Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and “tunes” the host inflammatory response so as to maximize persistent infection.
Helicobacter pylori is a bacterium that colonizes the stomach of about half the world's population, most of whom are asymptomatic. However, some strains of H. pylori express a bacterial secretion system, a sort of molecular syringe that injects a bacterial protein inside the gastric cells and causes inflammation that can lead to peptic ulcer disease or gastric cancer. One of the essential components of the H. pylori secretion system is CagY, which is unusual because it contains a series of repetitive amino acid motifs that are encoded by a very large number of direct DNA repeats. Here we have shown that DNA recombination in cagY changes the protein motif structure and alters the function of the secretion system—turning it on or off. Using mouse and non-human primate models, we have demonstrated that CagY is a molecular switch that “tunes” the host inflammatory response, and likely contributes to persistent infection. Determining the mechanism by which CagY functions will enhance our understanding of the effects of H. pylori on human health, and could lead to novel applications for the modulation of host cell function.
Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyze the membranes of the pathogen-containing vacuole (“inclusion”). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of >54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient Type III secretion (T3S) in the RB stage at 18 hpi, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a “per organism” basis. Our results show that EB and RB proteomes are streamlined to fulfill their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.
Chlamydia; Pathogenesis; Elementary body; Reticulate body; quantitative proteomics
The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole (“inclusion”). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a pre-infected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive anti-chlamydial therapeutic strategy.
Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.
Vibrio parahaemolyticus is a bacterial pathogen that causes food-borne gastroenteritis and also wound infection and septicemia. It exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1) during the infection of mammalian cells. Although an effector VepA plays a major role in the cytotoxicity, the mechanism was unknown. Here we show that VepA targets subunit c of the vacuolar H+-ATPase (V-ATPase) and induces the rupture of host cell lysosomes. We found that VepA alone is cytotoxic in HeLa cells and also toxic in yeast Saccharomyces cerevisiae. Using a yeast genome-wide screening, we identified yeast V-ATPase subunit c as essential for the toxicity of VepA to yeast. We also demonstrated that knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity toward HeLa cells and that VepA interacted with subunit c of V-ATPase. During infection, lysosomal contents leaked into the cytosol prior to cell lysis, and VepA was necessary and sufficient for this leakage. Our data suggest that a bacterial effector VepA ruptures lysosomes, the “suicide bags” of host cells, by targeting the evolutionarily conserved V-ATPase, and elicits subsequent cell death.
Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms.
The marine bacterium Vibrio parahaemolyticus is a leading cause worldwide of gastroenteritis linked to the consumption of contaminated seafood. Despite the prevalence of V. parahaemolyticus-induced gastroenteritis, there is limited understanding of how this pathogen causes disease in the intestine. In part, the paucity of knowledge results from the absence of an oral infection-based animal model of the human disease. We developed a simple oral infection-based infant rabbit model of V. parahaemolyticus-induced intestinal pathology and diarrhea. This experimental model enabled us to define several previously unknown but key features of the pathology elicited by this organism. We found that V. parahaemolyticus chiefly colonizes the distal small intestine and that the organism's second type III secretion system is essential for colonization. The epithelial surface of the distal small intestine is also the major site of V. parahaemolyticus-induced damage, which arises via a characteristic sequence of events culminating in the formation of V. parahaemolyticus-filled cavities in the epithelial surface. This experimental model will transform future studies aimed at deciphering the bacterial and host factors/processes that contribute to disease, as well as enable testing of new therapeutics to prevent and/or combat infection.
CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.
The Chlamydiae are obligate intracellular pathogen that replicate within a membrane-bound vacuole, termed the “inclusion”. From this compartment, bacteria acquire essential nutrients by selectively redirecting transport vesicles and hijacking intracellular organelles. Re-routing is achieved by several mechanisms including proteolysis-mediated fragmentation of the Golgi apparatus, recruitment of Rab GTPases and SNAREs, and translocation of cytoplasmic organelles into the inclusion lumen. Given Chlamydiae’s extended co-evolution with eukaryotic cells, it is likely that co-option of multiple cellular pathways is a strategy to provide redundancy in the acquisition of essential nutrients from the host and has contributed to the success of these highly adapted pathogens.
The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread.
Chlamydia trachomatis is an obligate intracellular bacterium that is an important cause of human disease, including sexually transmitted diseases and acquired blindness in developing countries. The inability to carry out conventional genetic manipulations limits our understanding of the mechanisms of C. trachomatis binding, entry, and spread. Previous studies have shown that heparan sulfate proteoglycans (HSPGs) play a role in early binding events. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated whether HSPG-associated growth factors affect C. trachomatis binding or entry. Here, we report the novel finding that Fibroblast Growth Factor 2 (FGF2), a ubiquitously expressed growth factor, enhances C. trachomatis binding to host cells in an HSPG-dependent manner. Furthermore, C. trachomatis infection stimulates production and release of FGF2 through distinct signaling pathways. Released FGF2 is sufficient to enhance the subsequent rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway that sets up a positive feedback loop to enhance bacterial infection and spread.
Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.
C. trachomatis is a major human pathogen for which there is no means of genetically manipulating its DNA. It is an obligate intracellular bacterium which has a complex developmental cycle that takes place in a specialized host cell cytoplasmic vacuole known as an inclusion. We have constructed a shuttle vector based on the chlamydial plasmid and developed a new approach to select genetically modified bacteria. It uses rescue by selection of stable infectious, penicillin-resistant C. trachomatis from a pool of non-dividing, non-infectious C. trachomatis (induced by penicillin arrest of the developmental cycle). The transformed C. trachomatis is also cured of its endogenous plasmid by the selection of the transforming vector. The vector was modified to express the green fluorescent protein (GFP) in both Chlamydia and E. coli. We have genetically manipulated a plasmid-free recipient strain of C. trachomatis and shown restoration of the ability of this recipient strain to synthesize and accumulate glycogen within inclusions upon acquisition of the shuttle vector. The ability to transform and manipulate C. trachomatis using a complementation based vector is an important advance that opens up the field of Chlamydia research.
The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.
C. trachomatis is the leading cause of non-congenital blindness in developing countries and is the number one cause of sexually transmitted disease and non-congenital infertility in Western countries. The capacity of Chlamydia infections to lead to infertility and blindness, their association with chronic diseases, and the extraordinary prevalence and array of these infections make them public concerns of primary importance. This pathogen must establish a protective membrane-bound niche and acquire essential lipids from the host cell during infection in order to survive and replicate. This study identifies novel mechanisms by which C. trachomatis hijacks various lipid trafficking proteins for distinct roles during intracellular development. Disruption of these lipid trafficking pathways results in alterations in the growth and stability of its protective niche as well as a defect in replication. Understanding the molecular mechanisms of these host-pathogen interactions will lead to rational approaches for the development of novel therapeutics, diagnostics, and preventative strategies.
L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.
L. monocytogenes is a food-born pathogen responsible for listeriosis, a severe illness with a high mortality rate, which mainly affects immunocompromised patients and pregnant women. The bacterium is a facultative intracellular pathogen able to invade, survive and multiply in large variety of cells. Although the infectious process at the cellular level has been extensively studied, the role of InlK, a surface protein specific to L. monocytogenes, remains unknown. Here we established that L. monocytogenes use InlK to interact with a mammalian cytoplasmic protein named Major Vault Protein (MVP). Although MVP has been implicated in diverse cellular processes, its role remains elusive. Here we demonstrate that, inside the cell, L. monocytogenes is able, via InlK, to decorate its surface with MVP to escape autophagy, an innate immune defense system that protects the cell from invading pathogens. L. monocytogenes uses this camouflage strategy to efficiently survive inside cells.
Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.
Separate analysis of host and pathogen metabolic changes in intracellular C. trachomatis infections is arduous and has not been comprehensively realized so far. A more detailed understanding about the metabolic activity and needs of C. trachomatis and its specific interactions with the host cell would be the basis for the development of novel treatment strategies. We therefore applied fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NAD(P)H using two-photon microscopy to directly visualize metabolic changes of host cells and pathogens in living cells. NAD(P)H fluorescence was detected both on the chlamydial inclusion membrane and inside the inclusion. Interestingly, changes in chlamydial growth and progeny induced by glucose starvation and IFNγ treatment were directly linked to significant changes of the NAD(P)H fluorescence lifetimes inside the inclusions. Furthermore, measurement of the NAD(P)H fluorescence lifetime in the host cell nucleus revealed that infected cells were programmed for starvation during the metabolically active phase of intracellular chlamydial growth. Our findings highlight for the first time a direct interaction between host and pathogen metabolism in intracellular bacterial infections that exceeds sole competition for nutrients. In conclusion, fluorescence lifetime imaging of NAD(P)H by two-photon microscopy enables real-time analysis of metabolic host-pathogen interactions in intracellular infections with high spatial and temporal resolution.
Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development.
The obligate intracellular bacterial pathogen Chlamydia has developed strategies to invade, survive and replicate within the host genital, ocular and pulmonary epithelial surfaces. Chlamydia developmental cycle occurs in a membrane bound vacuole, the inclusion. The Chlamydia-dependent remodeling of the inclusion membrane leads to a unique and specialized compartment that allows for the specific interaction with cellular organelles and the acquisition of molecules and nutrients required for bacterial survival and replication. This study provides an example of how the insertion of C. trachomatis proteins into the inclusion membrane may allow the bacteria to manipulate the host cells to its own benefit. We showed that the lipid transfer protein CERT localized to C. trachomatis inclusion membrane and partly co-localized with the endoplasmic reticulum (ER) protein VAPB. Moreover, VAPB positive tubules made close contact with the inclusion membrane, suggesting the formation of ER-Inclusion membrane contact sites upon C. trachomatis infection. Finally, we have shown that CERT interacted with the C. trachomatis inclusion protein IncD. We propose that the presence of CERT, VAPB and IncD at ER-Inclusion membrane contact sites allow C. trachomatis to exploit the non-vesicular lipid transport machinery of the host cell and generate platforms specialized in metabolism and signaling events favorable to bacterial development.
The outer membrane (OM) of Gram-negative bacteria provides a barrier to the passage of hydrophobic and hydrophilic compounds into the cell. The OM has embedded proteins that serve important functions in signal transduction and in the transport of molecules into the periplasm. The OmpW family of OM proteins, of which P. aeruginosa OprG is a member, is widespread in Gram-negative bacteria. The biological functions of OprG and other OmpW family members are still unclear.
In order to obtain more information about possible functions of OmpW family members we have solved the X-ray crystal structure of P. aeruginosa OprG at 2.4 Å resolution. OprG forms an eight-stranded β-barrel with a hydrophobic channel that leads from the extracellular surface to a lateral opening in the barrel wall. The OprG barrel is closed off from the periplasm by interacting polar and charged residues on opposite sides of the barrel wall.
The crystal structure, together with recent biochemical data, suggests that OprG and other OmpW family members form channels that mediate the diffusion of small hydrophobic molecules across the OM by a lateral diffusion mechanism similar to that of E. coli FadL.
The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI.
Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI.
Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.
Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.
Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.
Chlamydia trachomatis is a particularly prevalent human pathogen responsible for loss of eyesight through trachoma and is the most common sexually transmitted disease of bacterial origin. Unlike most other bacterial pathogens, chlamydiae survive only within another cell and thus must develop sophisticated mechanisms to subvert immune clearance by their host. To this end, the bacteria secrete proteins into the cells they occupy which disrupt normal cellular function. In this work, we identify one such protein, NUE, which is injected into the nucleus of human cells during infection. Sequence analysis of NUE revealed the presence of a SET domain which suggests its involvement in chromatin remodeling. Indeed, we found the protein associated with chromatin both during infection and when transfected into human cells. Importantly, we demonstrate NUE is an active histone methyltransferase that targets host cell histones but does not modify bacterial histone-like proteins. This is the first bacterial protein identified which is able to penetrate the nucleus and directly modify mammalian histones.
A large number of intracellular pathogens survive in vacuolar niches composed of host-derived membranes modified extensively by pathogen proteins and lipids. Although intracellular lifestyles offer protection from humoral immune responses, vacuole-bound pathogens nevertheless face powerful intracellular innate immune surveillance pathways that can trigger fusion with lysosomes, autophagy and host cell death. While many of the strategies used by vacuole-bound pathogens to invade and establish a replicative vacuole are well described, how the integrity and stability of these parasitic vacuoles are maintained is poorly understood. Here we identify potential mechanisms of pathogenic vacuole maintenance and the consequences of vacuole disruption by highlighting a select subset of bacterial and protozoan parasites.
The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies.
Methods and Findings
We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative of subsp. holarctica) and subsp. novicida. Within BMDM, subsp. tularensis and LVS showed minimal replication, subsp. novicida showed marginal replication, and subsp. holartica replicated robustly. The limited intramacrophage replication of subsp. tularensis and novicida strains was correlated with the induction of nitric oxide production. Importantly, Fischer 344 rats that survived pulmonary infection with subsp. novicida were markedly protected against subsequent pulmonary challenge with subsp. tularensis, suggesting that subsp. novicida may be a useful platform for the development of vaccines against subsp. tularensis.
The Fischer 344 rat exhibits similar sensitivity to F. tularensis strains as that reported for humans, and thus the Fischer 344 ray may serve as a better animal model for tularemia vaccine development.
Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria.
Anaplasma phagocytophilum is the pathogen that causes human granulocytic anaplasmosis, an emerging infectious disease. As an obligate intracellular organism, this bacterium cannot reproduce outside of eukaryotic cells due to the loss of many genes that are present in free-living bacteria. Paradoxically, it specifically infects short-lived white blood cells that play critical roles in anti-microbial defense, by subverting a number of host innate immune responses including programmed cell death (apoptosis). A. phagocytophilum factors that are involved in this process are largely unknown. In this study, we first searched A. phagocytophilum proteins that are secreted by its specialized secretion system into eukaryotic cells. We found a protein of unknown function, here named Ats-1, which is abundantly produced by A. phagocytophilum and traverses five membranes to enter the mitochondria of human cells. Our further study showed that Ats-1 reduces the sensitivity of mitochondria to respond to apoptosis-inducing factors, leading to the inhibition of host cell apoptosis. Thus, present findings identified a bacterial protein that allows infected white blood cells to live longer to support bacterial growth. The absence of similarity of the sequence or the mode of action to any other known cell death suppressor suggests that Ats-1 defines a previously undescribed class of anti-apoptotic protein. This protein and the mechanism thereof may provide insight regarding a new therapeutic target for treatment of human granulocytic anaplasmosis.
Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-κB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-κB chaperone IκBα NleH1 repressed the transcription of a RPS3/NF-κB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter. We identified a region of NleH1 (N40-K45) that is at least partially responsible for the inhibitory activity of NleH1 toward RPS3. Deleting nleH1 from E. coli O157:H7 produced a hypervirulent phenotype in a gnotobiotic piglet model of Shiga toxin-producing E. coli infection. We suggest that NleH may disrupt host innate immune responses by binding to a cofactor of host transcriptional complexes.
The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor complex plays an essential role in regulating the immune response to infection. Bacterial pathogens inject effector proteins into host cells to modulate the host innate immune response by interfering with NF-κB activation. We have discovered that Escherichia coli O157:H7, an important cause of hemorrhagic colitis, injects into mammalian cells an effector protein termed NleH1, which inhibits NF-κB-dependent transcription through a novel mechanism. NleH1 binds directly to a subunit of NF-κB, the ribosomal protein S3 (RPS3). One of the functions of RPS3 is to guide the recruitment of the p65 NF-κB subunit to specific promoters in response to different stimuli. NleH1, but not a closely related effector, NleH2, functions by reducing the nuclear abundance of RPS3 to dampen host transcriptional outputs. Our findings highlight a previously unappreciated mechanism by which bacterial effector proteins are able to alter host cell functions.
With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive.
We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA.
This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.
Chlamydia sp. are responsible for a wide range of diseases of significant clinical and public health importance. In this review, we highlight how recent cellular and functional genomic approaches have significantly increased our knowledge of the pathogenic mechanisms employed by these genetically intractable bacteria. As the extensive repertoire of chlamydial proteins that are translocated into the mammalian host are identified and characterized, a molecular understanding of how Chlamydiae co-opt host cellular functions and block innate immune pathways is beginning to emerge.