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
Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.
Essential for the ability of Salmonella Typhimurium to cause disease is the function of a type III secretion system (T3SS) encoded within its pathogenicity island 1 (SPI-1), which through the delivery of bacterial effector proteins modulates a variety of cellular functions. This study reports that the infection of mammalian cells with Salmonella Typhimurium results in a profound reprogramming of gene expression that changes over time. The stimulation of this response requires the activity of a specific subset of bacterial T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for its intracellular replication. Targeting the mechanisms described in this study may lead to the development of novel anti-infective strategies.
Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.
Chlamydia trachomatis replicates within a large vacuole (“inclusion”) that sequesters bacterial components from direct contact with the host cytoplasm. Here, we report that the inclusion is encased in a network of F-actin and intermediate filaments (IF), scaffolding structures that provide mechanical support to metazoan cells, and that these cytoskeletal structures act cooperatively to stabilize the pathogen-containing vacuole. Formation of F-actin at the inclusion was dependent on RhoA and its disruption led to the disassembly of IFs, loss of inclusion integrity and leakage of inclusion contents into the host cytoplasm. In addition, IF proteins were processed by the secreted chlamydial protease CPAF to form filamentous structures at the inclusion surface with altered structural properties. We propose that Chlamydia has co-opted the function of F-actin and IFs to stabilize the inclusion with a dynamic, structural scaffold while minimizing the exposure of inclusion contents to cytoplasmic innate immune surveillance pathways.
Chlamydiae are obligate intracellular pathogens that cause a wide range of human diseases. Chlamydia resides in a membrane bound vacuole (“inclusion”) that expands to accommodate replicating bacteria. We recently reported that Chlamydia remodels and recruit two major cytoskeletal components of the host cell- F-actin and Intermediate filaments-to form a dynamic scaffold that provides structural stability to the inclusion. As the inclusion expands, a secreted chlamydial protease progressively modifies the intermediate filaments scaffold, presumably to increase the inclusion's flexibility and accommodate the increased bacterial load. This represents a unique mechanism employed by an intracellular pathogen to support its intracellular niche and may be linked to immune evasion by this pathogen. Here, we discuss the potential consequences of Chlamydia-mediated alteration of host cytoskeletal dynamics on the pathogenesis of chlamydial infections.
Chlamydia; intermediate filaments; Rho GTPases; cell motility; inflammation
Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.
Cell-autonomous host defense pathways directed against vacuolar pathogens constitute an essential arm of the mammalian innate immune defense system. Underlying most of these defense strategies is the ability of the host cell to recognize foreign or pathogen-modified structures and to deliver antimicrobial molecules specifically to these sites. Specific targeting of molecules to pathogen-containing vacuoles (PVs) requires host cells to recognize PVs as “non-self” structures that are distinct from intact “self” structures like organelles and other endomembrane components. In this work, we develop a new framework for understanding a critical principle that guides the mammalian immune system in the recognition of PVs as “non-self” structures. Our data indicates that so-called IRGM proteins function as markers of “self” compartments. We find that IRGM proteins act as “guards” that prevent a set of antimicrobial GTPases from stable association with “self” membranes. Because IRGM proteins are largely absent from “non-self” PVs, we propose that intracellular immune recognition of PVs can occur via the missing of “self” IRGM proteins.
STING (stimulator of interferon [IFN] genes) initiates type I IFN responses in mammalian cells through the detection of microbial nucleic acids. The membrane-bound obligate intracellular bacterium Chlamydia trachomatis induces a STING-dependent type I IFN response in infected cells, yet the IFN-inducing ligand remains unknown. In this report, we provide evidence that Chlamydia synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite not previously identified in Gram-negative bacteria, and that this metabolite is a prominent ligand for STING-mediated activation of IFN responses during infection. We used primary mouse lung fibroblasts and HEK293T cells to compare IFN-β responses to Chlamydia infection, c-di-AMP, and other type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment induced type I IFN responses in cells expressing STING but not in cells expressing STING variants that cannot sense cyclic dinucleotides but still respond to cytoplasmic DNA. The failure to induce a type I IFN response to Chlamydia and c-di-AMP correlated with the inability of STING to relocalize from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing such a response in Chlamydia-infected cells.
This study shows that the Gram-negative obligate pathogen Chlamydia trachomatis, a major cause of pelvic inflammatory disease and infertility, synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite that thus far has been described only in Gram-positive bacteria. We further provide evidence that the host cell employs an endoplasmic reticulum (ER)-localized cytoplasmic sensor, STING (stimulator of interferon [IFN] genes), to detect c-di-AMP synthesized by Chlamydia and induce a protective IFN response. This detection occurs even though Chlamydia is confined to a membrane-bound vacuole. This raises the possibility that the ER, an organelle that innervates the entire cytoplasm, is equipped with pattern recognition receptors that can directly survey membrane-bound pathogen-containing vacuoles for leaking microbe-specific metabolites to mount type I IFN responses required to control microbial infections.
We have characterized a host-induced virulence gene, mig-14, that is required for fatal infection in the mouse model of enteric fever. mig-14 is present in all Salmonella enterica subspecies I serovars and maps to a region of the chromosome that appears to have been acquired by horizontal transmission. A mig-14 mutant replicated in host tissues early after infection but was later cleared from the spleens and livers of infected animals. Bacterial clearance by the host occurred concomitantly with an increase in gamma interferon levels and recruitment of macrophages, but few neutrophils, to the infection foci. We hypothesize that the mig-14 gene product may repress immune system functions by interfering with normal cytokine expression in response to bacterial infections.
During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease CPAF into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Here we report the development of an in vitro assay for the enzyme and the discovery of a cell-permeable CPAF zymogen-based peptide inhibitor with nanomolar inhibitory affinity. Treating C. trachomatis-infected HeLa cells with this inhibitor prevented CPAF cleavage of the intermediate filament vimentin, and led to loss of vimentin-cage surrounding the intracellular vacuole. Because Chlamydia is a genetically intractable organism, this inhibitor may serve as a tool to understand the role of CPAF in pathogenesis.
Chlamydia trachomatis; inhibitor; pathogenesis; protease
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.
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.
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.
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.
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.
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.
Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.
In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole (“inclusion”). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.
The obligate intracellular bacteria Chlamydia trachomatis is a common sexually transmitted pathogen and the leading cause of preventable blindness worldwide. Chlamydia co-opts host cells by secreting virulence factors directly into target cells through a multi-protein complex termed a type III secretion system or “injectisome”. The lack of a system for molecular genetic manipulation in these pathogens has hindered our understanding of how the Chlamydia injectisome is assembled and how secreted factors are recognized and translocated. In this study, a yeast two-hybrid approach was used to identify networks of Chlamydia proteins that interact with components of the secretion apparatus. CdsQ, a conserved structural component predicted to be at the base of the injectisome, interacted with multiple proteins, including a new chaperone that binds to and stabilizes secretory cargo destined for the membrane of the pathogenic vacuole. These results suggest that the base of the secretion apparatus serves as a docking site for a chaperone and a subset of chaperone-cargo complexes. Because the chlamydial injectisome represents a unique and ancestral lineage of these virulence-associated secretion systems, findings made in Chlamydia should provide unique insights as to how effector proteins are recognized and stabilized, and how a hierarchy of virulence protein secretion may be established by Gram-negative bacterial pathogens.
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.
Chlamydia is a large burden on global health. It is the most common cause of infectious blindness, and the CDC (Centers for Disease Control and Prevention) estimates that in the United States alone there are more than 2 million people with sexually transmitted Chlamydia infections. Chlamydia is an obligate intracellular bacteria; thus, attachment and subsequent invasion of cells are key steps in Chlamydia pathogenesis. While strides have been made in understanding the molecular mechanism of Chlamydia infection, fundamental aspects of this process still remain elusive. We have identified a host protein, protein disulfide isomerase (PDI), that is essential for Chlamydia attachment as well as for entry into cells. Cell-surface PDI-mediated disulfide reduction is required for Chlamydia entry into cells, whereas bacterial attachment is independent of PDI enzymatic activity. Although PDI is necessary for Chlamydia attachment, the bacteria apparently does not bind directly to cell-associated PDI, suggesting that Chlamydia attaches to a host protein(s) associated with PDI. This study advances our understanding of Chlamydia pathogenesis by the characterization of a host factor essential for independent stages of bacterial attachment and entry.
The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of β-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.
Sodalis glossinidius, a maternally transmitted bacterial endosymbiont of tsetse flies (Glossina spp.), uses an acylated homoserine lactone (AHL)-based quorum sensing system to modulate gene expression in accordance with bacterial cell density. The S. glossinidius quorum sensing system relies on the function of two regulatory proteins; SogI (a LuxI homolog) synthesizes a signaling molecule, characterized as N-(3-oxohexanoyl) homoserine lactone (OHHL), and SogR1 (a LuxR homolog) interacts with OHHL to modulate transcription of specific target genes.
We used a tiling microarray to analyze the S. glossinidius transcriptome in the presence and absence of exogenous OHHL. The major finding is that OHHL increases transcription of a large number of genes that are known to be involved in the oxidative stress response. We also show that the obligate symbiont of the rice weevil, Sitophilus oryzae (SOPE), maintains copies of the quorum sensing regulatory genes that are found in S. glossinidius. Molecular evolutionary analyses indicate that these sequences are evolving under stabilizing selection, consistent with the maintenance of their functions in the SOPE symbiosis. Finally, the expression studies in S. glossinidius also reveal that quorum sensing regulates the expression of a cryptic, degenerate gene (carA) that arose from an ancient deletion in the last common ancestor of S. glossinidius and SOPE.
This oxidative stress response is likely mandated under conditions of dense intracellular symbiont infection, when intense metabolic activity is expected to generate a heavy oxidative burden. Such conditions are known to arise in the bacteriocytes of grain weevils, which harbor dense intracellular infections of symbiotic bacteria that are closely related to S. glossinidius. The presence of a degenerate carA sequence in S. glossinidius and SOPE indicates the potential for neofunctionalization to occur during the process of genome degeneration.
The sst1, “supersusceptibility to tuberculosis,” locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1S) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1S mice. Infection of sst1S macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-β and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-β. Intriguingly macrophages from the sst1S mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.
Chlamydia pneumoniae is a highly adapted intracellular pathogen and a common cause of atypical, community acquired pneumonia. It has also been suggested as a trigger or promoter of asthma and atherosclerosis. In this study, we examined the role of a genetic locus on mouse chromosome 1 that has been associated with susceptibility to another intracellular pathogen, Mycobacterium tuberculosis, in the pathogenesis of respiratory infections secondary to Chlamydia pneumoniae. We have determined that a variant at this locus, known as sst1 and associated with destructive pulmonary tuberculosis, makes mice dramatically more sensitive in vivo to the inflammatory changes following respiratory infection with C. pneumoniae. This appears to arise from activation of type I interferons and apoptotic cell death, two signaling pathways that are normally silent during productive C. pneumoniae infection. Despite a noted inability of sst1 susceptible macrophages to support chlamydial development, exuberant lung tissue damage resulted in overall more severe disease in vivo. We conclude the sst1-mediated control of lung tissue damage is an important determinant of the genetic susceptibility of a given host to a number of diverse intracellular bacterial pathogens, which may provide predictors of outcomes to infectious diseases as well as possible target for novel therapeutics.
The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis.
Many intracellular parasites ensure their survival and proliferation within host cells by secreting an array of effector molecules that modulate various cellular functions. Among these, Brucella abortus, the causative agent of the worldwide zoonosis brucellosis, controls the intracellular trafficking of its vacuole, the Brucella-containing vacuole (BCV), towards compartments of the secretory pathway via the expression of a Type IV secretion system (T4SS), VirB, which is thought to translocate effector proteins. Here, we have used bioinformatic algorithms and protein translocation reporter assays to identify novel Brucella proteins translocated into host cells, some of which are VirB T4SS substrates and targeted secretory pathway compartments when ectopically expressed in mammalian cells. Three VirB effectors, BspA, BspB and BspF, inhibited protein secretion and contributed to varying degrees to bacterial inhibition of host protein secretion, pathogen intracellular growth and persistence in the liver of infected mice. These findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins to promote Brucella pathogenesis.