Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Introduction: Chlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression. Methods: We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site. Results: The 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorectal" clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains. Discussion: The absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Chlamydia trachomatis , a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression. We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site. The 78 C. trachomatis genomes from participants were from two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorecta"l clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains. The absence of large numbers of fixed SNPs between C. trachomatis strains within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant variation in the different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage. Importance: Chlamydia trachomatis is a bacterial pathogen that causes millions of sexually transmitted infections (STIs) annually across the globe. Because C. trachomatis lives inside human cells, it has historically been hard to study. We know little about how the bacterium spreads between body sites. Here, samples from 26 study participants who had simultaneous infections in their vagina, rectum and endocervix were genetically analyzed using an improved method to extract C. trachomatis DNA directly from clinical samples for genome sequencing. By analyzing patterns of mutations in the genomes, we found that 21 participants shared very similar C. trachomatis strains in all three anatomic sites, suggesting recent infection and spread. For five participants two C. trachomatis strains were evident, indicating multiple infections. This study is significant in that improved enrichment methods for genome sequencing provides robust data to genetically trace patterns of C. trachomatis infection and transmission within an individual for epidemiologic and pathogenesis interrogations.

Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs.

Chlamydia trachomatis, an obligately intracellular bacterium, is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. Numbers of U.S. infections of the urogenital tract and rectum have increased annually. Because C. trachomatis is not easily cultured, comparative genomic studies are limited, restricting our understanding of strain diversity and emergence among populations globally. While Agilent SureSelectXT target enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal samples, public access to these libraries is not available. We therefore designed an RNA bait library (34,795 120-mer probes based on 85 genomes, versus 33,619 probes using 74 genomes in a previous one) to augment organism sequencing from clinical samples that can be shared with the scientific community, enabling comparison studies. We describe the library and limit of detection for genome copy input, and we present results of 100% efficiency and high-resolution determination of recombination and identical genomes within vaginal-rectal specimen pairs in women. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, which limits our understanding of urogenital and rectal C. trachomatis transmission and impact on morbidity. To provide a publicly available workflow for whole-genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we developed and report on an RNA bait library to enrich the organism from clinical samples for sequencing. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis and identified recombinant and identical C. trachomatis genomes in paired vaginal-rectal samples from women. Our workflow provides a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital, and rectal infections and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

Clinical Persistence of Chlamydia trachomatis Sexually Transmitted Strains Involves Novel Mutations in the Functional αßßα Tetramer of the Tryptophan Synthase Operon.

Clinical persistence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and ß subunits that encode TrpA and TrpB, respectively, and form a functional αßßα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αßßα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism's tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis.

High expression of IDO1 and TGF-ß1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response.

BACKGROUND: Chlamydia trachomatis infections in women continue to be a major public health concern due to their high prevalence and consequent reproductive morbidities. While antibiotics are usually efficient to clear the Chlamydia, repeat infections are common and may contribute to pathological outcomes. Interferon-gamma (IFN-γ)-mediated immunity has been suggested to be protective against reinfection, and represent an important anti-chlamydial agent, primarily via the induction of indoleamine-2,3 dioxygenase 1 (IDO1) enzyme. IDO1 catalyzes the degradation of tryptophan, which can eliminate C. trachomatis infection in vitro. Here, we sought to measure IDO1 expression levels and related immune markers during different C. trachomatis infection statuses (repeated vs single infection vs post antibiotic treatment), in vitro and in vivo. METHODS: In this study, we measured the expression levels of IDO1 and immune regulatory markers, transforming growth factor ß1 (TGF-ß1) and forkhead box P3 (FoxP3), in vaginal swab samples of C. trachomatis-infected women, with either single or repeated infection. In addition, we used an in vitro co-culture model of endometrial carcinoma cell-line and peripheral blood mononuclear cells (PBMCs) to measure the same immune markers. RESULTS: We found that in women with repeated C. trachomatis infections vaginal IDO1 and TGF-ß1 expression levels were significantly increased. Whereas, women who cleared their infection post antibiotic treatment, had increased levels of IDO1 and TGF-ß1, as well as FoxP3. Similarly, using the in vitro model, we found significant upregulation of IDO1 and TGF-ß1 levels in the co-culture infected with C. trachomatis. Furthermore, we found that in PBMCs infected with C. trachomatis there was a significant upregulation in IDO1 levels, which was independent of IFN-γ. In fact, C. trachomatis infection in PBMCs failed to induce IFN-γ levels in comparison to the uninfected culture. CONCLUSIONS: Our data provide evidence for a regulatory immune response comprised of IDO1, TGF-ß1 and FoxP3 in women post antibiotic treatment. In this study, we demonstrated a significant increase in IDO1 expression levels in response to C. trachomatis infection, both in vivo and in vitro, without elevated IFN-γ levels. This study implicates IDO1 and TGF-ß1 as part of the immune response to repeated C. trachomatis infections, independently of IFN-γ.

Dysbiosis of the Vaginal Microbiota and Higher Vaginal Kynurenine/Tryptophan Ratio Reveals an Association with Chlamydia trachomatis Genital Infections.

The natural course of Chlamydia trachomatis urogenital tract infections varies between individuals. While protective immunity can occur, some women can become reinfected, contributing to the development of severe pathology. While the reasons for these differences are unknown, an individual's response to induced interferon-γ (IFN-γ) is suggested to be critical. IFN-γ induction of the enzyme indoleamine 2,3-dioxygenase, which depletes tryptophan, may be the key. One hypothesis suggests that indole-producing bacteria in the vaginal microbiota can provide a substrate for the Chlamydia to synthesize tryptophan, rescuing the Chlamydia from host IFN-γ attack. We studied a cohort of 25 women who were either, Chlamydia negative, Chlamydia positive with a single infection, or Chlamydia positive with repeated infection, to test our hypothesis. We characterized their vaginal microbiota, cytokine response, as well as their tryptophan, kynurenine and indole concentrations directly in vaginal secretions. We found that C. trachomatis urogenital tract infections either initial or repeat infections, were associated with elevated vaginal kynurenine/tryptophan ratios, primarily as a result of elevated kynurenine levels. In addition, vaginal microbiota of community state type (CST) IV showed significantly lower vaginal tryptophan levels compared to CST I and III, which might be related to a higher abundance of indole producers found within this group. Furthermore, we found a higher abundance of indole producers in women who cleared their Chlamydia infection post antibiotic treatment. This study demonstrates for the first time in vivo, the association between high vaginal kynurenine/tryptophan ratios and C. trachomatis infections. In addition, tryptophan depletion was associated with vaginal microbiota of CST IV.

Systemic antibody response to Chlamydia Trachomatis infection in patients either infected or reinfected with different Chlamydia serovars.

INTRODUCTION: Chlamydia trachomatis is the etiological agent for the most prevalent bacterial sexually transmitted infection in both developed and developing countries. The aim of present study was to characterize the antibody response between two groups of individuals, having either a single C. trachomatis infection and or repeated infections. MATERIAL AND METHODS: Current study consisted of two groups, one with an initial Chlamydia infection and a second with repeated infections. A titre based estimation of specific serum (IgG and IgA) levels using ELISA were performed, which further validated by western blot. In vitro neutralizing ability of each patient's serum against both homologous and heterologous strains was also determined. RESULTS: Individuals infected with one of the C. trachomatis serovars D, E or K exhibited a strong systemic antibody response as characterized by ELISA and western blot. These individuals may have developed at least some level of protection as they only represented single infection. By comparison, individuals infected with serovar D, E or F that exhibited low systemic antibody response often presented repeated C. trachomatis infections, suggesting an association with poor immune response. An in vitro neutralizing level of 60-90% was observed in the human sera against homologous serovar D and two heterologous C. trachomatis serovars E and K, compared to <40% against heterologous serovars F. CONCLUSION: Individuals infected with serovars D and K showed a potential association between circulating antibody response and re-infection risk. While the patients infected with serovars E showed a disconnection between systemic antibody response and re-infection risk.

In vitro rescue of genital strains of Chlamydia trachomatis from interferon-γ and tryptophan depletion with indole-positive, but not indole-negative Prevotella spp.

BACKGROUND: The natural course of sexually transmitted infections caused by Chlamydia trachomatis varies between individuals. In addition to parasite and host effects, the vaginal microbiota might play a key role in the outcome of C. trachomatis infections. Interferon-gamma (IFN-γ), known for its anti-chlamydial properties, activates the expression of indoleamine 2,3-dioxygenase (IDO1) in epithelial cells, an enzyme that catabolizes the amino acid L- tryptophan into N-formylkynurenine, depleting the host cell's pool of tryptophan. Although C. trachomatis is a tryptophan auxotroph, urogenital strains (but not ocular strains) have been shown in vitro to have the ability to produce tryptophan from indole using the tryptophan synthase (trpBA) gene. It has been suggested that indole producing bacteria from the vaginal microbiota could influence the outcome of Chlamydia infection. RESULTS: We used two in vitro models (treatment with IFN-γ or direct limitation of tryptophan), to study the effects of direct rescue by the addition of exogenous indole, or by the addition of culture supernatant from indole-positive versus indole-negative Prevotella strains, on the growth and infectivity of C. trachomatis. We found that only supernatants from the indole-positive strains, P. intermedia and P. nigrescens, were able to rescue tryptophan-starved C. trachomatis. In addition, we analyzed vaginal secretion samples to determine physiological indole concentrations. In spite of the complexity of vaginal secretions, we demonstrated that for some vaginal specimens with higher indole levels, there was a link to higher recovery of the Chlamydia under tryptophan-starved conditions, lending preliminary support to the critical role of the IFN-γ-tryptophan-indole axis in vivo. CONCLUSIONS: Our data provide evidence for the ability of both exogenous indole as well as supernatant from indole producing bacteria such as Prevotella, to rescue genital C. trachomatis from tryptophan starvation. This adds weight to the hypothesis that the vaginal microbiota (particularly from women with lower levels of lactobacilli and higher levels of indole producing anaerobes) may be intrinsically linked to the outcome of chlamydial infections in some women.

Chlamydia trachomatis Genital Tract Infections: When Host Immune Response and the Microbiome Collide.

Genital infections with Chlamydia trachomatis continue to be a major health problem worldwide. While some individuals clear their infection (presumed to be the result of an effective Th1/interferon-γ response), others develop chronic infections and some are prone to repeat infections. In females in particular, chronic asymptomatic infections are common and can lead to pelvic inflammatory disease and infertility. Recent studies suggest that the genital tract microbiota could be a significant factor and explain person-to-person variation in C. trachomatis infections. One hypothesis suggests that C. trachomatis can use its trpBA genes to rescue tryptophan from indole, which is a product of anaerobic members of the genital tract microbiota. Women with particular microbiota types, such as seen in bacterial vaginosis, have increased numbers of anaerobes, and this would enable the chlamydia in these individuals to overcome the host's interferon-γ attempts to eliminate it, resulting in more repeat and/or chronic infections.

An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato.

Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.