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Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last 10 years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Objectives: Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) cause the majority of non-gonococcal urethritis (NGU). The role of Ureaplasma urealyticum (UU) in NGU is unclear. Prior case-control studies that examined the association of UU and NGU may have been confounded by mixed infections and less stringent criteria for controls. The objective of this case-control study was to determine the prevalence and aetiology of mixed infections in men and assess if UU monoinfection is associated with NGU., Methods: We identified 155 men with NGU and 103 controls. Behavioural and clinical information was obtained and men were tested for Neisseria gonorrhoeae and CT, MG, UU and Trichomonas vaginalis (TV). Men who were five-pathogen negative were classified as idiopathic urethritis (IU)., Results: Twelve per cent of NGU cases in which a pathogen was identified had mixed infections, mostly UU coinfections with MG or CT; 27% had IU. In monoinfected NGU cases, 34% had CT, 17% had MG, 11% had UU and 2% had TV. In controls, pathogens were rarely identified, except for UU, which was present in 20%. Comparing cases and controls, NGU was associated with CT and MG monoinfections and mixed infections. UU monoinfection was not associated with NGU and was almost twice as prevalent in controls. Men in both the case and control groups who were younger and who reported no prior NGU diagnosis were more likely to have UU (OR 0.97 per year of age, 95% CI 0.94 to 0.998 and OR 6.3, 95% CI 1.4 to 28.5, respectively)., Conclusions: Mixed infections are common in men with NGU and most of these are UU coinfections with other pathogens that are well-established causes of NGU. UU monoinfections are not associated with NGU and are common in younger men and men who have never previously had NGU. Almost half of NGU cases are idiopathic., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Rectal infection with Chlamydia trachomatis (CT) is frequent in women who deny receptive anal sex and is thought to arise from autoinoculation of the rectum from vaginal secretions. An alternate hypothesis is that oral sex inoculates and establishes gastrointestinal tract infection. Distinguishing these hypotheses is difficult in women. In men, autoinoculation is unlikely and heterosexual men frequently perform oral sex, but rarely participate in receptive anal exposure behaviors., Methods: We enrolled high-risk men with and without nongonococcal urethritis who presented to a sexually transmitted infection clinic in Indianapolis, Indiana. Urine and rectal swabs were collected and tested for urogenital and rectal CT, Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG). Men completed surveys concerning symptoms, sexual orientation, and detailed recent and lifetime oral and anal sexual behaviors., Results: Rectal CT was detected in 2/84 (2.4%) heterosexual men who reported cunnilingus, but no lifetime receptive anal behaviors. All of the men who denied receptive anal behaviors were negative for rectal NG and MG. In homosexual and bisexual men, rectal CT prevalence was high (9.7%), and rectal NG (4.8%) and MG (4.8%) were also detected., Conclusions: We detected rectal CT infections in heterosexual men who reported cunnilingus but denied receptive anal behaviors. Oral sex may be a risk factor for rectal CT infection via oral inoculation of the gastrointestinal tract.

The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host., Importance: Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and responsible for significant morbidity, including pelvic inflammatory disease, infertility, and ectopic pregnancies in women. As an obligate intracellular pathogen, C. trachomatis is in perpetual conflict with cell-intrinsic defense programs executed by its human host. Our study defines a novel anti-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system., (Copyright © 2016 Haldar et al.)

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle., Importance: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

Chlamydiae are obligate intracellular bacterial parasites that infect a wide range of metazoan hosts. Some Chlamydia species are important causes of chronic inflammatory diseases of the ocular, genital and respiratory tracts in humans. Genes located in a variable region of chlamydial genomes termed the plasticity zone are known to be key determinants of pathogenic diversity. The plasticity zone protein CT153, present only in select species, contains a membrane attack complex/perforin (MACPF) domain, which may mediate chlamydial interactions with the host cell. CT153 is present throughout the C. trachomatis developmental cycle and is processed into polypeptides that interact with membranes differently than does the parent protein. Chlamydiae interact extensively with membranes from the time of invasion until they eventually exit host cells, so numerous roles for a MACPF protein in pathogenesis of these pathogens are conceivable. Here, we present an overview of what is known about CT153 and highlight potential roles of a MACPF family protein in a group of pathogens whose intracellular development is marked by a series of interactions with host cell membranes and organelles. Finally, we identify new strategies for identifying CT153 functions made feasible by the recent development of a basic toolset for genetic manipulation of chlamydiae.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

Chlamydia trachomatis strains are obligate intracellular human pathogens that share near genomic synteny but have distinct infection and disease organotropisms. The genetic basis for differences in the pathogen-host relationship among chlamydial strains is linked to a variable region of chlamydial genomes, termed the plasticity zone (PZ). Two groups of PZ-encoded proteins, the membrane attack complex/perforin (MACPF) domain protein (CT153) and members of the phospholipase D-like (PLD) family, are related to proteins that modify membranes and lipids, but the functions of CT153 and the PZ PLDs (pzPLDs) are unknown. Here, we show that full-length CT153 (p91) was present in the elementary bodies (EBs) of 15 C. trachomatis reference strains. CT153 underwent a rapid infection-dependent proteolytic cleavage into polypeptides of 57 and 41 kDa that was independent of de novo chlamydial protein synthesis. Following productive infection, p91 was expressed during the mid-developmental cycle and was similarly processed into p57 and p41 fragments. Infected-cell fractionation studies showed that insoluble fractions contained p91, p57, and p41, whereas only p91 was found in the soluble fraction, indicating that unprocessed CT153 may be secreted. Finally, CT153 localized to a distinct population of reticulate bodies, some of which were in contact with the inclusion membrane.

Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. To assess the role played by pathogen variation in disease outcome, we analyzed the genomes of 4 trachoma strains representative of the 3 major trachoma serotypes, using microarray-based comparative genome sequencing. Outside of ompA, trachoma strains differed primarily in a very small subset of genes (n = 22). These subtle genetic variations were manifested in profound differences in virulence as measured by in vitro growth rate, burst size, plaque morphology, and interferon-gamma sensitivity but most importantly in virulence as shown by ocular infection of nonhuman primates. Our findings are the first to identify genes that correlate with differences in pathogenicity among trachoma strains.

Chlamydia trachomatis and C. muridarum, human and mouse pathogens, respectively, share more than 99% of open reading frames (ORFs) but differ in a cytotoxin locus. Presence or absence of cytotoxin gene(s) in these strains correlates with their ability to grow in IFN-gamma treated mouse cells. Growth of toxin-positive C. muridarum is not affected in IFN-gamma treated cells, whereas growth of toxin-negative C. trachomatis is inhibited. We previously reported that this difference in IFN-gamma sensitivity is important to the in vivo infection tropism of these pathogens. Here we describe a phenotypic rescue assay that utilizes C. muridarum gamma irradiated killed elementary bodies (iEB) to rescue C. trachomatis infectivity in IFN-gamma treated mouse cells. Rescue by iEB was temporal, maximal early post infection, directly related to multiplicity of iEB infection, and was independent of de novo chlamydial transcription. Lastly, C. muridarum iEB vacuoles and C. trachomatis inclusions were not fusogenic, suggesting the factor(s) responsible for rescue was secreted or exposed to the cytosol where it inactivated IFN-gamma induced effectors. Chlamydial phenotypic rescue may have broader utility for the study of other EB associated virulence factors that function early in the interaction of chlamydiae with host cells.

Members of the genus Chlamydia are obligate intracellular pathogens that have a unique biphasic developmental cycle and interactions with host cells. Many genes that dictate host infection tropism and, putatively, pathogenic manifestations of disease are clustered in a hypervariable region of the genome termed the plasticity zone (PZ). Comparative genomics studies have determined that an uncharacterized family of PZ genes encoding orthologs of eukaryotic and prokaryotic members of the phospholipase D (PLD) enzyme family varies among chlamydiae. Here, we show that the PZ PLD (pzPLD) of Chlamydia trachomatis are transcribed during both normal and persistent infection and that the corresponding PLD proteins are predominantly localized in reticulate bodies on the inner leaflet of the inclusion membrane. Further, we show that strains of chlamydiae encoding the pzPLD, but not a strain lacking these genes, are inhibited by primary alcohols, potent PLD inhibitors, during growth in HeLa 229 cells. This inhibitory effect is amplified approximately 5,000-fold during recovery from persistent infection. These findings suggest that the chlamydial pzPLD may be important, strain-specific, pathogenesis factors in vivo.

Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host-pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host-pathogen niche revolves around a restricted repertoire of host species-specific IFN-gamma-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-gamma induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-gamma induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (Iigp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-gamma-mediated immunity.

Chlamydia trachomatis is an obligatory intracellular prokaryotic parasite that causes a spectrum of clinically important chronic inflammatory diseases of humans. Persistent infection may play a role in the pathophysiology of chlamydial disease. Here we describe the chlamydial transcriptome in an in vitro model of IFN-gamma-mediated persistence and reactivation from persistence. Tryptophan utilization, DNA repair and recombination, phospholipid utilization, protein translation, and general stress genes were up-regulated during persistence. Down-regulated genes included chlamydial late genes and genes involved in proteolysis, peptide transport, and cell division. Persistence was characterized by altered but active biosynthetic processes and continued replication of the chromosome. On removal of IFN-gamma, chlamydiae rapidly reentered the normal developmental cycle and reversed transcriptional changes associated with cytokine treatment. The coordinated transcriptional response to IFN-gamma implies that a chlamydial response stimulon has evolved to control the transition between acute and persistent growth of the pathogen. In contrast to the paradigm of persistence as a general stress response, our findings suggest that persistence is an alternative life cycle used by chlamydiae to avoid the host immune response.

Chlamydiae are obligate intracellular pathogens that rely on secreted effector proteins to establish their intracellular niche. In this issue of the Journal of Bacteriology, Yanatori et al. describe a screen for Chlamydia pneumoniae effectors, performed in Chlamydia trachomatis, which identified several new proteins that are translocated during infection (I. Yanatori, K. Miura, Y. S. Chen, R. H. Valdivia, F. Kishi, J Bacteriol 203:e00511-20, 2021, https://doi.org/10.1128/JB.00511-20). More broadly, they demonstrate how new genetic approaches in C. trachomatis can be used to characterize the virulence factors of other Chlamydia species. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis and C. muridarum, human and mouse pathogens, respectively, share more than 99% of open reading frames (ORFs) but differ in a cytotoxin locus. Presence or absence of cytotoxin gene(s) in these strains correlates with their ability to grow in IFN-γ treated mouse cells. Growth of toxin-positive C. muridarum is not affected in IFN-γ treated cells, whereas growth of toxin-negative C. trachomatis is inhibited. We previously reported that this difference in IFN-γ sensitivity is important to the in vivo infection tropism of these pathogens. Here we describe a phenotypic rescue assay that utilizes C. muridarum gamma irradiated killed elementary bodies (iEB) to rescue C. trachomatis infectivity in IFN-γ treated mouse cells. Rescue by iEB was temporal, maximal early post infection, directly related to multiplicity of iEB infection, and was independent of de novo chlamydial transcription. Lastly, C. muridarum iEB vacuoles and C. trachomatis inclusions were not fusogenic, suggesting the factor(s) responsible for rescue was secreted or exposed to the cytosol where it inactivated IFN-γ induced effectors. Chlamydial phenotypic rescue may have broader utility for the study of other EB associated virulence factors that function early in the interaction of chlamydiae with host cells. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is a human pathogen and Chlamydia muridarum is a mouse pathogen but paradoxically, they share near genomic synteny. The majority of strain-variable genes are located primarily in a hyper-variable region termed the plasticity zone. Tryptophan synthase and cytotoxin are plasticity zone genes unique to the human and murine strains, respectively. Tryptophan synthase is a virulence factor that differentiates C. trachomatis strains into genital and ocular disease pathotypes, whereas cytotoxin(s) is a virulence factor linked to murine infection tropism. Divergence in these loci is strongly correlated with host-specific interferon gamma effector activities, suggesting that these virulence genes have co-evolved with their respective hosts as a primary mechanism to evade innate immunity. These findings have important implications for chlamydial animal modeling studies. [Copyright &y& Elsevier]