In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection.
Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.
MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.
Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes a range of human diseases of significant public health importance. To create a suitable replicative niche within its host, Chlamydia delivers effector proteins across mammalian membranes via a syringe-like apparatus termed a Type III secretion (T3S) system. The lack of a robust system for the molecular genetic manipulation of these pathogens has hindered progress in identifying and characterizing T3S effectors. In this study, we took a mass spectrometry-based approach to identify Chlamydia effector proteins based on their interaction with Slc1, an abundant T3S chaperone. We identified a previously uncharacterized protein, Ct875/TepP, as a new T3S effector and determined that TepP is phosphorylated upon translocation into host cells, leading to the recruitment of the host scaffolding protein Crk and presumably manipulating Crk-dependent signaling functions. Finally, we provide genetic confirmation of the role of TepP in recruiting Crk and in modulating the expression of genes involved in innate immune responses to Chlamydia. This study is the first example of genetic validation of the function of a T3S effector in Chlamydia and a new example of a bacterial effector that directly co-opts the oncoprotein Crk to modulate host cell signaling events.
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.
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.
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
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.
Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.
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.
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.
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.
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 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.
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.
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
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.