A Narrative Review on Spontaneous Clearance of Urogenital Chlamydia trachomatis: Host, Microbiome, and Pathogen-Related Factors.

ABSTRACT: Chlamydia trachomatis (CT) is the most commonly reported sexually transmitted infection in the United States. Untreated urogenital infection in women can result in adverse sequelae such as pelvic inflammatory disease and infertility. Despite national screening and treatment guidelines, rates continue to rise; because most infections are asymptomatic, the actual prevalence of CT infection is likely significantly higher than reported. Spontaneous clearance of CT in women (in the absence of antibiotic treatment) has been described in multiple epidemiologic studies. Given the serious consequences and high prevalence of CT infection, there is growing interest in understanding this phenomenon and factors that may promote CT clearance in women. Spontaneous CT clearance is likely the result of complex interactions between CT, the host immune system, and the vaginal microbiota (i.e., the communities of bacteria inhabiting the vagina), which has been implicated in CT acquisition. Herein, we briefly review current literature regarding the role of each of these factors in spontaneous CT clearance, identify knowledge gaps, and discuss future directions and possible implications for the development of novel interventions that may protect against CT infection, facilitate clearance, and prevent reproductive sequelae.

Nonoptimal Vaginal Microbiota After Azithromycin Treatment for Chlamydia trachomatis Infection.

We characterized the composition and structure of the vaginal microbiota in a cohort of 149 women with genital Chlamydia trachomatis infection at baseline who were followed quarterly for 9 months after antibiotic treatment. At time of diagnosis, the vaginal microbiota was dominated by Lactobacillus iners or a diverse array of bacterial vaginosis-associated bacteria including Gardnerella vaginalis. Interestingly, L. iners-dominated communities were most common after azithromycin treatment (1 g monodose), consistent with the observed relative resistance of L. iners to azithromycin. Lactobacillus iners-dominated communities have been associated with increased risk of C. trachomatis infection, suggesting that the impact of antibiotic treatment on the vaginal microbiota could favor reinfections. These results provide support for the dual need to account for the potential perturbing effect(s) of antibiotic treatment on the vaginal microbiota, and to develop strategies to protect and restore optimal vaginal microbiota.

Does Active Oral Sex Contribute to Female Infertility?

Based on recent, historical, and circumstantial evidence, we present a multifactorial hypothesis that has potential direct implications on the epidemiology and management of chlamydial infection and disease in humans. We propose that (1) like its veterinary relatives, the oculogenital pathogen Chlamydia trachomatis evolved as a commensal organism of the human gastrointestinal (GI) tract primarily transmissible via the fecal-oral route; (2) in the modern era, C. trachomatis causes "opportunistic" infection at non-GI sites under conditions driven by improved sanitation/hygiene and reduced fecal-oral transmission; and (3) the rise in the practice of oral sex is contributing to the increased prevalence of C. trachomatis in the human GI tract. Infectious organisms produced in the GI tract and reaching the rectum may then chronically contaminate and infect the female urogenital tract, thereby potentially contributing to the most serious sequelae of chlamydial infection in women: pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility.

A novel co-infection model with Toxoplasma and Chlamydia trachomatis highlights the importance of host cell manipulation for nutrient scavenging.

Toxoplasma and Chlamydia trachomatis are obligate intracellular pathogens that have evolved analogous strategies to replicate within mammalian cells. Both pathogens are known to extensively remodel the cytoskeleton, and to recruit endocytic and exocytic organelles to their respective vacuoles. However, how important these activities are for infectivity by either pathogen remains elusive. Here, we have developed a novel co-infection system to gain insights into the developmental cycles of Toxoplasma and C. trachomatis by infecting human cells with both pathogens, and examining their respective ability to replicate and scavenge nutrients. We hypothesize that the common strategies used by Toxoplasma and Chlamydia to achieve development results in direct competition of the two pathogens for the same pool of nutrients. We show that a single human cell can harbour Chlamydia and Toxoplasma. In co-infected cells, Toxoplasma is able to divert the content of host organelles, such as cholesterol. Consequently, the infectious cycle of Toxoplasma progresses unimpeded. In contrast, Chlamydia's ability to scavenge selected nutrients is diminished, and the bacterium shifts to a stress-induced persistent growth. Parasite killing engenders an ordered return to normal chlamydial development. We demonstrate that C. trachomatis enters a stress-induced persistence phenotype as a direct result from being barred from its normal nutrient supplies as addition of excess nutrients, e.g. amino acids, leads to substantial recovery of Chlamydia growth and infectivity. Co-infection of C. trachomatis with slow growing strains of Toxoplasma or a mutant impaired in nutrient acquisition does not restrict chlamydial development. Conversely, Toxoplasma growth is halted in cells infected with the highly virulent Chlamydia psittaci. This study illustrates the key role that cellular remodelling plays in the exploitation of host intracellular resources by Toxoplasma and Chlamydia. It further highlights the delicate balance between success and failure of infection by intracellular pathogens in a co-infection system at the cellular level.

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Use of gene sequences as type for naming prokaryotes: Recommendations of the international committee on the taxonomy of chlamydiae.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Full genome sequences of all nine Chlamydia psittaci genotype reference strains.

Chlamydia psittaci primarily infects birds, but zoonotic transmission occurs in people in close contact with infected birds. The clinical outcome ranges from inapparent disease to pneumonia. Here we report the genome sequences of all 9 Chlamydia psittaci genotype reference strains.

Unity in variety--the pan-genome of the Chlamydiae.

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Altered developmental expression of polymorphic membrane proteins in penicillin-stressed Chlamydia trachomatis.

Late Chlamydia trachomatis inclusions express each member of the surface-exposed polymorphic membrane protein family (Pmp subtypes A through I) with a reproducible distribution of fully-on, fully-off and intermediate phenotypes. This observation is consistent with observed variable Pmp antibody profiles in C. trachomatis-infected patients and has led to the hypothesis that the pmp gene family forms the basis of a phase variation-like mechanism of antigenic variation. Here we investigate and compare the developmental expression of each of the nine pmp genes under conditions of optimal in vitro growth with that under conditions that promote prolonged survival of chlamydiae when exposed to penicillin-induced stress. We demonstrate that the pmp gene family includes distinct transcriptional units that are differentially expressed along development and differentially responsive to stress. In particular, our results indicate that expression of pmpA, pmpD and pmpI is uniquely unaffected by stress, suggesting that the PmpA, PmpD and PmpI proteins play a critical role in the pathogenesis of C. trachomatis.

Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections.

2D cell culture systems have historically provided controlled, reproducible means to analyze host-pathogen interactions observed in the human reproductive tract. Although inexpensive, straightforward, and requiring a very short time commitment, these models recapitulate neither the functionality of multilayered cell types nor the associated microbiome that occurs in a human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications of animal models are required to recreate interactions that resemble those in the human reproductive tract. 3D cell culture models have emerged as alternative means of reproducing vital elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments. Here, we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extracellular matrix (ECM)-like layer. The model produced tissues with morphologic and physiological resemblance to human cervical and vaginal epithelia, including mucus levels produced by cervical cells. Infection by Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated, as well as the growth of bacterial species observed in the human vaginal microbiota. This enabled controlled mechanistic analyses of the interactions between host cells, the vaginal microbiota, and STI pathogens. Affordable and semi high-throughput 3D models of the cervicovaginal epithelia that are physiologically relevant by sustaining vaginal bacterial colonization, and facilitate studies of chlamydial and gonococcal infections.

Serum antibodies to surface proteins of Chlamydia trachomatis as candidate biomarkers of disease: results from the Baltimore Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort.

We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis-infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, Hsp60, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. More C. trachomatis-infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9%-40.7% of females vs. 24.2%-30.0% of males), with statistical significance for PmpC, F and H (P < 0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis-infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis (P< 0.05) was observed. Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs. urethra in males) stimuli may control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum.

Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58.

Chlamydia pecorum is an obligate intracellular bacterial pathogen that causes diverse disease in a wide variety of economically important mammals. We report the finished complete genome sequence of C. pecorum E58, the type strain for the species.

Genome sequences of the zoonotic pathogens Chlamydia psittaci 6BC and Cal10.

Chlamydia psittaci is a highly prevalent avian pathogen and the cause of a potentially lethal zoonosis, causing life-threatening pneumonia in humans. We report the genome sequences of C. psittaci 6BC, the prototype strain of the species, and C. psittaci Cal10, a widely used laboratory strain.

Variable expression of surface-exposed polymorphic membrane proteins in in vitro-grown Chlamydia trachomatis.

The hypothesized variable expression of polymorphic membrane proteins (PmpA-PmpI) in Chlamydia trachomatis-infected patients was tested by examination of the expression of each Pmp subtype in in vitro-grown C. trachomatis. A panel of monospecific polyclonal and monoclonal antibodies was used to demonstrate surface exposure of Pmps of each subtype by differential immunofluorescence (IF) with and without prior detergent permeabilization of paraformaldehyde-fixed inclusions and for selected Pmps by immunogold labelling. Although specific transcript was detected for each pmp gene late in development, IF experiments with Pmp subtype-specific antibodies reveal that a number of inclusions in a single infection do not express Pmps of a given subtype. Coexpression experiments suggest that pmp genes are shut off independently from one another in non-expressing inclusions, i.e. different inclusions are switched off for different Pmps. Overall, these studies establish the existence of an efficient shutoff mechanism independently affecting the expression of each member of the pmp gene family in in vitro-grown C. trachomatis. Like other paralogous gene families of bacterial pathogens, the pmp gene family of C. trachomatis may serve the critical dual function of a highly adaptable virulence factor also providing antigenic diversity in the face of the host adaptive immune response.

The role of Chlamydia trachomatis polymorphic membrane proteins in inflammation and sequelae among women with pelvic inflammatory disease.

Chlamydia trachomatis polymorphic membrane proteins (Pmps) may increase genital tract inflammation and play a role in virulence. Antibody levels for PmpA, PmpD, and PmpI, measured in densitometric units, were assessed among a pilot sample of 40 C. trachomatis-infected women with mild-to-moderate clinical PID. Women who expressed antibodies to PmpA were less likely to achieve pregnancy (40.0% versus 85.7%; P = 0.042) and less likely to have a live birth (0.0% versus 80.0%; P = 0.005) compared to women who did not express antibody to PmpA. Women who expressed antibodies to PmpI were more likely to have upper genital tract infection (61.5% versus 20.0%; P = 0.026). However, seropositivity to PmpI and PmpD did not modify the risk of reproductive sequelae or inflammation. Seropositivity to chlamydial PmpA may represent a biomarker of increased risk of sequelae secondary to infection with C. trachomatis.

Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species.

BACKGROUND: Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species. RESULTS: We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes). CONCLUSIONS: We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.

The association of Chlamydia trachomatis and Mycoplasma genitalium infection with the vaginal metabolome.

Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) are two highly prevalent bacterial sexually transmitted infections (STIs) with a significant rate of co-infection in some populations. Vaginal metabolites are influenced by resident vaginal microbiota, affect susceptibility to sexually transmitted infections (STIs), and may impact local inflammation and patient symptoms. Examining the vaginal metabolome in the context of CT mono (CT+) and CT/MG co-infection (CT+/MG+) may identify biomarkers for infection or provide new insights into disease etiology and pathogenesis. Yet, the vaginal metabolome in the setting of CT infection is understudied and the composition of the vaginal metabolome in CT/MG co-infected women is unknown. Therefore, in this analysis, we used an untargeted metabolomic approach combined with 16S rRNA gene amplicon sequencing to characterize the vaginal microbiota and metabolomes of CT+, CT+/MG+, and uninfected women. We found that CT+ and CT+/MG+ women had distinct vaginal metabolomic profiles as compared to uninfected women both before and after adjustment for the vaginal microbiota. This study provides important foundational data documenting differences in the vaginal metabolome between CT+, CT+/MG+ and uninfected women. These data may guide future mechanistic studies that seek to provide insight into the pathogenesis of CT and CT/MG infections.

Kinematics of intracellular chlamydiae provide evidence for contact-dependent development.

A crucial process of chlamydial development involves differentiation of the replicative reticulate body (RB) into the infectious elementary body (EB). We present experimental evidence to provide support for a contact-dependent hypothesis for explaining the trigger involved in differentiation. We recorded live-imaging of Chlamydia trachomatis-infected McCoy cells at key times during development and tracked the temporospatial trajectories of individual chlamydial particles. We found that movement of the particles is related to development. Early to mid-developmental stages involved slight wobbling of RBs. The average speed of particles increased sharply at 24 h postinfection (after the estimated onset of RB to EB differentiation). We also investigated a penicillin-supplemented culture containing EBs, RBs, and aberrantly enlarged, stressed chlamydiae. Near-immobile enlarged particles are consistent with their continued tethering to the chlamydial inclusion membrane (CIM). We found a significantly negative, nonlinear association between speed and size/type of particles, providing further support for the hypothesis that particles become untethered near the onset of RB to EB differentiation. This study establishes the relationship between the motion properties of the chlamydiae and developmental stages, whereby wobbling RBs gradually lose contact with the CIM, and RB detachment from the CIM is coincidental with the onset of late differentiation.

Effect of Chlamydiaphage phiCPG1 on the course of conjunctival infection with "Chlamydia caviae" in guinea pigs.

Over the last several years, four different phages of chlamydiae, in addition to a phage associated with Chlamydia psittaci isolated from an ornithosis infection in ducks over 25 years ago, have been described and characterized. While these phages and their chlamydial host specificities have been characterized in vitro, there is virtually nothing known about the interaction of the phage with chlamydiae in their natural animal host. phiCPG1 is a lytic phage specific for "Chlamydia caviae," which is a natural parasite of the guinea pig. In this study, guinea pigs were inoculated in the conjunctiva with suspensions of phiCPG1 and C. caviae and the effect on the development of pathology and on the course of chlamydial infection in the conjunctiva was determined. The presence of phage delayed the appearance of the peak level of chlamydiae in the animal and decreased the pathological response. Evidence for replication of the phage in C. caviae in the conjunctival tissue was observed. Modifying the ratio of phage to chlamydiae altered the course of infection and affected phage replication. There was no evidence for the phage increasing the virulence of C. caviae infection.