Trachomatis Serovar L2 Research Articles

Chlamydia trachomatis Inc Ct226 is vital for FLI1 and LRRF1 recruitment to the chlamydial inclusion.

The obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a host membrane-derived vacuole called the chlamydial inclusion. From within this inclusion, C. trachomatis orchestrates numerous host-pathogen interactions, in part, by utilizing a family of type III secreted effectors, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). Previously, LRRF1 has been shown to interact with Inc protein Ct226/CTL0478. This is the first study to examine interactions of FLI1 with candidate Incs or with LRRF1 during infection. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. We demonstrated that FLI1 co-immunoprecipitated with Ct226 but only in the presence of LRRF1. Furthermore, FLI1 localized to the inclusion when LRRF1 was depleted via small interfering RNA, suggesting that FLI1 may have an alternative recruitment mechanism. We further developed a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 targeting ct226 and co-transcribed candidate Incs, ct225 and ct224. Simultaneous knockdown of ct226, ct225, and ct224 prevented localization of both FLI1 and LRRF1 to the inclusion, and only complementation of ct226 restored their localization. Thus, we demonstrated Ct226 is critical for FLI1 and LRRF1 localization to the inclusion. Our results also indicate an LRRF1-independent localization mechanism for FLI1, which likely influence their mechanism(s) of action during chlamydial infection.IMPORTANCEChlamydia trachomatis is a leading cause of both bacterial sexually transmitted infections and preventable infectious blindness worldwide. As an obligate intracellular pathogen, C. trachomatis has evolved multiple ways of manipulating the host to establish a successful infection. As such, it is important to understand host-chlamydial protein-protein interactions as these reveal strategies that C. trachomatis uses to shape its intracellular environment. This study looks in detail at interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of CRISPR inference knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.

Metabolic model guided CRISPRi identifies a central role for phosphoglycerate mutase in Chlamydia trachomatis persistence.

This study uses a metabolic model to investigate factors that contribute to the persistence of Chlamydia trachomatis serovar L2 (CTL) under tryptophan and iron starvation conditions. As CTL lacks many canonical transcriptional regulators, the model was used to assess two prevailing hypotheses on persistence-that the chlamydial response to nutrient starvation represents a passive response due to the lack of regulators or that it is an active response by the bacterium. K-means clustering of stress-induced transcriptomics data revealed striking evidence in favor of the lack of adaptive (i.e., a passive) response. To find the metabolic signature of this, metabolic modeling pin-pointed pgm as a potential regulator of persistence. Thermodynamic driving force, enzyme cost, and CRISPRi knockdown of pgm supported this finding. Overall, this work introduces thermodynamic driving force and enzyme cost as a tool to understand chlamydial persistence, demonstrating how systems biology-guided CRISPRi can unravel complex bacterial phenomena.

Evaluation of Anti‐Chlamydial Effect of a Synthetic Linear Peptide

AbstractInspired by the broad spectrum of antimicrobial activity exhibited by Magainins and SMAP‐28, and based on their chemical structures, several linear peptides were designed and synthesized with the aim of achieving peptides possessing promising anti‐chlamydial activity with lower cytotoxicity towards human normal cell lines. We found these peptides to be cytotoxic against human normal cell lines, except for one designated as DJ‐7, which was utilized in subsequent experiments, while the others were excluded. Peptide DJ‐7 was readily synthesized using standard Fmoc‐SPPS, and its anti‐chlamydial activity was investigated against HeLa cells (ATCC CCL‐2) infected with Chlamydia trachomatis L2 (ATCC VR‐902B) at MOI 1 for 2 hours, followed by treatment with increasing concentrations of peptide DJ‐7 (15–60 μg/mL). Microscopic examination revealed a significant reduction in the total number of bacterial inclusions in cells by around 50 % and 80 % after treatment with 15 μg/mL (5.5 μM) and 30 μg/mL (11 μM) of peptide DJ‐7, respectively, compared to control untreated infected cells. The impact of peptide DJ‐7 treatment on the development of infectious C. trachomatis serovar L2 progeny was investigated, demonstrating a significant decrease in infectious chlamydia after treatment with peptide DJ‐7. This suggests that chlamydia failed to complete its typical developmental cycle, indicating that peptide DJ‐7 exhibits anti‐chlamydial properties, by disrupting the normal bacterial development process. Our results indicate that peptide DJ‐7 is a promising lead peptide for further development as a potential anti‐chlamydial agent.

B-273 Lymphogranuloma Venereum: Validation of a Real-Time PCR Kit for Its Rapid Diagnosis

Abstract Background Chlamydia trachomatis serovars L1, L2 and L3 are responsible for lymphogranuloma venereum (LGV), a sexually transmitted infection that causes ulcerated lesions and adenopathy. Following the procedures in Clinical Microbiology recommendations, rectal samples positive for C. trachomatis are processed to detect the LGV-producing serovars in order to provide appropriate treatment. Since European regulations require for CE and IVD certification that an in vitro diagnostic product must be validated by an external body, the aim of this study was to validate the VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit from Certest Biotec SL, using DNAs characterized as positive or negative for LGV extracted from clinical samples previously analyzed in the Laboratory. Methods A total of 214 DNAs from clinical samples collected between January 2021 and November 2022 were analyzed. Based on the initial diagnosis performed with the Allplex™ Genital ulcer Assay (Seegene®), 37 samples were positive for LGV and 177 were negative. The use of all data and samples was approved by the Aragon research ethics committee (CEICA) (PI20/426). The clinical sensitivity and specificity of the kit and the positive and negative predictive values (95% CI) were calculated with MetaDisc 1.4 software. Results Results are summarized in Table 1. Conclusion The comparative analysis of the “VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit” proved to be as sensitive and specific as the reference molecular assay. The main advantage is that VIASURE products feature a stabilized, ready-to-use format, reducing the number of time-consuming laboratory steps, avoiding possible contamination and allowing transport and storage at room temperature.

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion.

The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors. To identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting. RNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation - which was instead sensitive to inhibition of Abl. Our results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP.

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.

Shifting proteomes: limitations in using the BioID proximity labeling system to study SNARE protein trafficking during infection with intracellular pathogens.

We hypothesize that intracellular trafficking pathways are altered in chlamydial infected cells to maximize the ability of Chlamydia to scavenge nutrients while not overtly stressing the host cell. Previous data demonstrated the importance of two eukaryotic SNARE proteins, VAMP4 and syntaxin 10 (Stx10), in chlamydial growth and development. Although, the mechanism for these effects is still unknown. To interrogate whether chlamydial infection altered these proteins' networks, we created BirA*-VAMP4 and BirA*-Stx10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between Stx10 and VAPB, we also identified caveats in using the BioID system to study the impact of infection by an obligate intracellular pathogen on SNARE protein networks. The addition of the BirA* altered the localization of VAMP4 and Stx10 during infection with Chlamydia trachomatis serovars L2 and D and Coxiella burnetii Nine Mile Phase II. We also discovered that BirA* traffics to and biotinylates Coxiella-containing vacuoles and, in general, has a propensity for labeling membrane or membrane-associated proteins. While the BioID system identified a novel association for Stx10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.

Primary ectocervical epithelial cells display lower permissivity to Chlamydia trachomatis than HeLa cells and a globally higher pro-inflammatory profile

The tumoral origin and extensive passaging of HeLa cells, a most commonly used cervical epithelial cell line, raise concerns on their suitability to study the cell responses to infection. The present study was designed to isolate primary epithelial cells from human ectocervix explants and characterize their susceptibility to C. trachomatis infection. We achieved a high purity of isolation, assessed by the expression of E-cadherin and cytokeratin 14. The infectious progeny in these primary epithelial cells was lower than in HeLa cells. We showed that the difference in culture medium, and the addition of serum in HeLa cultures, accounted for a large part of these differences. However, all things considered the primary ectocervical epithelial cells remained less permissive than HeLa cells to C. trachomatis serovar L2 or D development. Finally, the basal level of transcription of genes coding for pro-inflammatory cytokines was globally higher in primary epithelial cells than in HeLa cells. Transcription of several pro-inflammatory genes was further induced by infection with C. trachomatis serovar L2 or serovar D. In conclusion, primary epithelial cells have a strong capacity to mount an inflammatory response to Chlamydia infection. Our simplified purification protocol from human explants should facilitate future studies to understand the contribution of this response to limiting the spread of the pathogen to the upper female genital tract.

Effective Treatment of Lymphogranuloma venereum Proctitis With Azithromycin.

Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis (CT) serovars L1, L2, and L3 and is endemic among men who have sex with men (MSM) in Europe. We evaluated weekly oral azithromycin 1 g for 3 weeks as a treatment for LGV proctitis. This is an open clinical trial with convenience allocation according to treating physician preferences. Adults with clinical proctitis received a single dose of 1 g of intramuscular ceftriaxone and were subsequently allocated to receive (i) doxycycline 100 mg twice daily for 21 days (Doxycycline group) or (ii) azithromycin 1 g orally once weekly for 3 weeks (Azithromycin group). LGV cure (primary endpoint) was defined as resolution of symptoms at week 6 (clinical cure, LGV-CC), with an additional supporting negative rectal polymerase chain reaction (PCR) at week 4 (microbiological cure, LGV-MC), if available. One hundred and twenty-five individuals with LGV clinical proctitis were included. All were MSM, and 96% were living with human immunodeficiency virus (HIV). Eighty-two were in the Azithromycin group, and 43 were in the Doxycycline group. LGV cure on a modified intention-to-treat analysis (primary endpoint), occurred in 80 of 82 (98%) in the Azithromycin group versus 41 of 43 (95%) in the Doxycycline group (treatment difference [95% confidence interval {CI}] 2.2% [-3.2, 13.2]). LGV-MC occurred in 70 of 72 (97%) vs 15 of 15 (100%) in the Azithromycin group and Doxycycline group, respectively (treatment difference [95% CI] -2.8% [-9.6; 17.7]). Adverse events were similar in both treatment groups. Our findings support extended azithromycin dosing as an alternative treatment option for symptomatic LGV proctitis and provides the rationale for future randomized trials.

Lymphogranuloma Venereum in a Public Health Service Hospital in Southern Spain: A Clinical and Epidemiologic Study

Lymphogranuloma venereum (LGV) is an emerging disease in men who have sex with men (MSM): the incidence was 1.15 cases per 100 000 population in Spain in 2017. Patients with LGV characteristically have severe proctitis that can cause abscesses, fistulas, and anal stenosis. Genital ulcers and inflammatory inguinal adenopathy may occasionally be present. The aim of this study was to describe a series of patients with LGV treated in a public health service hospital in Andalusia, Spain. Materials and methodsRetrospective, observational description of a series of patients diagnosed with LGV. We gathered epidemiologic, clinical, microbiologic, and treatment data. Patients’ sexual behaviors were also noted. ResultsWe found 17 cases of LGV diagnosed in MSM between October 2016 and May 2019. Twelve of the patients were also infected with the human immunodeficiency virus, and 13 had severe proctitis with ulcers in the anal canal and rectum. Four patients had genital or inguinal manifestations. The following high-risk sexual behaviors were on record: a high number of sexual partners, receptive anal sex with strangers and without a condom, seeking sexual partners online, participation in group sex, and sex with partners from outside Andalusia. Chlamydia trachomatis serovar L2 was identified in all cases, and the infection responded well to oral doxycycline. Two patients with the most characteristic form of LGV required longer treatment cycles. Three required surgery. ConclusionsWhen symptomatic proctitis is found in MSM who engage in high-risk sex, the LGV exudate should be sampled and the C trachomatis serovar identified. Genital ulcers or inguinal buboes are also highly suggestive of LGV. Clinical suspicion and early treatment are the keys to preventing complications and disease transmission.

Estudio clínico y epidemiológico del linfogranuloma venéreo en un hospital público del sur de España

Lymphogranuloma venereum (LGV) is an emerging disease in men who have sex with men (MSM): the incidence was 1.15 cases per 100,000 population in Spain in 2017. Patients with LGV characteristically have severe proctitis that can cause abscesses, fistulas, and anal stenosis. Genital ulcers and inflammatory inguinal adenopathy may occasionally be present. The aim of this study was to describe a series of patients with LGV treated in a public health service hospital in Andalusia, Spain. Material and methodsRetrospective, observational description of a series of patients diagnosed with LGV. We gathered epidemiologic, clinical, microbiologic, and treatment data. Patients’ sexual behaviors were also noted. ResultsWe found 17 cases of LGV diagnosed in MSM between October 2016 and May 2019. Twelve of the patients were also infected with the human immunodeficiency virus, and 13 had severe proctitis with ulcers in the anal canal and rectum. Four patients had genital or inguinal manifestations. The following high-risk sexual behaviors were on record: a high number of sexual partners, receptive anal sex with strangers and without a condom, seeking sexual partners online, participation in group sex, and sex with partners from outside Andalusia. Chlamydia trachomatis serovar L2 was identified in all cases, and the infection responded well to oral doxycycline. Two patients with the most characteristic form of LGV required longer treatment cycles. Three required surgery. ConclusionsWhen symptomatic proctitis is found in MSM who engage in high-risk sex, the LGV exudate should be sampled and the C trachomatis serovar identified. Genital ulcers or inguinal buboes are also highly suggestive of LGV. Clinical suspicion and early treatment are the keys to preventing complications and disease transmission.

Penicillin-binding proteins regulate multiple steps in the polarized cell division process of Chlamydia

Chlamydia trachomatis serovar L2 and Chlamydia muridarum, which do not express FtsZ, undergo polarized cell division. During division, peptidoglycan assembles at the pole of dividing Chlamydia trachomatis cells where daughter cell formation occurs, and peptidoglycan regulates at least two distinct steps in the polarized division of Chlamydia trachomatis and Chlamydia muridarum. Cells treated with inhibitors that prevent peptidoglycan synthesis or peptidoglycan crosslinking by penicillin-binding protein 2 (PBP2) are unable to initiate polarized division, while cells treated with inhibitors that prevent peptidoglycan crosslinking by penicillin-binding protein 3 (PBP3/FtsI) initiate polarized division, but the process arrests at an early stage of daughter cell growth. Consistent with their distinct roles in polarized division, peptidoglycan organization is different in cells treated with PBP2 and PBP3-specific inhibitors. Our analyses indicate that the sequential action of PBP2 and PBP3 drives changes in peptidoglycan organization that are essential for the polarized division of these obligate intracellular bacteria. Furthermore, the roles we have characterized for PBP2 and PBP3 in regulating specific steps in chlamydial cell division have not been described in other bacteria.

Protamine sulfate may prevent infections by pathogens that require heparan sulfate proteoglycan interactions, including high- and low-risk Human Papillomaviruses and Chlamydia trachomatis..

e13065 Background: Human papillomaviruses (HPVs) are the most common pathogens of the reproductive tract with 80% of the population acquiring an infection at least once in their lifetime. Globally, high-risk (oncogenic) HPVs cause 630,000 new cancer cases per year and are responsible for 99% of cervical cancers. Regardless of available screening and preventive measures, cervical cancer is the fourth most common malignancy in women worldwide causing more than 445,000 new cases and 270,000 deaths annually. Like many pathogens of the reproductive tract, initiation of HPV infections require attachment to negatively-charged heparan sulfate proteoglycans (HSPG) present in the extracellular matrix and plasma membrane of host cells. To better define the role of HSPGs in HPV infections, we used protamine sulfate (PrS), a polycationic peptide that is FDA approved to neutralize heparin. Structural similarities between HSPGs and heparin allow PrS to bind HSPGs preventing HPV-HSPG interactions and infection. In order to further evaluate proof of concept for PrS we expanded our investigation to Chlamydia trachomatis, another genital tract pathogen whose mechanism of infection has been reported to rely on HSPGs. Methods: High and low risk HPV viruses were used in prophylactic and post exposure cell-based assays and a preclinical prophylactic mouse vaginal challenge model to evaluate the effects of PrS on infection. Chlamydia trachomatis serovar L2 was also used in prophylactic cell-based assays to evaluate the effects of PrS on infection. Results: A single dose prophylactic application of PrS reduced infection by high- and low-risk HPV genotypes by up to 95% in vitro (p-value < 0.001) in a dose response manner and 60% in vivo (p-value 0.05). In addition, post-HPV exposure PrS treatment significantly decreased infection. PrS showed the greatest effectiveness in the first two hours after HPV exposure. In line with our data on HPV, Chlamydia trachomatis infection showed a statistically significant decrease in cells pretreated with PrS (p-value 0.05). Conclusions: Inhibition of attachment by PrS presents a unique opportunity to provide a new cheap prophylactic modality for HPV infection, which would reduce cervical cancer global burden.

Lymphogranuloma venereum proctitis mimicking inflammatory bowel diseases in 11 patients: a 4-year single-center experience

ABSTRACT Lymphogranuloma venereum (LGV) is a sexually transmitted disease caused by Chlamydia trachomatis (CT) serovars L1–L3. Our study wants to underline the similarities between rectal LGV and idiopathic inflammatory bowel diseases (IBD), which can share clinical, endoscopic and histopathological findings.

Propagation and Purification of Chlamydia trachomatis Serovar L2 Transformants and Mutants.

Chlamydia trachomatis (C.t.) is an obligate intracellular pathogen that cannot be cultured axenically and must be propagated within eukaryotic host cells. There are at least 15 distinct chlamydial serovariants that belong to 2 major biovars commonly referred to as trachoma and lymphogranuloma venereum (LGV). The invasive chlamydia LGV serovar L2 is the most widely used experimental model for studying C.t. biology and infection and is the only strain with reliable genetic tools available. New techniques to genetically manipulate C.t. L2 have provided opportunities to make mutants using TargeTron and allelic exchange as well as strains overexpressing epitope-tagged proteins, in turn necessitating the regular purification of transformant and mutant clones. Purification of C.t. is a labor-intensive exercise and one of the most common reagents classically used in the purification process, Renografin, is no longer commercially available. A similar formulation of diatrizoate meglumine called Gastrografin is readily available and we as well as others have had great success using this in place of Renografin for chlamydial purifications. Here, we provide a detailed general protocol for infection, propagation, purification, and titering of Chlamydia trachomatis serovar L2 with additional notes specifically pertaining to mutants or recombinant DNA carrying clones.

Feasibility of a Conditional Knockout System for Chlamydia Based on CRISPR Interference.

Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C. trachomatis. CRISPRi has been developed and used successfully in a variety of bacterial organisms including E. coli and Mycobacterium tuberculosis. I first describe the creation of a single plasmid system for CRISPRi in Chlamydia, targeted to a non-essential gene, incA, that expresses a dispensable inclusion membrane protein. Control transformations of C. trachomatis serovar L2 with plasmids encoding only the dCas9, under the control of an inducible promoter, or only the guide RNA (gRNA) targeted to the 5' UTR of incA, expressed constitutively, failed to prevent expression of IncA. Importantly, expression of dCas9 alone did not have a deleterious effect on chlamydiae. Transformation of C. trachomatis with a plasmid combining the dCas9 and a gRNA targeting incA and induction of expression of the dCas9 resulted in the reversible inhibition of IncA expression. Consequently, conditional knockout mediated by CRISPRi is feasible in Chlamydia. Potential improvements and experimental concerns in using the system are also discussed.

Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane.

Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncATM-APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.

Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.

ABSTRACTAlthough progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.

P08.06 Lactic acid isomers differentially reducechlamydia trachomatisinfection in a ph dependent manner

Introduction Epidemiological studies indicate that the vaginal microbiota can significantly impact the risk of acquiring sexually transmitted infections, including chlamydia. Lactobacillus spp. are the most common commensal bacteria in the healthy human vagina; they produce lactic acid to create an acidic environment with pH ranging between 3.5 and 4, thought to reduce vaginal colonisation by STI agents. However, not all species of Lactobacillus are believed to perform this function equally, and we hypothesised that species that produce low amounts or no D-lactic acid, while achieving low pH do not fully protect women. Methods A 3D model of cervical epithelial cells (A2EN) developed in our lab was exposed to D(-), L(+) or a D/L racemic mixture of lactic acid at various concentrations to produce pH 7, 5.5 and 4 or to several Lactobacillus spp. conditioned media (LCM). Cells were infected with C. trachomatis serovar L2 for 48 h, stained and imaged by confocal microscopy. Analysis of the resultant IFUs was used to determine the number of infected host cells. Results We observed a reduction of Chlamydia trachomatis infectivity in a pH dependent manner. Further, at pH 4, D(-) lactic acid afforded maximal protection compared to L(+) lactic acid. Interestingly, 50% infectivity is still observed with HCL at pH 4, indicating that pH alone is not responsible for this protection. Exposure of cells to conditioned media from the various Lactobacillus spp. showed that high D(-) lactic acid producing bacteria ( Lactobacillus crispatus and Lactobacillus jensenii) afforded significantly greater protection against C. trachomatis than did Lactobacillus iners, which produces predominantly L(+) lactic acid. Conclusion These results suggest a differential role for specific species of Lactobacillus in driving resistance to C. trachomatis infections and potentially other STIs. Lactic acid isomers production should be considered when developing Lactobacillus vaginal probiotics. Disclosure of interest statement Project funded by NIH-NIAID-STI-CRC U19 AI084044 “Eco-Pathogenomics of Chlamydial Reproductive Tract Infection”. No pharmaceutical grants were received in the development of this study.

P08.14 Lymphogranuloma venereumin the czech republic

Introduction Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis serovars L1 – L3. LGV was considered as tropical disease with typical inguinal syndrome and it wasn’t usual in Europe until 2003, when an outbreak was observed in the Netherlands. This was followed by series of outbreaks emerging in different European countries and North America. A common feature for this epidemic is men who have sex with men (MSM) with signs of severe proctocolitis. Most of the patients are co-infected with HIV and/or other sexually transmitted infections (STI). Methods The National Reference Laboratory for Chlamydia Infections offers a diagnostic service to clinicians. The disease is confirmed by the presence of Chlamydia trachomatis and L1 – L3 serovars from multiplex PCR (Seegene). Multiplex PCR is very useful, because multiple infections are observed in many cases. Results First case of LGV was diagnosed from a lymph node puncture in 2010. Then the number of patients was slowly increasing (5–10 patients per year) and the most cases were diagnosed in 2014 (23 patients). Until March 2015, a total of 56 patients with LGV were confirmed. Characteristics of these cases were similar to those in other European countries. LGV was confirmed among MSM with high prevalence of other STI. Forty-eight patients (85%) were co-infected with HIV. In some cases, HIV and LGV were diagnosed at approximately the same time. Forty-three patients (77%) were co-infected with syphilis. The data on other STI are not completed. The vast majority of patients manifested proctocolitis. Only in few cases the inguinal syndrome was observed. Conclusion Lymphogranuloma venereum is also present among MSM in the Czech Republic. We observed, that the number of cases increases. Certainly, it is necessary to expand testing of chlamydial infection in MSM, because this disease could facilitate HIV transmission. Disclosure of interest statement Nothing to declare.