The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate ...intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.
The family Chlamydiaceae (order Chlamydiales, phylum Chlamydiae) comprises important, obligate intracellular bacterial pathogens of humans and animals. Subdivision of the family into the two genera ...Chlamydia and Chlamydophila has been discussed controversially during the past decade. Here, we have revisited the current classification in the light of recent genomic data and in the context of the unique biological properties of these microorganisms. We conclude that neither generally used 16S rRNA sequence identity cut-off values nor parameters based on genomic similarity consistently separate the two genera. Notably, no easily recognizable phenotype such as host preference or tissue tropism is available that would support a subdivision. In addition, the genus Chlamydophila is currently not well accepted and not used by a majority of research groups in the field. Therefore, we propose the classification of all 11 currently recognized Chlamydiaceae species in a single genus, the genus Chlamydia. Finally, we provide emended descriptions of the family Chlamydiaceae, the genus Chlamydia, as well as the species Chlamydia abortus, Chlamydia caviae and Chlamydia felis.
The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this ...restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein beta-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. beta-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS beta-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to beta-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the beta-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the ...mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI) N-terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane-associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Chlamydia are gram-negative obligate intracellular bacteria that replicate within a discrete cellular vacuole, called an inclusion. Although it is known that Chlamydia require essential ...nutrients from host cells to support their intracellular growth, the molecular mechanisms for acquiring these macromolecules remain uncharacterized. In the present study, it was found that the expression of mammalian cell glucose transporter proteins 1 (GLUT1) and glucose transporter proteins 3 (GLUT3) were up-regulated during chlamydial infection. Up-regulation was dependent on bacterial protein synthesis and Chlamydia-induced MAPK kinase activation. GLUT1, but not GLUT3, was observed in close proximity to the inclusion membrane throughout the chlamydial developmental cycle. The proximity of GLUT1 to the inclusion was dependent on a brefeldin A-sensitive pathway. Knockdown of GLUT1 and GLUT3 with specific siRNA significantly impaired chlamydial development and infectivity. It was discovered that the GLUT1 protein was stabilized during infection by inhibition of host-dependent ubiquitination of GLUT1, and this effect was associated with the chlamydial deubiquitinase effector protein CT868. This report demonstrates that Chlamydia exploits host-derived transporter proteins altering their expression, turnover and localization. Consequently, host cell transporter proteins are manipulated during infection as a transport system to fulfill the carbon source requirements for Chlamydia.
Manipulation of GLUT1 and GLUT3 by Chlamydia.
Exit strategies of intracellular pathogens Stephens, Richard S; Hybiske, Kevin
Nature reviews. Microbiology,
200802, 2008-Feb, 2008-2-00, 20080201, Letnik:
6, Številka:
2
Journal Article
Recenzirano
The exit of intracellular pathogens from host cells is an important step in the infectious cycle, but is poorly understood. It has recently emerged that microbial exit is a process that can be ...directed by organisms from within the cell, and is not simply a consequence of the physical or metabolic burden that is imposed on the host cell. This Review summarizes our current knowledge on the diverse mechanisms that are used by intracellular pathogens to exit cells. An integrated understanding of the diversity that exists for microbial exit pathways represents a new horizon in the study of host-pathogen interactions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to ...provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies.
Methods
Baseline urine samples from Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network study participants with UCPPS (n = 259), positive controls (PCs; chronic pain without pelvic pain, n = 107) and healthy controls (HCs, n = 125) were analysed for the presence of proteins that are suggested in the literature to be associated with UCPPS. Matrix metalloproteinase (MMP)‐2, MMP‐9, MMP‐9/neutrophil gelatinase‐associated lipocalin (NGAL) complex (also known as Lipocalin 2), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF‐R1) and NGAL were assayed and quantitated using mono‐specific enzyme‐linked immunosorbent assays for each protein. Log‐transformed concentration (pg/mL or ng/mL) and concentration normalized to total protein (pg/μg) values were compared among the UCPPS, PC and HC groups within sex using the Student's t‐test, with P values adjusted for multiple comparisons. Multivariable logistic regression and receiver‐operating characteristic curves assessed the utility of the biomarkers in distinguishing participants with UCPPS and control participants. Associations of protein with symptom severity were assessed by linear regression.
Results
Significantly higher normalized concentrations (pg/μg) of VEGF, VEGF‐R1 and MMP‐9 in men and VEGF concentration (pg/mL) in women were associated with UCPPS vs HC. These proteins provided only marginal discrimination between UCPPS participants and HCs. In men with UCCPS, pain severity was significantly positively associated with concentrations of MMP‐9 and MMP‐9/NGAL complex, and urinary severity was significantly positively associated with MMP‐9, MMP‐9/NGAL complex and VEGF‐R1. In women with UCPPS, pain and urinary symptom severity were associated with increased normalized concentrations of MMP‐9/NGAL complex, while pain severity alone was associated with increased normalized concentrations of VEGF, and urinary severity alone was associated with increased normalized concentrations of MMP‐2. Pain severity in women with UCPPS was significantly positively associated with concentrations of all biomarkers except NGAL, and urinary severity with all concentrations except VEGF‐R1.
Conclusion
Altered levels of MMP‐9, MMP‐9/NGAL complex and VEGF‐R1 in men, and all biomarkers in women, were associated with clinical symptoms of UCPPS. None of the evaluated candidate markers usefully discriminated UCPPS patients from controls. Elevated VEGF, MMP‐9 and VEGF‐R1 levels in men and VEGF levels in women may provide potential new insights into the pathophysiology of UCPPS.
Diseases caused by
Chlamydia are based on intense and chronic inflammation elicited and maintained by reinfection or persistent infection. The traditional view in the field is that disease is ...mediated by antigen-dependent delayed-type hypersensitivity or autoimmunity. This immunological paradigm has served as the basis for years of chlamydial research but the mechanism or the antigen that causes pathology has yet to be unequivocally revealed. Recent research on responses elicited in
Chlamydia-infected cells defines a new direction for our understanding of this microorganism–host interaction and provides the basis for a reassessment of disease mechanisms.
Chlamydia-infected non-immune mammalian cells produce proinflammatory chemokines, cytokines, growth factors and other cellular modulators. This cellular response to infection supports an alternative hypothesis for chlamydial pathogenesis: the inflammatory processes of chlamydial pathogenesis are elicited by infected host cells and are necessary and sufficient to account for chronic and intense inflammation and the promotion of cellular proliferation, tissue remodeling and scarring, the ultimate cause of disease sequelae.
Study objective We evaluated the efficacy of an emergency department (ED)–based lung-protective mechanical ventilation protocol for the prevention of pulmonary complications. Methods This was a ...quasi-experimental, before-after study that consisted of a preintervention period, a run-in period of approximately 6 months, and a prospective intervention period. The intervention was a multifaceted ED-based mechanical ventilator protocol targeting lung-protective tidal volume, appropriate setting of positive end-expiratory pressure, rapid oxygen weaning, and head-of-bed elevation. A propensity score–matched analysis was used to evaluate the primary outcome, which was the composite incidence of acute respiratory distress syndrome and ventilator-associated conditions. Results A total of 1,192 patients in the preintervention group and 513 patients in the intervention group were included. Lung-protective ventilation increased by 48.4% in the intervention group. In the propensity score–matched analysis (n=490 in each group), the primary outcome occurred in 71 patients (14.5%) in the preintervention group compared with 36 patients (7.4%) in the intervention group (adjusted odds ratio 0.47; 95% confidence interval CI 0.31 to 0.71). There was an increase in ventilator-free days (mean difference 3.7; 95% CI 2.3 to 5.1), ICU-free days (mean difference 2.4; 95% CI 1.0 to 3.7), and hospital-free days (mean difference 2.4; 95% CI 1.2 to 3.6) associated with the intervention. The mortality rate was 34.1% in the preintervention group and 19.6% in the intervention group (adjusted odds ratio 0.47; 95% CI 0.35 to 0.63). Conclusion Implementing a mechanical ventilator protocol in the ED is feasible and is associated with significant improvements in the delivery of safe mechanical ventilation and clinical outcome.
The mechanisms of entry for the obligate intracellular bacterium C. trachomatis were examined by functional disruption of proteins essential for various modes of entry. RNA interference was used to ...disrupt proteins with established roles in clathrin-mediated endocytosis (clathrin heavy chain, dynamin-2, heat shock 70-kDa protein 8, Arp2, cortactin, and calmodulin), caveola-mediated endocytosis (caveolin-1, dynamin-2, Arp2, NSF, and annexin II), phagocytosis (RhoA, dynamin-2, Rac1, and Arp2), and macropinocytosis (Pak1, Rac1, and Arp2). Comparative quantitative PCR analysis was performed on small interfering RNA-transfected HeLa cells to accurately determine the extent of C. trachomatis entry after these treatments. Key structural and regulatory factors associated with clathrin-mediated endocytosis were found to be involved in Chlamydia entry, whereas those for caveola-mediated endocytosis, phagocytosis, and macropinocytosis were not. Thus, clathrin and its coordinate accessory factors were required for entry of C. trachomatis, although additional, uncharacterized mechanisms are also utilized.
PDF
