    Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection isa key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor Clq (gClq-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans(including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells,including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

  8. Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection

    PubMed Central

    Lee, Heather; Prince, Jessica; Stadnisky, Michael D.; Anderson, Monique; Nash, William; Rival, Claudia; Wei, Hairong; Gamache, Awndre; Farber, Charles R.; Tung, Kenneth; Brown, Michael G.

    2016-01-01

    The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust Dk-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen. PMID:26845690

  9. Factors affecting nonunion of the allograft-host junction.

    PubMed

    Hornicek, F J; Gebhardt, M C; Tomford, W W; Sorger, J I; Zavatta, M; Menzner, J P; Mankin, H J

    2001-01-01

    Nonunion of allograft-host junction after bone transplantation is not uncommon, and its treatment frequently is problematic. To improve the understanding of these nonunions, a retrospective review was performed of 163 nonunions in 945 patients who underwent allograft transplantation (17.3%) for various benign and malignant tumors at the authors' institution between 1974 and 1997. Of these 945 patients, 558 did not receive adjuvant therapy. Chemotherapy was administered to 354 patients and only 33 patients received radiation therapy alone. Seventy-one patients had radiation treatment and chemotherapy. Of the 163 patients who had nonunion develop at the allograft-host junction, there were 269 reoperations performed on the involved extremity. In 108 patients, treatment was successful resulting in union of the allograft-host junction. Forty-nine patients did not respond to multiple surgical treatment attempts. The greater the number of surgical procedures, the worse the outcome. The rate of nonunions increased to 27% for the patients who received chemotherapy as compared with 11% for the patients who did not receive chemotherapy. The order of allografts from highest rate of nonunion to lowest was as follows: alloarthrodesis, intercalary, osteoarticular, and alloprosthesis. Infection and fracture rates were higher in the patients with nonunions as compared with the patients without nonunions.

  10. Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae

    PubMed Central

    2011-01-01

    Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail. PMID:21489243

  11. Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

    PubMed

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor.

  12. Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

    PubMed Central

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  13. Lipoarabinomannan in urine during tuberculosis treatment: association with host and pathogen factors and mycobacteriuria

    PubMed Central

    2012-01-01

    Background Detection of lipoarabinomannan (LAM), a Mycobacterium tuberculosis (Mtb) cell wall antigen, is a potentially attractive diagnostic. However, the LAM-ELISA assay has demonstrated variable sensitivity in diagnosing TB in diverse clinical populations. We therefore explored pathogen and host factors potentially impacting LAM detection. Methods LAM-ELISA assay testing, sputum smear and culture status, HIV status, CD4 cell count, proteinuria and TB outcomes were prospectively determined in adults diagnosed with TB and commencing TB treatment at a South African township TB clinic. Sputum TB isolates were characterised by IS61110-based restriction fragment length polymorphism (RFLP) and urines were tested for mycobacteriuria by Xpert® MTB/RIF assay. Results 32/199 (16.1%) of patients tested LAM-ELISA positive. Median optical density and proportion testing LAM positive remained unchanged during 2 weeks of treatment and then declined over 24 weeks. LAM was associated with positive sputum smear and culture status, HIV infection and low CD4 cell counts but not proteinuria, RFLP strain or TB treatment outcome. The sensitivity of LAM for TB in HIV-infected patients with CD4 counts of ≥ 200, 100-199, 50-99, and < 50 cells/μl, was 15.2%, 32%, 42.9%, and 69.2% respectively. Mycobacteriuria was found in 15/32 (46.9%) of LAM positive patients and in none of the LAM negative controls. Conclusions Urinary LAM was related to host immune factors, was unrelated to Mtb strain and declined steadily after an initial 2 weeks of TB treatment. The strong association of urine LAM with mycobacteriuria is a new finding, indicating frequent TB involvement of the renal tract in advanced HIV infection. PMID:22369353

  14. Lipoarabinomannan in urine during tuberculosis treatment: association with host and pathogen factors and mycobacteriuria.

    PubMed

    Wood, Robin; Racow, Kimberly; Bekker, Linda-Gail; Middelkoop, Keren; Vogt, Monica; Kreiswirth, Barry N; Lawn, Stephen D

    2012-02-27

    Detection of lipoarabinomannan (LAM), a Mycobacterium tuberculosis (Mtb) cell wall antigen, is a potentially attractive diagnostic. However, the LAM-ELISA assay has demonstrated variable sensitivity in diagnosing TB in diverse clinical populations. We therefore explored pathogen and host factors potentially impacting LAM detection. LAM-ELISA assay testing, sputum smear and culture status, HIV status, CD4 cell count, proteinuria and TB outcomes were prospectively determined in adults diagnosed with TB and commencing TB treatment at a South African township TB clinic. Sputum TB isolates were characterised by IS61110-based restriction fragment length polymorphism (RFLP) and urines were tested for mycobacteriuria by Xpert® MTB/RIF assay. 32/199 (16.1%) of patients tested LAM-ELISA positive. Median optical density and proportion testing LAM positive remained unchanged during 2 weeks of treatment and then declined over 24 weeks. LAM was associated with positive sputum smear and culture status, HIV infection and low CD4 cell counts but not proteinuria, RFLP strain or TB treatment outcome. The sensitivity of LAM for TB in HIV-infected patients with CD4 counts of ≥ 200, 100-199, 50-99, and < 50 cells/μl, was 15.2%, 32%, 42.9%, and 69.2% respectively. Mycobacteriuria was found in 15/32 (46.9%) of LAM positive patients and in none of the LAM negative controls. Urinary LAM was related to host immune factors, was unrelated to Mtb strain and declined steadily after an initial 2 weeks of TB treatment. The strong association of urine LAM with mycobacteriuria is a new finding, indicating frequent TB involvement of the renal tract in advanced HIV infection.

  15. The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

    PubMed

    Bannister, L H

    1979-04-11

    Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the

  16. Stem cell-paved biobridges facilitate stem transplant and host brain cell interactions for stroke therapy.

    PubMed

    Duncan, Kelsey; Gonzales-Portillo, Gabriel S; Acosta, Sandra A; Kaneko, Yuji; Borlongan, Cesar V; Tajiri, Naoki

    2015-10-14

    Distinguished by an infarct core encased within a penumbra, stroke remains a primary source of mortality within the United States. While our scientific knowledge regarding the pathology of stroke continues to improve, clinical treatment options for patients suffering from stroke are extremely limited. Tissue plasminogen activator (tPA) remains the sole FDA-approved drug proven to be helpful following stroke. However, due to the need to administer the drug within 4.5h of stroke onset its usefulness is constrained to less than 5% of all patients suffering from ischemic stroke. One experimental therapy for the treatment of stroke involves the utilization of stem cells. Stem cell transplantation has been linked to therapeutic benefit by means of cell replacement and release of growth factors; however the precise means by which this is accomplished has not yet been clearly delineated. Using a traumatic brain injury model, we recently demonstrated the ability of transplanted mesenchymal stromal cells (MSCs) to form a biobridge connecting the area of injury to the neurogenic niche within the brain. We hypothesize that MSCs may also have the capacity to create a similar biobridge following stroke; thereby forming a conduit between the neurogenic niche and the stroke core and peri-infarct area. We propose that this biobridge could assist and promote interaction of host brain cells with transplanted stem cells and offer more opportunities to enhance the effectiveness of stem cell therapy in stroke. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Role of myeloid cells in HIV-1-host interplay.

    PubMed

    Stevenson, Mario

    2015-06-01

    The AIDS research field has embarked on a bold mission to cure HIV-1-infected individuals of the virus. To do so, scientists are attempting to identify the reservoirs that support viral persistence in patients on therapy, to understand how viral persistence is regulated and to come up with strategies that interrupt viral persistence and that eliminate the viral reservoirs. Most of the attention regarding the cure of HIV-1 infection has focused on the CD4+ T cell reservoir. Investigators are developing tools to probe the CD4+ T cell reservoirs as well as in vitro systems that provide clues on how to perturb them. By comparison, the myeloid cell, and in particular, the macrophage has received far less attention. As a consequence, there is very little understanding as to the role played by myeloid cells in viral persistence in HIV-1-infected individuals on suppressive therapy. As such, should myeloid cells constitute a viral reservoir, unique strategies may be required for their elimination. This article will overview research that is examining the role of macrophage in virus-host interplay and will discuss features of this interplay that could impact efforts to eliminate myeloid cell reservoirs.

  18. Role of the host cell in bacteriophage T4 development. I. Characterization of host mutants that block T4 head assembly.

    PubMed Central

    Revel, H R; Stitt, B L; Lielausis, I; Wood, W B

    1980-01-01

    To study the role of the host cell in bacteriophage T4 infection, we selected more than 600 mutant host-defective bacteria that absorbed and were killed by phage T4+ but were unable to support its growth. The mutants were grouped into seven classes by the growth patterns of T4 phages carrying compensating mutations (go mutants [grows on]), selected on four prototype host-defective strains. Lysis and DNA synthesis experiments indicated that classes A, AD, D, and B (the majority of the host-defective mutants) block T4+ development at an assembly step, class C mutants affect an early stage in phage development, and class F mutants appear to act at more than one stage. Analysis of T4+ infection in the assembly-defective mutants by in vitro complementation, electron microscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the host-defective mutations interfere with T4+ capsid formation at the level of phage gene 31 function, before assembly of any recognizable capsid structure. The mutations map near purA, but at two or possibly three different sites. The go mutant phages able to overcome the host defect carry mutations in either gene 31, as found by others for similar defective hosts, or in the gene for the major capsid protein (gene 23). The gene 23 go mutations do not bypass the requirement for gene 31 function. These results suggest that at least three components must interact to initiate T4 head assembly: gp31, gp23, and one or more host factors. The compensatory effects of mutational alterations in these components are highly allele specific, consistent with the view that phage and host components interact directly in protein complexes. Images PMID:6988606

  19. Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host

    PubMed Central

    Chiers, Koen; De Waele, Tine; Pasmans, Frank; Ducatelle, Richard; Haesebrouck, Freddy

    2010-01-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The virulence factors of this microorganism involved in colonization and the induction of lung lesions have been thoroughly studied and some have been well characterized. A. pleuropneumoniae binds preferentially to cells of the lower respiratory tract in a process involving different adhesins and probably biofilm formation. Apx toxins and lipopolysaccharides exert pathogenic effects on several host cells, resulting in typical lung lesions. Lysis of host cells is essential for the bacterium to obtain nutrients from the environment and A. pleuropneumoniae has developed several uptake mechanisms for these nutrients. In addition to persistence in lung lesions, colonization of the upper respiratory tract – and of the tonsils in particular – may also be important for long-term persistent asymptomatic infection. Information on virulence factors involved in tonsillar and nasal cavity colonization and persistence is scarce, but it can be speculated that similar features as demonstrated for the lung may play a role. PMID:20546697

  20. Homeostatic expansion and repertoire regeneration of donor T cells during graft versus host disease is constrained by the host environment

    PubMed Central

    Gorski, Jack; Chen, Xiao; Gendelman, Mariya; Yassai, Maryam; Krueger, Ashley; Tivol, Elizabeth; Logan, Brent; Komorowski, Richard; Vodanovic-Jankovic, Sanja

    2007-01-01

    Graft versus host disease (GVHD) typically results in impaired T-cell reconstitution characterized by lymphopenia and repertoire skewing. One of the major causes of inadequate T-cell reconstitution is that T-cell survival and expansion in the periphery are impaired. In this report, we have performed adoptive transfer studies to determine whether the quantitative reduction in T-cell numbers is due to an intrinsic T-cell defect or whether the environmental milieu deleteriously affects T-cell expansion. These studies demonstrate that T cells obtained from animals with graft-versus-host disease (GVHD) are capable of significant expansion and renormalization of an inverted CD4/CD8 ratio when they are removed from this environment. Moreover, these cells can generate complex T-cell repertoires early after transplantation and are functionally competent to respond to third-party alloantigens. Our data indicate that T cells from mice undergoing GVHD can respond to homeostatic signals in the periphery and are not intrinsically compromised once they are removed from the GVHD environment. We thereby conclude that the host environment and not an intrinsic T-cell defect is primarily responsible for the lack of effective T-cell expansion and diversification of complex T-cell repertoires that occurs during GVHD. PMID:17347406

  1. GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

    PubMed Central

    Shastri, Anjali J.; Marino, Nicole D.; Franco, Magdalena; Lodoen, Melissa B.

    2014-01-01

    The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between type II and type III Toxoplasma strains and quantitative trait locus (QTL) mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside the parasites and is found within the parasitophorous vacuole. In vitro experiments with a type II Δgra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 than those infected with the wild-type or complemented control parasites. In vivo experiments showed that mice infected with a type II Δgra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites and that complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the type II and type III versions of GRA25 differ in endogenous expression levels; however, both are able to promote parasite expansion in vivo when expressed in a type II Δgra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator. PMID:24711568

  2. SAMHD1 host restriction factor: a link with innate immune sensing of retrovirus infection.

    PubMed

    Sze, Alexandre; Olagnier, David; Lin, Rongtuan; van Grevenynghe, Julien; Hiscott, John

    2013-12-13

    SAMHD1 [sterile alpha motif and histidine-aspartic domain (HD) containing protein 1] is the most recent addition to a unique group of host restriction factors that limit retroviral replication at distinct stages of the viral life cycle. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase that degrades the intracellular pool of deoxynucleoside triphosphates available during early reverse transcription. SAMHD1 activity is blocked by the Vpx accessory function present in human immunodeficiency virus type 2 and SIVsm. Mutations in SAMHD1 are associated with the autoimmune disorder Aicardi-Goutières syndrome, thus emphasizing its role in regulation of the immune response. SAMHD1 antiretroviral activity is modulated by post-translational modifications, cell-cycle-dependent functions and cytokine-mediated changes. Innate receptors that sense retroviral DNA intermediates are the focus of intense study, and recent studies have established a link among SAMHD1 restriction, innate sensing of DNA and protective immune responses. Cell-cycle-dependent regulation of SAMHD1 by phosphorylation and the increasingly broad range of viruses inhibited by SAMHD1 further emphasize the importance of these mechanisms of host restriction. This review highlights current knowledge regarding SAMHD1 regulation and its impact on innate immune signaling and retroviral restriction. © 2013.

  3. Targeting Host Factors to Treat West Nile and Dengue Viral Infections

    PubMed Central

    Krishnan, Manoj N.; Garcia-Blanco, Mariano A.

    2014-01-01

    West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans. PMID:24517970

  4. Targeting host factors to treat West Nile and dengue viral infections.

    PubMed

    Krishnan, Manoj N; Garcia-Blanco, Mariano A

    2014-02-10

    West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.

  5. Quantitative detection of residual porcine host cell DNA by real-time PCR.

    PubMed

    Chang, Jen-Ting; Chen, Yu-Chen; Chou, Yu-Chi; Wang, Shih-Rong

    2014-03-01

    All biological products are derived from complex living systems and are often mixed with large numbers of impurities. For reasons of safety, residual host-cell DNA must be eliminated during processing. To assay host-cell DNA content in biopharmaceutical products derived from porcine sources, this study applies the quantitative real-time polymerase chain reaction (Q-PCR) method. The optimized assay in this study is based on the pol region of the porcine endogenous retrovirus (PERV). Assay validation results demonstrate that the proposed assay has appropriate accuracy, preciseness, reproducibility, and sensitivity. Primer and probe specificity are evaluated in real-time Q-PCR reactions using genomic DNA from rabbit, mouse, cat, hamster, monkey, human cell, yeast, and Escherichia coli as templates. The sensitivity of real-time Q-PCR is determined using genomic DNA from the porcine kidney cell line. The reliable detection range is within 0.5-10(5) pg/reaction. The limit of quantitation is 500 fg. The sensitivity of the assay meets the authority criterion. Moreover, the assay is applied to determine the level of host-cell DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs. The real-time Q-PCR assay is thus a promising new tool for quantitative detection and clearance validation of residual porcine DNA when manufacturing recombinant therapeutics.

  6. Science Signaling Podcast for 16 May 2017: Vibrio rewires host cells.

    PubMed

    De Nisco, Nicole J; Orth, Kim; VanHook, Annalisa M

    2017-05-16

    This Podcast features a conversation with Kim Orth and Nicole De Nisco, authors of a Research Resource that appears in the 16 May 2017 issue of Science Signaling, about how the marine bacterium Vibrio parahaemolyticus rewires host cell signaling networks. V. parahaemolyticus thrives in warm brackish waters and infects both shellfish and finfish. This bacterium causes gastroenteritis when humans consume contaminated seafood that is raw or undercooked. V. parahaemolyticus delivers virulence factors into host cells through two different type 3 secretion systems (T3SSes). Whereas T3SS2 mediates gastroenteritis, T3SS1 is required for the bacterium to survive in its natural environment and delivers virulence factors that target conserved cellular processes. De Nisco et al examined transcriptional changes in human cells infected with a strain of V. parahaemolyticus that lacked T3SS2 but had an intact T3SS1. They found that the virulence factors delivered through T3SS1 initially induced transcriptional changes that promoted cell survival, then later repressed prosurvival signaling to induce cell death.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

  7. Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.

    PubMed

    Ramirez, M I; Deolindo, P; de Messias-Reason, I J; Arigi, Emma A; Choi, H; Almeida, I C; Evans-Osses, I

    2017-04-01

    Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology. © 2016 John Wiley & Sons Ltd.

  8. Coxiella burnetii Infects Primary Bovine Macrophages and Limits Their Host Cell Response.

    PubMed

    Sobotta, Katharina; Hillarius, Kirstin; Mager, Marvin; Kerner, Katharina; Heydel, Carsten; Menge, Christian

    2016-06-01

    Although domestic ruminants have long been recognized as the main source of human Q fever, little is known about the lifestyle that the obligate intracellular Gram-negative bacterium Coxiella burnetii adopts in its animal host. Because macrophages are considered natural target cells of the pathogen, we established primary bovine monocyte-derived macrophages (MDM) as an in vitro infection model to study reservoir host-pathogen interactions at the cellular level. In addition, bovine alveolar macrophages were included to take cell type peculiarities at a host entry site into account. Cell cultures were inoculated with the virulent strain Nine Mile I (NMI; phase I) or the avirulent strain Nine Mile II (NMII; phase II). Macrophages from both sources internalized NMI and NMII. MDM were particularly permissive for NMI internalization, but NMI and NMII replicated with similar kinetics in these cells. MDM responded to inoculation with a general upregulation of Th1-related cytokines such as interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha (TNF-α) early on (3 h postinfection). However, inflammatory responses rapidly declined when C. burnetii replication started. C. burnetii infection inhibited translation and release of IL-1β and vastly failed to stimulate increased expression of activation markers, such as CD40, CD80, CD86, and major histocompatibility complex (MHC) molecules. Such capability of limiting proinflammatory responses may help Coxiella to protect itself from clearance by the host immune system. The findings provide the first detailed insight into C. burnetii-macrophage interactions in ruminants and may serve as a basis for assessing the virulence and the host adaptation of C. burnetii strains.

  9. Coxiella burnetii Infects Primary Bovine Macrophages and Limits Their Host Cell Response

    PubMed Central

    Sobotta, Katharina; Hillarius, Kirstin; Mager, Marvin; Kerner, Katharina; Heydel, Carsten

    2016-01-01

    Although domestic ruminants have long been recognized as the main source of human Q fever, little is known about the lifestyle that the obligate intracellular Gram-negative bacterium Coxiella burnetii adopts in its animal host. Because macrophages are considered natural target cells of the pathogen, we established primary bovine monocyte-derived macrophages (MDM) as an in vitro infection model to study reservoir host-pathogen interactions at the cellular level. In addition, bovine alveolar macrophages were included to take cell type peculiarities at a host entry site into account. Cell cultures were inoculated with the virulent strain Nine Mile I (NMI; phase I) or the avirulent strain Nine Mile II (NMII; phase II). Macrophages from both sources internalized NMI and NMII. MDM were particularly permissive for NMI internalization, but NMI and NMII replicated with similar kinetics in these cells. MDM responded to inoculation with a general upregulation of Th1-related cytokines such as interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha (TNF-α) early on (3 h postinfection). However, inflammatory responses rapidly declined when C. burnetii replication started. C. burnetii infection inhibited translation and release of IL-1β and vastly failed to stimulate increased expression of activation markers, such as CD40, CD80, CD86, and major histocompatibility complex (MHC) molecules. Such capability of limiting proinflammatory responses may help Coxiella to protect itself from clearance by the host immune system. The findings provide the first detailed insight into C. burnetii-macrophage interactions in ruminants and may serve as a basis for assessing the virulence and the host adaptation of C. burnetii strains. PMID:27021246

  10. Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins

    PubMed Central

    Meng, Xiangzhi; Krumm, Brian; Li, Yongchao; Deng, Junpeng; Xiang, Yan

    2015-01-01

    Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel β-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the β sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique “three-fingered molecular claw.” The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction. PMID:26578811

  11. Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

    PubMed Central

    Romano, Julia D.; de Beaumont, Catherine; Carrasco, Jose A.; Ehrenman, Karen; Bavoil, Patrik M.

    2013-01-01

    The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients. PMID:23243063

  12. Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

    PubMed

    Romano, Julia D; de Beaumont, Catherine; Carrasco, Jose A; Ehrenman, Karen; Bavoil, Patrik M; Coppens, Isabelle

    2013-02-01

    The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

  13. L-FABP is a critical host factor for successful malaria liver stage development.

    PubMed

    Mikolajczak, Sebastian A; Jacobs-Lorena, Vanessa; MacKellar, Drew C; Camargo, Nelly; Kappe, Stefan H I

    2007-04-01

    The malaria parasite liver stage produces tens of thousands of red cell-infectious forms within its host hepatocyte. It is thought that the vacuole-enclosed parasite completely depends on the host cell for successful development but the molecular parasite-host cell interactions underlying this remarkable growth have remained elusive. Using a yeast two-hybrid screen and a yeast overexpression system we show that UIS3, a parasite protein essential for liver stage development, interacts directly with liver-fatty acid binding protein, L-FABP. Down-regulation of L-FABP expression in hepatocytes severely impairs parasite growth and overexpression of L-FABP promotes growth. This is the first identified direct liver stage-host cell protein interaction, providing a possible explanation for the importance of UIS3 in liver infection.

  14. A probabilistic model for risk assessment of residual host cell DNA in biological products.

    PubMed

    Yang, Harry; Zhang, Lanju; Galinski, Mark

    2010-04-26

    Biological products such as viral vaccines manufactured in cells contain residual DNA derived from host cell substrates used in production. It is theoretically possible that the residual DNA could transmit activated oncogenes and/or latent infectious viral genomes to subjects receiving the product, and induce oncogenic or infective events. A probabilistic model to estimate the risks due to residual DNA is proposed. The model takes account of enzyme inactivation process. It allows for more accurate risk assessment when compared to methods currently in use. An application of the method to determine safety factor of a vaccine product is provided.

  15. Inhibition of host cell catalase by Mycoplasma pneumoniae: a possible mechanism for cell injury.

    PubMed Central

    Almagor, M; Yatziv, S; Kahane, I

    1983-01-01

    This study demonstrates that viable Mycoplasma pneumoniae cells inhibit catalase activity in several types of intact human cells as well as in solution. Human erythrocyte catalase was inhibited up to 72%, and the inhibition of catalase in human cultured skin fibroblasts, lung carcinoma epithelial cells, and ciliated epithelial cells from human nasal polyps ranged between 75 and 80%. UV light-killed mycoplasmas failed to inhibit catalase activity both in intact cells and in vitro. After M. pneumoniae infection of human cultured skin fibroblasts, the level of malonyldialdehyde, an indicator for membrane lipid peroxidation, was 3.5 times higher than in control fibroblasts. Virulent M. pneumoniae completely inhibited catalase activity in solution, whereas the nonvirulent strains had a lesser ability to inhibit catalase activity. These findings suggest that as a result of host cell catalase inhibition by M. pneumoniae, the toxicity of the hydrogen peroxide generated by the microorganism and the affected cell is enhanced, thereby inducing host cell damage. PMID:6407999

  16. Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis

    PubMed Central

    Kerr, Markus C.; Gomez, Guillermo A.; Ferguson, Charles; Tanzer, Maria C.; Murphy, James M.; Yap, Alpha S.; Parton, Robert G.; Huston, Wilhelmina M.; Teasdale, Rohan D

    2017-01-01

    Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death. PMID:28281536

  17. A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

    PubMed

    Delincé, Matthieu J; Bureau, Jean-Baptiste; López-Jiménez, Ana Teresa; Cosson, Pierre; Soldati, Thierry; McKinney, John D

    2016-08-16

    The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.

  18. Factors affecting suitability of Quercus rubra as hosts for Enaphalodes rufulus (Coleoptera: Cerambycidae).

    PubMed

    Haavik, L J; Fierke, M K; Stephen, F M

    2010-04-01

    Epidemic populations of Enaphalodes rufulus (Haldeman), red oak borer, a native longhorned wood boring beetle, were implicated as a major contributor to a recent widespread oak mortality event in the Ozark National Forest of Arkansas. We assessed potential factors affecting suitability of a primary host Quercus rubra L., northern red oak, which experienced dieback and mortality throughout two successive borer cohorts. We sampled trees with various E. rufulus infestation levels during 2001-2003 when populations were at outbreak levels and 2003-2005 when borer numbers were declining. We measured phloem thickness and calculated a vigor index; the ratio of the past 5-yr basal area increment to sapwood area. We also counted established first year larval feeding galleries and outer-bark adult emergence holes and measured surface area of feeding galleries on a subset of Q. rubra to assess the importance of host susceptibility versus suitability. Phloem thickness did not exhibit any patterns among hosts of varying infestation levels and was therefore not likely an important factor limiting larval success. Less vigorous Q. rubra appeared to be the most suitable hosts, although it is unclear whether reduced vigor was initially caused by stress of E. rufulus infestation or environmental factors. Host suitability seems to be more important than host susceptibility, as numbers of initiated galleries were not consistently different among host infestation classes and between both cohorts, whereas numbers of emerging adults did differ predictably among host infestation classes.

  19. Incidence, risk factors, and outcomes of sclerosis in patients with chronic graft-versus-host disease.

    PubMed

    Inamoto, Yoshihiro; Storer, Barry E; Petersdorf, Effie W; Nelson, J Lee; Lee, Stephanie J; Carpenter, Paul A; Sandmaier, Brenda M; Hansen, John A; Martin, Paul J; Flowers, Mary E D

    2013-06-20

    Sclerotic chronic graft-versus-host disease (GVHD) can result in disability after allogeneic hematopoietic cell transplantation. We assessed the incidence and risk factors of sclerosis and its association with transplant outcomes among 977 consecutive patients treated with systemic immunosuppression for chronic GVHD. Sclerosis was defined when cutaneous sclerosis, fasciitis, or joint contracture was first documented in the medical record. Seventy (7%) patients presented with sclerosis at the time of initial systemic treatment for chronic GVHD, and the cumulative incidence of sclerosis increased to 20% at 3 years. Factors associated with an increased risk of sclerosis included the use of a mobilized blood cell graft and a conditioning regimen with > 450 cGy total body irradiation. Factors associated with a decreased risk of sclerosis included the use of an HLA-mismatched donor and a major ABO-mismatched donor. Development of sclerosis was associated with longer time to withdrawal of immunosuppressive treatment but not with risks of overall mortality, nonrelapse mortality, or recurrent malignancy. We found a substantial incidence of sclerosis in patients with chronic GVHD. Development of sclerosis can cause disability but does not affect mortality or recurrent malignancy in patients with chronic GVHD.

  20. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

    PubMed

    John Von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

    2017-04-21

    Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

  1. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens

    PubMed Central

    John von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J.; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

    2017-01-01

    Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases. PMID:28430160

  2. Mast cells impair host defense during murine Streptococcus pneumoniae pneumonia.

    PubMed

    van den Boogaard, Florry E; Brands, Xanthe; Roelofs, Joris J T H; de Beer, Regina; de Boer, Onno J; van 't Veer, Cornelis; van der Poll, Tom

    2014-11-01

    Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Mast cells (MCs) are located mainly at the host-environment interface where they function as sentinels. Our goal was to study the role of MCs during pneumonia caused by S. pneumoniae. Lung tissue of patients who had died from pneumococcal pneumonia or a nonpulmonary cause was stained for MCs and tryptase. Wild-type (WT) and MC-deficient (Kit(W-sh/W-sh)) mice were observed or sacrificed after induction of pneumonia by intranasal inoculation of S. pneumoniae. In separate experiments, WT mice were treated with doxantrazole or cromoglycate, which are MC stabilizing agents. The constitutive presence of tryptase-positive MCs was reduced in affected lungs from pneumonia patients. Kit(W-sh/W-sh) mice showed a prolonged survival during the first few days after median lethal dose (LD)100 and LD50 infection, while overall mortality did not differ from that in WT mice. Relative to WT mice, Kit(W-sh/W-sh) mice showed reduced bacterial counts with less bacterial dissemination to distant organs and less inflammation. Neither doxantrazole nor cromoglycate influenced antibacterial defense or inflammatory responses after airway infection with S. pneumoniae. MCs exhibit an unfavorable role in host defense during pneumococcal pneumonia by a mechanism independent of degranulation. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection.

    PubMed

    Verplaetse, Emilie; Slamti, Leyla; Gohar, Michel; Lereclus, Didier

    2015-04-28

    Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host's death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment. Bt is an entomopathogen found ubiquitously in the environment and is a widely used biopesticide. Studies performed at the population level suggest that the infection process of Bt includes three successive steps (virulence, necrotrophism, and sporulation) controlled by different regulators. This study aimed to determine how these phenotypes are activated at the

  4. Some aspects of oncogenic virus-host cell and virus-tumor cell antigenic relationships.

    PubMed

    Nastac, E

    1982-01-01

    Some viewpoints are presented as regards the virus-host cell relationship within the framework of carcinogenesis. Data are reviewed which point out the possibility of the transfer of cellular antigenic fractions from the tumor cell to the virus that grows in it, as well as of a hybridization between the virus genome and the genome of the tumoral host cell. Such a hybridization may have multiple consequences, among which the appearance of new oncogenic variants of viruses so far known to be nononcogenic ones.

  5. Host factors important in immune surveillance against tumours.

    PubMed

    Ada, G L

    1982-01-01

    The nonspecific and specific arms of the immune response are described. The nonspecific components consist of three cell types - macrophages, natural killer and killer cells - and the specific components are antibody produced by B lymphocytes and regulatory and effector T lymphocytes. The evidence suggests that a surveillance system does operate, since at least some tumours are contained. Malignant lesions are more common in unselected autopsies and surgical biopsies of noncancer patients compared with the observed rate of clinical cases; and spontaneous cures of cancer do occur. There is general agreement that surveillance of all types of tumours by the specific components of the immune response does not occur; but there is direct evidence from some model systems and correlations from the incidence and class of tumours which arise in germ-free athymic mice, in immunosuppressed patients with transplants and in immunodeficient patients that the specific immune response, particularly by effector T cells, may control the expression of some tumours of lymphoreticular cells. Such surveillance may be superimposed on a more general system which is mediated by the nonspecific elements. One possible way of increasing the efficiency of the system would be to provide some of the soluble mediators of activated effector cells such as macrophages and T cells. Thus, administration of interferon has had some success, and there is a case for further purification of these and other lymphokines and monokines for administration to patients.

  6. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    SciTech Connect

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  7. Integration host factor is important for biofilm formation by Salmonella enterica Enteritidis.

    PubMed

    Leite, Bruna; Werle, Catierine Hirsch; Carmo, Camila Pinheiro do; Nóbrega, Diego Borin; Milanez, Guilherme Paier; Culler, Hebert Fabricio; Sircili, Marcelo Palma; Alvarez-Martinez, Cristina E; Brocchi, Marcelo

    2017-08-31

    Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments, infecting poultry and eggs. Bacteria in biofilms are difficult to eradicate compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated the role of ihfA and ihfB in biofilm formation by S. enterica Enteritidis by employing different microbiology techniques. Our data indicate that ihf mutant strains are impaired in biofilm formation, showing a reduction in matrix formation and a decrease in viability and metabolic activity. Phenotypic analysis also showed that deletion of ihf causes a deficiency in curli fimbriae expression, cellulose production and pellicle formation. These results show that integration host factor has an important regulatory role in biofilm formation by S. enterica Enteritidis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Microvesicles released during the interaction between Trypanosoma cruzi TcI and TcII strains and host blood cells inhibit complement system and increase the infectivity of metacyclic forms of host cells in a strain-independent process.

    PubMed

    Wyllie, M P; Ramirez, M I

    2017-09-29

    Extracellular vesicles, whether microvesicles (MVs) or exosomes, shed by pathogens transfer virulence factors and biomolecules to host cells, thereby altering the host's susceptibility to infection. We have previously demonstrated that MV release is increased during the interaction between the infective forms of Trypanosoma cruzi and host cells. MVs confer parasite resistance to complement-mediated lysis and enhance parasite invasion. In this study, we show that differences exist in the levels of MVs released during the interaction between metacyclic trypomastigotes of different T. cruzi strains (with varied sensitivity to complement-mediated lysis, namely sensitive G strain TcI and resistant Y strain TcII) and host cells. MVs produced during the interaction between TcII parasites and host cells increased parasite resistance to complement lysis from 50% to 80% and parasite invasion was increased to over 50%. MVs purified during the interaction between TcI parasites and host cells have a stronger effect, doubling complement resistance and parasite invasion. The complement-mediated lysis assays showed that all MVs inhibit mainly the lectin pathway. Interestingly, MVs derived from parasites of one class did not alter complement resistance and the invasion process of parasites from the other class. This is the first description of MVs from T. cruzi with strain-dependent phenotypic effects. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation.

    PubMed

    Kilsgård, Ola; Karlsson, Christofer; Malmström, Erik; Malmström, Johan

    2016-11-01

    Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could

  10. Proteome-scale identification and characterization of mitochondria targeting proteins of Mycobacterium avium subspecies paratuberculosis: Potential virulence factors modulating host mitochondrial function.

    PubMed

    Rana, Aarti; Kumar, Devender; Rub, Abdur; Akhter, Yusuf

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's Disease among ruminants. During the course of infection, it expresses a number of proteins for its successful persistence inside the host that cause variety of physiological abnormalities in the host. Mitochondrion is one of the attractive targets for pathogenic bacteria. Employing a proteome-wide sequence and structural signature based approach we have identified 46 M. avium subsp. paratuberculosis proteins as potential targets for the host mitochondrial targeting. These may act as virulence factors modulating mitochondrial physiology for bacterial survival and immune evasion inside the host cells.

  11. A Loss of Function Analysis of Host Factors Influencing Vaccinia virus Replication by RNA Interference

    PubMed Central

    Gonzalez, Orland; Haga, Ismar R.; Pechenick Jowers, Tali; Reynolds, Danielle K.; Wildenhain, Jan; Tekotte, Hille; Auer, Manfred; Tyers, Mike; Ghazal, Peter; Zimmer, Ralf; Haas, Jürgen

    2014-01-01

    Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics. PMID:24901222

  12. The Transforming Parasite Theileria Co-opts Host Cell Mitotic and Central Spindles to Persist in Continuously Dividing Cells

    PubMed Central

    von Schubert, Conrad; Xue, Gongda; Schmuckli-Maurer, Jacqueline; Woods, Kerry L.; Nigg, Erich A.; Dobbelaere, Dirk A. E.

    2010-01-01

    The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival. PMID:20927361

  13. Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

    PubMed Central

    Verplaetse, Emilie; Slamti, Leyla; Gohar, Michel

    2015-01-01

    ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host’s death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment. PMID:25922389

  14. Interaction between viral RNA silencing suppressors and host factors in plant immunity.

    PubMed

    Nakahara, Kenji S; Masuta, Chikara

    2014-08-01

    To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host-pathogen interactions. By analogy of the steps in virus-host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus-host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus-plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization

    PubMed Central

    Baranowski, Eric; Bergonier, Dominique; Sagné, Eveline; Hygonenq, Marie-Claude; Ronsin, Patricia; Berthelot, Xavier; Citti, Christine

    2014-01-01

    Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs. PMID:24699671

  16. Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization.

    PubMed

    Baranowski, Eric; Bergonier, Dominique; Sagné, Eveline; Hygonenq, Marie-Claude; Ronsin, Patricia; Berthelot, Xavier; Citti, Christine

    2014-01-01

    Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.

  17. Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

    PubMed

    Ortega, Fabian E; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S; Lauer, Peter; Nelson, W James; Theriot, Julie A

    2017-09-06

    An intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. L. monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here, we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required. © 2017 by The American Society for Cell Biology.

  18. TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host.

    PubMed

    Kang, Ye; Feng, Hongwei; Zhang, Jingxu; Chen, Shiguo; Valverde, Bernal E; Qiang, Sheng

    2017-06-01

    A toxin-deficient mutant strain, HP001 mutant of Alternaria alternata, whose mycelium is unable to infect its host, produces little tenuazonic acid (TeA) toxin. How TeA plays a role in initiating host infection by A. alternata remains unclear. In this research we use Imaging-PAM based on chlorophyll fluorescence parameters and transmission electron microscopy to explore the role of TeA toxin during the infection process of A. alternata. Photosystem II damage began even before wild type mycelium infected the leaves of its host, croftonweed (Ageratina adenophora). Compared with the wild type, HP001 mutant produces morphologically different colonies, hyphae with thinner cell walls, has higher reactive oxygen species (ROS) content and lower peroxidase activity, and fails to form appressoria on the host surface. Adding TeA toxin allows the mutant to partially recover these characters and more closely resemble the wild type. Additionally, we found that the mutant is able to elicit disease symptoms when its mycelium is placed on leaves whose epidermis has been manually removed, which indicates that TeA may be determinant in the fungus recognition of its plant host. Lack of TeA toxin appears responsible for the loss of pathogenicity of the HP001 mutant. As a key virulence factor, TeA toxin not only damages the host plant but also is involved in maintaining ROS content, host recognition, inducing appressoria to infect the host and for allowing completion of the infection process. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    SciTech Connect

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang; and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  20. Equine herpesvirus type 1 modulates inflammatory host immune response genes in equine endothelial cells.

    PubMed

    Johnstone, Stephanie; Barsova, Jekaterina; Campos, Isabel; Frampton, Arthur R

    2016-08-30

    Equine herpesvirus myeloencephalopathy (EHM), a disease caused by equine herpesvirus type 1 (EHV-1), is characterized by severe inflammation, thrombosis, and hypoxia in central nervous system (CNS) endothelial cells, which can result in a spectrum of clinical signs including urinary incontinence, ataxia, and paralysis. Strains of EHV-1 that contain a single point mutation within the viral DNA polymerase (nucleotide A2254>G2254: amino acid N752→D752) are isolated from EHM afflicted horses at higher frequencies than EHV-1 strains that do not harbor this mutation. Due to the correlation between the DNA Pol mutation and EHM disease, EHV-1 strains that contain the mutation have been designated as neurologic. In this study, we measured virus replication, cell to cell spread efficacy, and host inflammatory responses in equine endothelial cells infected with 12 different strains of EHV-1. Two strains, T953 (Ohio 2003) (neurologic) and Kentucky A (KyA) (non-neurologic), have well described disease phenotypes while the remaining strains used in this study are classified as neurologic or non-neurologic based solely on the presence or absence of the DNA pol mutation, respectively. Results show that the neurologic strains do not replicate better or spread more efficiently in endothelial cells. Also, the majority of the host inflammatory genes were modulated similarly regardless of EHV-1 genotype. Analyses of host gene expression showed that a subset of pro-inflammatory cytokines, including the CXCR3 ligands CXCL9, CXCL10, and CXCL11, as well as CCL5, IL-6 and TNF-α were consistently up-regulated in endothelial cells infected with each EHV-1 strain. The identification of specific pro-inflammatory cytokines in endothelial cells that are modulated by EHV-1 provides further insight into the factors that contribute to the immunopathology observed after infection and may also reveal new targets for disease intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  2. Characteristics of tumor and host cells in 3-D simulated microgravity environment

    NASA Astrophysics Data System (ADS)

    Chopra, V.; Dinh, T.; Wood, T.; Pellis, N.; Hannigan, E.

    Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in simulated microgravity environment have been used to investigate invasive properties of normal and malignant cell types. We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional (2-D) monolayer cultures. Histological preparations and immunohistochemical staining procedures of cocultured harvests demonstrated various markers of interest: like collagen vimentin, mucin, elastin, fibrin, fibrinogen, cytokeratin, adhesion molecules and various angiogenic factors by tumor cells from gynecological cancer patients along with fibroblasts, endothelial cells and patient-derived mononuclear cells (n=8). The growth rate was enhanced 10-15 folds by 3-D cocultures of patient-derived cells as compared with 2-D monolayer cultures and 3-D monocultures. The production of interleukin-2, interleukin-6, interleukin -8, vascular endothelial cell growth factor, basic fibroblast growth factor, and angiogenin was studied by using ELISA and RT- PCR. Human umbilical vein-derived endothelial cell (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures during proliferation and migration assays. The conditioned medium collected from 3-D cocultures of cancer cells also 1) increased the expression of message levels of vascular endothelial growth factor and its receptor flt-1 and KDR was observed by HUVEC, and 2) increased the expression of intracellular and vascular cell adhesion molecules on the surface of HUVEC, when measured by using Live cell ELISA assays and immunofluorescent staining as compared with 3-D monocultures of normal epithelial cells. There was an increase in production of 1) enzymatic activity that

  3. Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography.

    PubMed

    Sisodiya, Vikram N; Lequieu, Joshua; Rodriguez, Maricel; McDonald, Paul; Lazzareschi, Kathlyn P

    2012-10-01

    Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

  4. Eimeria bovis-induced modulation of the host cell proteome at the meront I stage.

    PubMed

    Lutz, Kathleen; Schmitt, Sigrid; Linder, Monica; Hermosilla, Carlos; Zahner, Horst; Taubert, Anja

    2011-01-01

    The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.

  5. How the growth rate of host cells affects cancer risk in a deterministic way

    NASA Astrophysics Data System (ADS)

    Draghi, Clément; Viger, Louise; Denis, Fabrice; Letellier, Christophe

    2017-09-01

    It is well known that cancers are significantly more often encountered in some tissues than in other ones. In this paper, by using a deterministic model describing the interactions between host, effector immune and tumor cells at the tissue level, we show that this can be explained by the dependency of tumor growth on parameter values characterizing the type as well as the state of the tissue considered due to the "way of life" (environmental factors, food consumption, drinking or smoking habits, etc.). Our approach is purely deterministic and, consequently, the strong correlation (r = 0.99) between the number of detectable growing tumors and the growth rate of cells from the nesting tissue can be explained without evoking random mutation arising during DNA replications in nonmalignant cells or "bad luck". Strategies to limit the mortality induced by cancer could therefore be well based on improving the way of life, that is, by better preserving the tissue where mutant cells randomly arise.

  6. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    DOEpatents

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  7. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    DOEpatents

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2017-08-22

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  8. Human Skeletal Muscle-derived CD133(+) Cells Form Functional Satellite Cells After Intramuscular Transplantation in Immunodeficient Host Mice.

    PubMed

    Meng, Jinhong; Chun, Soyon; Asfahani, Rowan; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer

    2014-05-01

    Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.

  9. Human skeletal muscle-derived CD133(+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice.

    PubMed

    Meng, Jinhong; Chun, Soyon; Asfahani, Rowan; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer

    2014-05-01

    Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.

  10. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus

    SciTech Connect

    Macnab, J.C.M.; Adams, R.L.P.; Rinaldi, A.; Orr, A.; Clark, L.

    1988-04-01

    Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

  11. Host Cell Interactions Are a Significant Barrier to the Clinical Utility of Peptide Antibiotics.

    PubMed

    Starr, Charles G; He, Jing; Wimley, William C

    2016-12-16

    Despite longstanding promise and many known examples, antimicrobial peptides (AMPs) have failed, thus far, to impact human medicine. On the basis of the physical chemistry and mechanism of action of AMPs, we hypothesized that host cell interactions could contribute to a loss of activity in vivo where host cells are highly concentrated. To test this idea, we characterized AMP activity in the presence of human red blood cells (RBC). Indeed, we show that most of a representative set of natural and synthetic AMPs tested are significantly inhibited by preincubation with host cells and would be effectively inactive at physiological cell density. We studied an example broad-spectrum AMP, ARVA (RRGWALRLVLAY), in a direct, label-free binding assay. We show that weak binding to host cells, coupled with their high concentration, is sufficient to account for a loss of useful activity, for at least some AMPs, because >1 × 10(8) peptides must be bound to each bacterial cell to achieve sterilization. The effect of host cell preincubation on AMP activity is comparable to that of serum protein binding. Feasible changes in host cell binding could lead to AMPs that do not lose activity through interaction with host cells. We suggest that the intentional identification of AMPs that are active in the presence of concentrated host cells can be achieved with a paradigm shift in the way AMPs are discovered.

  12. Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a “Bookmarking” Oncoprotein that Alters the Host Cell Epigenotype?

    PubMed Central

    Niller, Hans Helmut; Minarovits, Janos

    2012-01-01

    EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may “bookmark” active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis. PMID:25436603

  13. Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a "Bookmarking" Oncoprotein that Alters the Host Cell Epigenotype?

    PubMed

    Niller, Hans Helmut; Minarovits, Janos

    2012-09-17

    EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may "bookmark" active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis.

  14. Cutaneous graft-versus-host disease after hematopoietic stem cell transplant - a review*

    PubMed Central

    Villarreal, Cesar Daniel Villarreal; Alanis, Julio Cesar Salas; Pérez, Jose Carlos Jaime; Candiani, Jorge Ocampo

    2016-01-01

    Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplants (allo-HSCT) associated with significant morbidity and mortality. The earliest and most common manifestation is cutaneous graft-versus-host disease. This review focuses on the pathophysiology, clinical features, prevention and treatment of cutaneous graft-versus-host disease. We discuss various insights into the disease's mechanisms and the different treatments for acute and chronic skin graft-versus-host disease. PMID:27438202

  15. Making Bunyaviruses Talk: Interrogation Tactics to Identify Host Factors Required for Infection

    PubMed Central

    Riblett, Amber M.; Doms, Robert W.

    2016-01-01

    The identification of host cellular genes that act as either proviral or antiviral factors has been aided by the development of an increasingly large number of high-throughput screening approaches. Here, we review recent advances in which these new technologies have been used to interrogate host genes for the ability to impact bunyavirus infection, both in terms of technical advances as well as a summary of biological insights gained from these studies. PMID:27187446

  16. Host cell protein adsorption characteristics during protein A chromatography.

    PubMed

    Tarrant, Richard D R; Velez-Suberbie, M Lourdes; Tait, Andrew S; Smales, C Mark; Bracewell, Daniel G

    2012-07-01

    Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

  17. Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

    PubMed

    Mehlitz, Adrian; Banhart, Sebastian; Mäurer, André P; Kaushansky, Alexis; Gordus, Andrew G; Zielecki, Julia; Macbeath, Gavin; Meyer, Thomas F

    2010-07-12

    Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

  18. Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function.

    PubMed

    Zhu, Heng; Yang, Fei; Tang, Bo; Li, Xi-Mei; Chu, Ya-Nan; Liu, Yuan-Lin; Wang, Shen-Guo; Wu, De-Cheng; Zhang, Yi

    2015-01-01

    The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair.

  19. Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells

    PubMed Central

    2010-01-01

    Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains. PMID:20942914

  20. Eimeria bovis meront I-carrying host cells express parasite-specific antigens on their surface membrane.

    PubMed

    Badawy, Ahmed Ibrahem I; Lutz, Kathleen; Taubert, Anja; Zahner, Horst; Hermosilla, Carlos

    2010-02-01

    Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.

  1. How Stem Cells Speak with Host Immune Cells in Inflammatory Brain Diseases

    PubMed Central

    Pluchino, Stefano; Cossetti, Chiara

    2014-01-01

    Advances in stem cell biology have raised great expectations that diseases and injuries of the central nervous system (CNS) may be ameliorated by the development of non-hematopoietic stem cell medicines. Yet, the application of adult stem cells as CNS therapeutics is challenging and the interpretation of some of the outcomes ambiguous. In fact, the initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent cellular signaling between the graft and the host. Cellular signaling is the foundation of coordinated actions and flexible responses, and arises via networks of exchanging and interacting molecules that transmit patterns of information between cells. Sustained stem cell graft-to-host communication leads to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses in vivo, ultimately promoting the healing of the injured CNS. Among a number of adult stem cell types, mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their ability to signal to the immune system upon transplantation in experimental CNS diseases. Here, we focus on the main cellular signaling pathways that grafted MSCs and NPCs use to establish a therapeutically relevant cross talk with host immune cells, while examining the role of inflammation in regulating some of the bidirectionality of these communications. We propose that the identification of the players involved in stem cell signaling might contribute to the development of innovative, high clinical impact therapeutics for inflammatory CNS diseases. PMID:23633288

  2. Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology

    PubMed Central

    Raz, Assaf; Tanasescu, Ana-Maria; Zhao, Anna M.; Serrano, Anna; Alston, Tricia; Sol, Asaf; Bachrach, Gilad; Fischetti, Vincent A.

    2015-01-01

    Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed. PMID:26484774

  3. Obesity and endocrine therapy: host factors and breast cancer outcome.

    PubMed

    Goodwin, Pamela J

    2013-08-01

    Obesity is becoming increasingly prevalent and it has been linked to poor breast cancer outcomes. Because obesity is associated with increased adipose tissue mass and aromatase activity [the target of aromatase inhibitors (AIs)], there is concern that these agents may be less effective in women who are overweight or obese. Four of the randomized trials of AIs vs. tamoxifen conducted in the adjuvant breast cancer setting (ATAC, BIG 1-98 and TEAM in the postmenopausal setting and ABCSG-12 in the premenopausal setting) have reported effects of body mass index (BMI) on the relative effectiveness of an AI vs. tamoxifen. Obesity was confirmed as an adverse prognostic factor in ATAC and BIG 1-98 but not the TEAM study; in ABSCG-12, obesity was associated with poor outcomes in the anastrozole arm only. In the three postmenopausal trials, the use of an AI vs. tamoxifen was associated with better outcomes at all levels of BMI [all hazard ratios for recurrence <1, although 95% confidence intervals often included 1 due to lower power and smaller reductions in risk]. Of note, there was no significant interaction of BMI with letrozole (vs. tamoxifen) in the BIG 1-98 trial; while ATAC investigators concluded that the relative benefit of anastrozole (vs. tamoxifen) might be better in thinner (vs. heavier) women. In ABCSG-12, the use of anastrozole (vs. tamoxifen) was associated with significantly worse outcomes in women with BMI ≥25 kg/m(2) (similar to the detrimental effect of anastrozole on overall survival seen in the parent trial). These findings do not support the use of BMI as a predictor of AI (vs. tamoxifen) benefit in the adjuvant setting in postmenopausal breast cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Microbial Hub Taxa Link Host and Abiotic Factors to Plant Microbiome Variation

    PubMed Central

    Agler, Matthew T.; Ruhe, Jonas; Kroll, Samuel; Morhenn, Constanze; Kim, Sang-Tae; Weigel, Detlef; Kemen, Eric M.

    2016-01-01

    Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe–microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe–microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial “hubs,” are strongly interconnected and have a severe effect on communities. By documenting these microbe–microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on “hub” microbes, which, via microbe–microbe interactions, transmit the effects to the microbial community. We analyzed two “hub” microbes (the

  5. Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection

    PubMed Central

    Fan, Ying; Mok, Chris Ka-Pun; Chan, Michael Chi Wai; Zhang, Yang; Nal, Béatrice; Kien, François; Bruzzone, Roberto; Sanyal, Sumana

    2017-01-01

    To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection. PMID:28130444

  6. Cell Cycle Independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection.

    PubMed

    Fan, Ying; Mok, Chris Ka-Pun; Chan, Michael Chi Wai; Zhang, Yang; Nal-Rogier, Béatrice; Kien, François; Bruzzone, Roberto; Sanyal, Sumana

    2017-01-27

    To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se, and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into the S phase, failed to normalize virus production. Infection by IAV triggered redistribution of cyclin D3 from the nucleus to the cytoplasm followed by its proteasomal degradation. When over-expressed in HEK 293T cells cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.

  7. Host Genetic Factors Associated with Symptomatic Primary HIV Infection and Disease Progression among Argentinean Seroconverters

    PubMed Central

    Coloccini, Romina Soledad; Dilernia, Dario; Ghiglione, Yanina; Turk, Gabriela; Laufer, Natalia; Rubio, Andrea; Socías, María Eugenia; Figueroa, María Inés; Sued, Omar; Cahn, Pedro; Salomón, Horacio; Mangano, Andrea; Pando, María Ángeles

    2014-01-01

    Background Variants in HIV-coreceptor C-C chemokine receptor type 5 (CCR5) and Human leukocyte antigen (HLA) genes are the most important host genetic factors associated with HIV infection and disease progression. Our aim was to analyze the association of these genetic factors in the presence of clinical symptoms during Primary HIV Infection (PHI) and disease progression within the first year. Methods Seventy subjects diagnosed during PHI were studied (55 symptomatic and 15 asymptomatic). Viral load (VL) and CD4 T-cell count were evaluated. HIV progression was defined by presence of B or C events and/or CD4 T-cell counts <350 cell/mm3. CCR5 haplotypes were characterized by polymerase chain reaction and SDM-PCR-RFLP. HLA-I characterization was performed by Sequencing. Results Symptoms during PHI were significantly associated with lower frequency of CCR5-CF1 (1.8% vs. 26.7%, p = 0.006). Rapid progression was significantly associated with higher frequency of CCR5-CF2 (16.7% vs. 0%, p = 0.024) and HLA-A*11 (16.7% vs. 1.2%, p = 0.003) and lower frequency of HLA-C*3 (2.8% vs. 17.5%, p = 0.035). Higher baseline VL was significantly associated with presence of HLA-A*11, HLA-A*24, and absence of HLA-A*31 and HLA-B*57. Higher 6-month VL was significantly associated with presence of CCR5-HHE, HLA-A*24, HLA-B*53, and absence of HLA-A*31 and CCR5-CF1. Lower baseline CD4 T-cell count was significantly associated with presence of HLA-A*24/*33, HLA-B*53, CCR5-CF2 and absence of HLA-A*01/*23 and CCR5-HHA. Lower 6-month CD4 T-cell count was associated with presence of HLA-A*24 and HLA-B*53, and absence of HLA-A*01 and HLA-B*07/*39. Moreover, lower 12-month CD4 T-cell count was significantly associated with presence of HLA-A*33, HLA-B*14, HLA-C*08, CCR5-CF2, and absence of HLA-B*07 and HLA-C*07. Conclusion Several host factors were significantly associated with disease progression in PHI subjects. Most results agree with previous studies performed in other groups

  8. Life in cells, hosts, and vectors: parasite evolution across scales.

    PubMed

    Mideo, Nicole; Acosta-Serrano, Alvaro; Aebischer, Toni; Brown, Mark J F; Fenton, Andy; Friman, Ville-Petri; Restif, Olivier; Reece, Sarah E; Webster, Joanne P; Brown, Sam P

    2013-01-01

    Parasite evolution is increasingly being recognized as one of the most important issues in applied evolutionary biology. Understanding how parasites maximize fitness whilst facing the diverse challenges of living in cells, hosts, and vectors, is central to disease control and offers a novel testing ground for evolutionary theory. The Centre for Immunity, Infection, and Evolution at the University of Edinburgh recently held a symposium to address the question "How do parasites maximise fitness across a range of biological scales?" The symposium brought together researchers whose work looks across scales and environments to understand why and how parasites 'do what they do', tying together mechanism, evolutionary explanations, and public health implications. With a broad range of speakers, our aim was to define and encourage more holistic approaches to studying parasite evolution. Here, we present a synthesis of the current state of affairs in parasite evolution, the research presented at the symposium, and insights gained through our discussions. We demonstrate that such interdisciplinary approaches are possible and identify key areas for future progress. Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  9. Host Cell Invasion and Virulence Mediated by Candida albicans Ssa1

    PubMed Central

    Sun, Jianing N.; Solis, Norma V.; Phan, Quynh T.; Bajwa, Jashanjot S.; Kashleva, Helena; Thompson, Angela; Liu, Yaoping; Dongari-Bagtzoglou, Anna; Edgerton, Mira; Filler, Scott G.

    2010-01-01

    Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease. PMID:21085601

  10. Non-alloreactive T Cells Prevent Donor Lymphocyte Infusion-Induced Graft-vs.-Host Disease by Controlling Microbial Stimuli

    PubMed Central

    Li, Hao Wei; Sachs, Jessica; Pichardo, Clarimel; Bronson, Roderick; Zhao, Guiling; Sykes, Megan

    2012-01-01

    In mice, graft-versus-host reactions (GVHR), associated with powerful graft-versus-tumor effects, can be achieved without graft-versus-host disease (GVHD) by delayed administration of donor lymphocyte infusions (DLI) to established mixed chimeras (MCs). However, GVHD sometimes occurrs after DLI in established mixed chimeric patients. In contrast to mice, in which T cell recovery from the thymus occurs prior to DLI administration, human T cell reconstitution following T cell-depleted hematopoietic cell transplantation is slow, resulting in lymphopenia at the time of DLI. We demonstrate here that T cell lymphopenia is an independent risk factor for GVHD following DLI in the absence of known inflammatory stimuli. DLI-induced GVHD was prevented in lymphopenic recipients by prior administration of a small number of non-alloreactive polyclonal T cells, insufficient to prevent lymphopenia-associated expansion of subsequently administered T cells, through a Treg-independent mechanism, but not by T cells with irrelevant specificity. Moreover, administration of antibiotics reduced the severity of GVHD in lymphopenic hosts. Accumulation of DLI-derived effector T cells and host hematopoietic cell elimination were markedly diminished by Treg-depleted, non-alloreactive T cells. Finally, thymectomized mixed chimeras showed increased GVHD following delayed DLI. Collectively, our data demonstrate that in the absence of known conditioning-induced inflammatory stimuli, T cell lymphopenia is a risk factor for GVHD in MCs receiving delayed DLI and suggest that the predisposition to GVHD can at least in part be explained by the presence of occult inflammatory stimuli due to the absence of T cells to control microbial infections. PMID:23136200

  11. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

    PubMed Central

    Flaherty, Rebecca A.; Lee, Shaun W.

    2016-01-01

    Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection. PMID:27585035

  12. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.

    PubMed

    Flaherty, Rebecca A; Lee, Shaun W

    2016-08-19

    Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

  13. Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells.

    PubMed

    Tsou, Lun K; Yount, Jacob S; Hang, Howard C

    2017-03-10

    Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells.

  14. Identifying Francisella tularensis genes required for growth in host cells

    USDA-ARS?s Scientific Manuscript database

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  15. Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice

    SciTech Connect

    Nueesch, Juerg P.F. . E-mail: jpf.nuesch@dkfz-heidelberg.de; Lachmann, Sylvie; Rommelaere, Jean

    2005-01-05

    During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of {alpha}/{beta} tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott-Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production.

  16. Invasive Primary Colonic Aspergillosis in the Immunocompetent Host without Classical Risk Factors.

    PubMed

    Cha, Seon Ah; Kim, Mi Hee; Lim, Tae Seok; Kim, Hyun Ho; Chang, Kyung Yoon; Park, Hoon Suk; Kim, Hyung Wook; Wie, Seong Heon; Jin, Dong Chan

    2015-09-01

    Invasive aspergillosis (IA), generally considered an opportunistic infection in immunocompromised hosts, is associated with high morbidity and mortality. IA commonly occurs in the respiratory tract with isolated reports of aspergillosis infection in the nasal sinuses, central nervous system, skin, liver, and urinary tract. Extra-pulmonary aspergillosis is usually observed in disseminated disease. To date, there are a few studies regarding primary and disseminated gastrointestinal (GI) aspergillosis in immunocompromised hosts. Only a few cases of primary GI aspergillosis in non-immunocompromised hosts have been reported; of these, almost all of them involved the upper GI tract. We describe a very rare case of IA involving the lower GI tract in the patient without classical risk factors that presented as multiple colon perforations and was successfully treated by surgery and antifungal treatment. We also review related literature and discuss the characteristics and risk factors of IA in the immunocompetent hosts without classical risk factors. This case that shows IA should be considered in critically ill patients, and that primary lower GI aspergillosis may also occur in the immunocompetent hosts without classical risk factors.

  17. Host pigment epithelium-derived factor (PEDF) prevents progression of liver metastasis in a mouse model of uveal melanoma.

    PubMed

    Lattier, John M; Yang, Hua; Crawford, Susan; Grossniklaus, Hans E

    2013-12-01

    Uveal melanoma (UM) has a 30 % 5-year mortality rate, primarily due to liver metastasis. Both angiogenesis and stromagenesis are important mechanisms for the progression of liver metastasis. Pigment epithelium-derived factor (PEDF), an anti-angiogenic and anti-stromagenic protein, is produced by hepatocytes. Exogenous PEDF suppresses metastasis progression; however, the effects of host-produced PEDF on metastasis progression are unknown. We hypothesize that host PEDF inhibits liver metastasis progression through a mechanism involving angiogenesis and stromagenesis. Mouse melanoma cells were injected into the posterior ocular compartment of PEDF-null mice and control mice. After 1 month, the number, size, and mean vascular density (MVD) of liver metastases were determined. The stromal component of hepatic stellate cells (HSCs) and the type III collagen they produce was evaluated by immunohistochemistry. Host PEDF inhibited the total area of liver metastasis and the frequency of macrometastases (diameter >200 μm) but did not affect the total number of metastases. Mice expressing PEDF exhibited significantly lower MVD and less type III collagen production in metastases. An increase in activated HSCs was seen in the absence of PEDF, but this result was not statistically significant. In conclusion, host PEDF inhibits the progression of hepatic metastases in a mouse model of UM, and loss of PEDF is accompanied by an increase in tumor blood vessel density and type III collagen.

  18. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis

    PubMed Central

    Quigley, Jeff; Hughitt, V. Keith; Velikovsky, Carlos A.; Mariuzza, Roy A.

    2017-01-01

    ABSTRACT The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. PMID:28270579

  19. Mesenchymal stem cells in stem cell transplant recipients are damaged and remain of host origin.

    PubMed

    Wang, Jing; Liu, Kaiyan; Lu, Dao-Pei

    2005-08-01

    The aim of this study was to investigate the expansion capacity and origin of bone marrow-derived mesenchymal stem cells (MSCs) in 34 patients who received a sex-mismatched stem cell transplant (SCT). Polymerase chain reaction (PCR) analysis of the amelogenin gene (AMEL) was used to detect donor-derived MSCs. Cultured MSCs were hybridized with fluorescence in situ hybridization (FISH) probes for chromosomes X and Y to distinguish cells of donor origin from those of host origin. The MSCs of 31 of the 34 patients showed confluent stroma, and the MSCs from 24 of these 31 patients were successfully passaged more than 5 times and were able to be used for PCR and FISH analyses. The colony-forming unit-fibroblast, confluence time, and passage numbers of the MSCs and the colony-forming capacity of the hematopoietic progenitor cells of the patients were significantly different from those of 30 healthy control subjects. Flow cytometry results showed that the proportion of CD14(+)CD45(+) cells, which are regarded as monocytes/macrophages, in cultured MSCs (fifth passage) was less than 0.04%. PCR and FISH analyses revealed that the MSC-derived cells in all 24 patients were from the host. In conclusion, the expansion capacity of MSCs in patients who receive an SCT is damaged, and the MSCs originate from the host.

  20. Host cell protein dynamics in recombinant CHO cells: impacts from harvest to purification and beyond.

    PubMed

    Hogwood, Catherine Em; Bracewell, Daniel G; Smales, C Mark

    2013-01-01

    During the production of recombinant protein products, such as monoclonal antibodies, manufacturers must demonstrate clearance of host cell impurities and contaminants to appropriate levels prior to use in the clinic. These include host cell DNA and RNA, product related contaminants such as aggregates, and importantly host cell proteins (HCPs). Despite the importance of HCP removal, the identity and dynamics of these proteins during cell culture and downstream processing (DSP) are largely unknown. Improvements in technologies such as SELDI-TOF mass spectrometry alongside the gold standard technique of ELISA has allowed semi-quantification of the total HCPs present. However, only recently have techniques been utilized in order to identify those HCPs present and align this with the development of approaches to monitor the dynamics of HCPs during both fermentation and downstream processing. In order to enable knowledge based decisions with regards to improving HCP clearance it is vital to identify potential problematic HCPs on a cell line and product specific basis. Understanding the HCP dynamics will in the future help provide a platform to rationally manipulate and engineer and/or select suitable recombinant CHO cell lines and downstream processing steps to limit problematic HCPs.

  1. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    PubMed

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

  2. [Ultrastructure of the initial stages of interaction between halprowiae (Chlamydiae) and host cells].

    PubMed

    Popov, V L; Beskina, S R; Shatkin, A A; Avakian, A A

    1980-08-01

    The initial stages of the interaction of halprowiae (strain CP-1), isolated in Reiter's syndrome, with a monolayer culture of cells L-929 were studied. The electron-microscopic examination of the cells every 2 hours during the first 24 hours after the inoculation allowed to follow the processes of adsorption, endocytosis of elementary bodies and their transformation into reticulate bodies by the "decondensation" of nucleoid, which indicated the beginning of the vegetative (reproductive) stage of the developmental cycle. During all the periods of observation halprowiae were enclosed into the membranes of phagocytic vacuoles formed by host cells. Besides, in other vacuoles (probably, phagolysosomes) halprowiae at various stages of destruction could be observed. 8--10 hours after inoculation the phagosomes containing reticulate bodies began to merge, thus forming an inoculation the phagosomes containing reticulate bodies began to merge, thus forming an intracytoplasmic inclusion. In this inclusion a new generation of the agent was formed by binary-like fission. At the beginning of the vegetative stage halprowiae seem to be highly sensitive to the action of exo- and endogenic factors which may cause their L-transformation. Depending on the reaction of the host cell at the initial stages of interaction with halprowiae, 3 ways of such interaction are seemingly possible: developmental cycle, destruction in phagolysosomes. L-transformation.

  3. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification

    PubMed Central

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the MetacoreTM database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection. PMID:27406444

  4. CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

    PubMed Central

    Slater, Louise H.; Hett, Erik C.; Clatworthy, Anne E.; Mark, Kevin G.; Hung, Deborah T.

    2013-01-01

    Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to β-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore. PMID:23716698

  5. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

    PubMed

    Skoog, Emma C; Sjöling, Åsa; Navabi, Nazanin; Holgersson, Jan; Lundin, Samuel B; Lindén, Sara K

    2012-01-01

    Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

  6. Predicting bacteriophage proteins located in host cell with feature selection technique.

    PubMed

    Ding, Hui; Liang, Zhi-Yong; Guo, Feng-Biao; Huang, Jian; Chen, Wei; Lin, Hao

    2016-04-01

    A bacteriophage is a virus that can infect a bacterium. The fate of an infected bacterium is determined by the bacteriophage proteins located in the host cell. Thus, reliably identifying bacteriophage proteins located in the host cell is extremely important to understand their functions and discover potential anti-bacterial drugs. Thus, in this paper, a computational method was developed to recognize bacteriophage proteins located in host cells based only on their amino acid sequences. The analysis of variance (ANOVA) combined with incremental feature selection (IFS) was proposed to optimize the feature set. Using a jackknife cross-validation, our method can discriminate between bacteriophage proteins located in a host cell and the bacteriophage proteins not located in a host cell with a maximum overall accuracy of 84.2%, and can further classify bacteriophage proteins located in host cell cytoplasm and in host cell membranes with a maximum overall accuracy of 92.4%. To enhance the value of the practical applications of the method, we built a web server called PHPred (〈http://lin.uestc.edu.cn/server/PHPred〉). We believe that the PHPred will become a powerful tool to study bacteriophage proteins located in host cells and to guide related drug discovery.

  7. Mechanism of host restriction of adenovirus-associated virus replication in African green monkey kidney cells.

    PubMed

    Buller, R M; Straus, S E; Rose, J A

    1979-06-01

    Human adenovirus (Ad) serotypes provide an early factor(s) that is necessary for adenovirus-associated virus (AAV) multiplication in human cell lines. However, little, if any, AAV production occurs in primary African green monkey kidney (AGMK) cells co-infected with AAV and a helper human Ad (non-permissive infection), unless cells are additionally infected with SV40 (permissive infection). To determine the basis of the host restriction of AAV replication in AGMK cells, AAV DNA, RNA and protein synthesis were analyzed under various conditions of infection. Hybridization reactions revealed no detectable AAV-specific DNA or RNA in infections with AAV alone or in combination with SV40. In co-infections with AAV and Ad5 or Ad7, the synthesis of both AAV- and Ad-specific DNA and RNA occurred without a significant rise in titre of either virus. During non-permissive infection, however, AAV DNA synthesis was abnormal in that an expected accumulation of single-stranded progeny molecules was not observed. Finally, although intact 20S AAV transcripts were present in the cytoplasm of AGMK cells during non-permissive infection (in amounts ranging from 50 to 80% of that found during permissive infection), AAV-specific polypeptides were not demonstrable by polyacrylamide gel electrophoresis. Taken together, these experiments indicate that the host restriction of AAV replication in AGMK cells is exerted at the level of translation of the single AAV messenger RNA. In addition, it appears that one or more of the AAV polypeptides specified by this message is required for the production of single-stranded AAV progeny DNA.

  8. [What is the most important factor for gastric carcinogenesis in Koreans: Helicobacter pylori, host factor or environmental factor?].

    PubMed

    Kim, Hak Yang

    2007-02-01

    Epidemiological data including our studies demonstrated the association between Helicobacter pylori (H. pylori) infection and gastric cancer. However, this significant clinical outcome happens only in a small portion of infected person. This suggests that other contributors including host genetic and environmental factors might be involved in the disease process. Studies on the association between virulent strains of H. pylori and clinical outcomes failed to show significant results in Korea. Cytokine gene polymorphism such as interleukin-1 (IL-1) has been thought to play a role in gastric carcinogenesis. Our studies showed the controversial role of IL-1, TNF-A, IL-10 and IL-2 gene polymorphisms in the development of gastric cancer in Korea. Chronic infection and inflammation leading to tumorigenesis are mediated in part through the recognition of various stimuli by toll-like receptors (TLRs). Our studies on the polymorphisms of TLR4 and TLR2 showed no mutant form in Koreans. These discrepancies might reflect the genetic differences between Caucasians and Koreans or might be due to prevalent genetic polymorphisms with masked effect in gastric carcinogenesis in Koreans. As other candidate risk factors, there are constant or inconsistent results on the effect of dietary intake in gastric cancer. There are numerous similar risk for gastric carcinogenesis with different risk ratio including environmental factors in Caucasians and Koreans. Under the background of prevalent H. pylori infection and genetic polymorphisms, environmental factors including diet may potentiate their role in gastric carcinogenesis in Koreans.

  9. How do cell-free HIV virions avoid infecting dead-end host cells and cell fragments?

    PubMed

    Lyengar, Sujatha; Schwartz, David H

    2004-01-01

    HIV faces the challenge of identifying and entering suitable host cells (i.e. activated and viable) among a wide array of receptor-positive but unsuitable targets. Lymph nodes contain resting cells, activated cells destined for apoptosis within 24 h, and cell fragments, all of which represent replicative dead ends. We postulate that 1) HIV virions have evolved the ability to probe the internal status of potential host cells from the external cell membrane by assessing the ability of cells to co-cap CD4 and chemokine receptors, and 2) the requirement for dual receptor binding in a concerted manner by three gp120 molecules is the molecular mechanism by which virions stochastically ensure high density co-capping of receptors. Cell-associated HIV accomplishes the same selective process by targeting cells capable of participating in immunological synapse formation.

  10. Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

    SciTech Connect

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2016-04-19

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  11. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

    DOEpatents

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2014-09-30

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  12. Induction of human host cell apoptosis by Trichomonas vaginalis cysteine proteases is modulated by parasite exposure to iron.

    PubMed

    Kummer, Shelley; Hayes, Gary R; Gilbert, Robert O; Beach, David H; Lucas, John J; Singh, Bibhuti N

    2008-03-01

    Trichomonas vaginalis is an understudied parasitic organism whose mechanisms of pathogenesis remain unclear. The adherence to host cells, the induction of host cell cytotoxicity and protease activity are all, however, thought to be contributing factors towards the development of the disease. T. vaginalis CP30 is an extracellular fraction containing four cysteine proteases, CP2, CP3, CP4 and CPT that induce apoptosis in primary human vaginal epithelial cells (HVECs) [Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ, Singh BN. Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 2005; 280: 23853-60]. We now show that CP30, and the induction of HVEC apoptosis are modulated by iron availability in the parasite growth medium. Growth of parasites under high iron conditions results in a decrease in levels of CP30 found in an extracellular soluble fraction (SF), a concomitant decline in protease activity, and a decreased ability of SF to induce host cell death. Conversely, iron restriction leads to an increase in CP30 levels, an increase in CP30 protease activity, and an increased ability to induce HVEC cell death. Iron-loaded lactoferrin, but not transferrin is an effective iron source for parasites. We hypothesize that CP30 induction of host cell apoptosis is crucial for the development of a persistent infection, and that iron plays a determining role in parasite pathogenesis.

  13. The stress-response factor SigH modulates the interaction between Mycobacterium tuberculosis and host phagocytes.

    PubMed

    Dutta, Noton K; Mehra, Smriti; Martinez, Alejandra N; Alvarez, Xavier; Renner, Nicole A; Morici, Lisa A; Pahar, Bapi; Maclean, Andrew G; Lackner, Andrew A; Kaushal, Deepak

    2012-01-01

    The Mycobacterium tuberculosis stress response factor SigH plays a crucial role in modulating the pathogen's response to heat, oxidative-stress, envelope damage and hypoxia. We hypothesized that the lack of this key stress response factor would alter the interaction between the pathogen and its host cells. We compared the interaction of Mtb, Mtb:Δ-sigH and a strain where the mutation had been genetically complemented (Mtb: Δ-sigH:CO) with primary rhesus macaque bone marrow derived macrophages (Rh-BMDMs). The expression of numerous inducible and homeostatic (CCL) β-chemokines and several apoptotic markers was induced to higher levels in the cells infected with Mtb:Δ-sigH, relative to Mtb or the complemented strain. The differential expression of these genes manifested into functional differences in chemotaxis and apoptosis in cells infected with these two strains. The mutant strain also exhibited reduced late-stage survival in Rh-BMDMs. We hypothesize that the product of one or more SigH-dependent genes may modulate the innate interaction of Mtb with host cells, effectively reducing the chemokine-mediated recruitment of immune effector cells, apoptosis of infected monocytes and enhancing the long-term survival and replication of the pathogen in this milieu The significantly higher induction of Prostaglandin Synthetase 2 (PTGS2 or COX2) in Rh-BMDMs infected with Mtb relative to Mtb: Δ-sigH may explain reduced apoptosis in Mtb-infected cells, as PTGS2 is known to inhibit p53-dependent apoptosis.The SigH-regulon modulates the innate interaction of Mtb with host phagocytes, perhaps as part of a strategy to limit its clearance and prolong its survival. The SigH regulon appears to be required to modulate innate immune responses directed against Mtb.

  14. Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles.

    PubMed

    Jung, Anna Lena; Herkt, Christina Elena; Schulz, Christine; Bolte, Kathrin; Seidel, Kerstin; Scheller, Nicoletta; Sittka-Stark, Alexandra; Bertrams, Wilhelm; Schmeck, Bernd

    2017-07-24

    Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1β in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.

  15. Host cell reactivation studies with epidermal cells of mice sensitive and resistant to carcinogenesis

    SciTech Connect

    Strickland, J.E.; Strickland, A.G.

    1984-03-01

    Primary epidermal cells from AKR, BALB/c, CD-1, and SENCAR mice, listed in order of least to most sensitive to epidermal carcinogenesis by initiation and promotion protocols, were found to be equally competent to ''reactivate'' herpes simplex virus type 1 irradiated by germicidal ultraviolet radiation. Nontumorigenic BALB/c epidermal cell lines selected in vitro for resistance to terminal differentiation after in vivo or in vitro treatment with initiating doses of carcinogens showed virus survival curves similar to those of primary cells. Similarly, primary cultures which were allowed to grow to confluency following a single treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml) retained normal host cell reactivation. Host cell reactivation studies with mouse dermal fibroblasts could not be done because of the failure of the herpes simplex virus to infect these cells and produce plaques. These results demonstrate that survival of ultraviolet light-damaged virus in primary epidermal cells in culture is unrelated to whether the cells are derived from mice sensitive or resistant to epidermal carcinogenesis. Furthermore, virus survival is not changed by tumor promoter treatment or by treatment with initiating doses of carcinogens which results in differentiation-resistant cells.

  16. Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria

    PubMed Central

    Inamoto, Yoshihiro; Carpenter, Paul A.; Lee, Stephanie J.; Kiem, Hans-Peter; Petersdorf, Effie W.; Pereira, Shalini E.; Nash, Richard A.; Mielcarek, Marco; Fero, Matthew L.; Warren, Edus H.; Sanders, Jean E.; Storb, Rainer F.; Appelbaum, Frederick R.; Storer, Barry E.; Martin, Paul J.

    2011-01-01

    Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD. PMID:21263156

  17. Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria.

    PubMed

    Flowers, Mary E D; Inamoto, Yoshihiro; Carpenter, Paul A; Lee, Stephanie J; Kiem, Hans-Peter; Petersdorf, Effie W; Pereira, Shalini E; Nash, Richard A; Mielcarek, Marco; Fero, Matthew L; Warren, Edus H; Sanders, Jean E; Storb, Rainer F; Appelbaum, Frederick R; Storer, Barry E; Martin, Paul J

    2011-03-17

    Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.

  18. Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration

    PubMed Central

    Arutyunyan, Irina; Elchaninov, Andrey; Fatkhudinov, Timur; Makarov, Andrey; Kananykhina, Evgeniya; Usman, Natalia; Bolshakova, Galina; Glinkina, Valeria; Goldshtein, Dmitry; Sukhikh, Gennady

    2015-01-01

    Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation. PMID:26191137

  19. Host Epithelial Cell Invasion by Campylobacter jejuni: Trigger or Zipper Mechanism?

    PubMed Central

    Ó Cróinín, Tadhg; Backert, Steffen

    2012-01-01

    Campylobacter jejuni, a spiral-shaped Gram-negative pathogen, is a highly frequent cause of gastrointestinal foodborne illness in humans worldwide. Clinical outcome of C. jejuni infections ranges from mild to severe diarrheal disease, and some other complications including reactive arthritis and Guillain–Barré syndrome. This review article highlights various C. jejuni pathogenicity factors, host cell determinants, and proposed signaling mechanisms involved in human host cell invasion and their potential role in the development of C. jejuni-mediated disease. A model is presented which outlines the various important interactions of C. jejuni with the intestinal epithelium, and we discuss the pro’s and con’s for the “zipper” over the “trigger” mechanism of invasion. Future work should clarify the contradictory role of some previously identified factors, and should identify and characterize novel virulence determinants, which are crucial to provide fresh insights into the diversity of strategies employed by this pathogen to cause disease. PMID:22919617

  20. Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

    PubMed Central

    Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

    2016-01-01

    The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

  1. Infection of U937 monocytic cells with Chlamydia pneumoniae induces extensive changes in host cell gene expression.

    PubMed

    Virok, Dezso; Loboda, Andrey; Kari, Laszlo; Nebozhyn, Michael; Chang, Celia; Nichols, Calen; Endresz, Valeria; Gonczol, Eva; Berencsi, Klara; Showe, Michael K; Showe, Louise C

    2003-11-01

    The effect of infection with Chlamydia pneumoniae on host messenger RNA expression in human monocytic cells with complement DNA microarrays was studied. The data chronicle a cascade of transcriptional events affecting 128 genes, many of which have not previously been reported to be affected by C. pneumoniae infection. Down-regulated genes are primarily associated with RNA and DNA metabolism, chromosomal stability, and cell-cycle regulation. Up-regulated messages include those for a variety of genes with important proinflammatory functions. Many of the up-regulated genes-including the hyaluron receptor CD44, vasoconstrictor endothelin-1, smooth muscle growth factor heparin-binding EGF-like growth factor, and fatty acid binding protein-4-had been previously described as linked to the development of atherosclerosis and other chronic inflammatory diseases. C. pneumoniae-infected monocytes can contribute to the development and progression of diseases for which acute or chronic inflammation has been shown to be important, such as ather