Trachomatis Serovar Research Articles

Host nectin-1 is required for efficient Chlamydia trachomatis serovar E development.

Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

Differential infection outcome of Chlamydia trachomatis in human blood monocytes and monocyte-derived dendritic cells.

BackgroundChlamydia trachomatis is an intracellular bacteria which consist of three biovariants; trachoma (serovars A-C), urogenital (serovars D-K) and lymphogranuloma venereum (L1-L3), causing a wide spectrum of disease in humans. Monocytes are considered to disseminate this pathogen throughout the body while dendritic cells (DCs) play an important role in mediating immune response against bacterial infection. To determine the fate of C. trachomatis within human peripheral blood monocytes and monocyte-derived DCs, these two sets of immune cells were infected with serovars Ba, D and L2, representative of the three biovariants of C. trachomatis.ResultsOur study revealed that the different serovars primarily infect monocytes and DCs in a comparable fashion, however undergo differential infection outcome, serovar L2 being the only candidate to inflict active infection. Moreover, the C. trachomatis serovars Ba and D become persistent in monocytes while the serovars predominantly suffer degradation within DCs. Effects of persistence gene Indoleamine 2, 3-dioxygenase (IDO) was not clearly evident in the differential infection outcome. The heightened levels of inflammatory cytokines secreted by the chlamydial infection in DCs compared to monocytes seemed to be instrumental for this consequence. The immune genes induced in monocytes and DCs against chlamydial infection involves a different set of Toll-like receptors, indicating that distinct intracellular signalling pathways are adopted for immune response.ConclusionOur results demonstrate that the host pathogen interaction in chlamydia infection is not only serovar specific but manifests cell specific features, inducing separate immune response cascade in monocytes and DCs.

Transient exposure to Chlamydia trachomatis can induce alteration of sperm function which cannot be stopped by sperm washing

Abstract Previous experiments have shown that Chlamydia trachomatis can directly affect sperm function and therefore may be an unrecognized contributor to male sub-fertility. However, the precise mechanism of sperm–chlamydial interaction remains unknown. Aim This experimental study attempted to replicate a transient exposure of sperm to C. trachomatis that might occur prior to assisted conception. Methods Six ejaculates from sperm donors were spiked with C. trachomatis serovar E and subjected to density centrifugation one hour later using a standard sperm washing technique. At 0, 6, and 24 h post-wash, the recovered motile suspension was evaluated for sperm motility, viability, phosphatidylserine externalization, DNA fragmentation and tyrosine phosphorylation. Results The results show that even after a relatively short exposure to C. trachomatis , changes in sperm motility, viability, phosphatidylserine externalization and sperm DNA fragmentation were detected up to 24 h later. Only tyrosine phosphorylation was unaffected. Conclusion These results suggest that sperm washing cannot protect sperm from the deleterious effects of C. trachomatis exposure and this may explain some cases of poor IVF outcome or fertilization failure. We suggest that all patients should be screened and treated for C. trachomatis prior to assisted conception. This is one of the current guidelines also recommended by the British Fertility Society.

Plasmid-encoded pgp3 protein of Chlamydia trachomatis serovar D: cloning, expression, identification, and evaluation of immunogenicity

Objective To express and identify the plasmid-encoded pgp3 protein of Chlamydia trachomatis (Ct) serovar D,and to evaluate its immunogenicity.Methods The pgp3 gene of Ct serovar D was amplified by PCR and directly inserted into the prokaryotic expression plasmid vector pGEX-6P-1 through the pZeroBack vector.Then,the recombinant plasmid pGEX-6P-1/pgp3 was transformed into E.coli DH5α followed by enzymatic digestion,sequencing and PCR identification.After the identification,the recombinant plasmid was transformed into E.coli BL21 followed by isopropyl-β-D-thiogalactoside (IPTG) induction.The expressed protein was identified by Western blot,and purified by glutathione S-transferase (GST) MagBeads.Male New Zealand rabbits were immunized with the purified protein for three times.Blood samples were collected from these rabbits one week after the last immunization and subjected to the qualification and quantification of anti-pgp3 polyclonal antibodies by Western blot and enzymelinked immunosorbent assay (ELISA) respectively.Results The length of the cloned gene was 795 bp,which was consistent with the sequence of the pgp3 gene in GenBank.The recombinant fusion protein,whose relative molecular weight was 54 000,was successfully purified.The serum of immunized rabbits could react with the pgp3 protein,with the titer of anti-pgp3 polyclonal antibodies being 1 ∶ 25 600.Conclusions The pgp3-GST fusion protein with high immunogenicity is successfully expressed and purified,which may lay a foundation for further studies. Key words: Chlamydia trachomatis; Protein, pgp3; Recombinant fusion proteins; Immunization; Vaccines,synthetic

Commonly prescribed β-lactam antibiotics induce C. trachomatis persistence/stress in culture at physiologically relevant concentrations.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other β-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

Chlamydia Infections in Children and Adolescents

1. Lorena M. Siqueira, MD, MSPH* 1. *Division of Adolescent Medicine, Miami Children’s Hospital, Miami, FL. * Abbreviations: CDC: : Centers for Disease Control and Prevention FDA: : Food and Drug Administration HIV: : human immunodeficiency virus LGV: : lymphogranuloma venereum MIF: : microimmunofluorescent MSM: : men who have sex with men NAAT: : nucleic acid amplification tests PID: : pelvic inflammatory disease STI: : sexually transmitted infection 1. The need for annual screening for chlamydia in sexually active adolescents and young adults is underrecognized because this infection is asymptomatic in most females (75%) and males (50%). 2. Difficulty in diagnosis arises from the range of possible symptoms at presentation of symptomatic infections and the type of testing that can be used in each situation. After completing this article the reader should be able to: 1. Recognize the spectrum of clinical manifestations of chlamydia infection in neonates, children, and adolescents. 2. Discuss the appropriate use of culture and nonculture diagnostic tests for chlamydia infections in various age groups. 3. List the current recommendations for screening and treatment of chlamydia infections. A 17-year-old girl presents with prolonged menstrual bleeding associated with severe menstrual cramping pain. Her menarche was at age 13 years. Subsequent menstrual cycles were monthly, lasting 5 days, with average flow associated with mild to moderate pain relieved by over-the-counter pain medications. The current cycle began a month ago and is associated with severe lower abdominal pain. She admits to sexual activity with one partner and uses condoms intermittently. On examination she is afebrile and in discomfort, with a heart rate of 120 beats per minute. She does not appear anemic and has good capillary refill. She has diffuse abdominal pain with significant tenderness in both the right lower and upper quadrants. Chlamydiae are small obligate, gram-negative, intracellular bacteria that contain both DNA and RNA and were formerly considered to be viruses. There are 3 species that are pathogenic to humans: Chlamydia trachomatis , Chlamydophilia pneumoniae, and Chlamydophilia psittaci . They fall within 2 genera in the family Chlamydiaceae . C trachomatis is in …

Chlamydia trachomatis infection increases the expression of inflammatory tumorigenic cytokines and chemokines as well as components of the Toll-like receptor and NF-κB pathways in human prostate epithelial cells

Inflammation has been reported to play a major role in prostate carcinogenesis. Several bacterial infections can lead to prostate inflammation; however, until now, the precise molecular and cellular mechanisms linking inflammation to carcinogenesis have remained unclear. We therefore investigated the initiation of inflammation induced by Chlamydia trachomatis (C. trachomatis) infection in human prostate epithelial cells using an in vitro culture system in which human androgen-independent PC-3 prostate cancer epithelial cells were infected with C. trachomatis serovar L2. The expression levels of VEGF, ICAM-1, IL-6, IL-8, IL-1β, TNFα, CCL5, CCL2 and iNOS inflammation-related genes, as well as genes involved in the Toll-like receptor (TLR) pathway (TLR2, TLR4, CD14 and MyD88), were evaluated at the mRNA level in infected PC-3 cells 24 h after infection with C. trachomatis serovar L2. The expression levels of components of the NF-κB pathway (p65 and IκBα) were evaluated at the mRNA level in infected PC-3 cells at different time points (1, 6, 12 and 24 h) after infection. The expression levels of inflammation-related genes, components of the Toll-like receptor pathway and genes involved in NF-κB activation were analyzed in infected and uninfected cells using semi-quantitative RT-PCR. We detected a significant increase (p < 0.001) in inflammation-related cytokines in infected PC-3 cells. During infection, PC-3 cells elicited a proinflammatory response, as shown by NF-κB activation, TLR2 and TLR4 upregulation and the increased expression of inflammation-related genes. Furthermore, we observed significant upregulation of the adhesion molecules ICAM-1 and VEGF, which are two biomarkers correlated with tumor progression and immune system evasion. The present study suggests that human prostate cancer epithelial cells are susceptible to C. trachomatis infection and upregulate proinflammatory markers during infection.

Identification of lymphogranuloma venereum‐associated Chlamydia trachomatis serovars by fluorescence in situ hybridisation – a proof‐of‐principle analysis

We describe a proof-of-principle evaluation of a fluorescence in situ hybridisation (FISH) procedure to identify Chlamydia trachomatis serovars L1-L3, the causative agents of lymphogranuloma venereum, in cell cultures based on newly designed DNA probes. Rapid and easy-to-perform FISH could facilitate the diagnosis of lymphogranuloma venereum without nucleic acid amplification or serotyping, but requires broader evaluation studies, for example, in tropical high-endemicity regions.

Infectivity of urogenitalChlamydia trachomatisplasmid-deficient, CT135-null, and double-deficient strains in female mice

Chlamydia trachomatis is the most common cause of human bacterial sexually transmitted infections and is the world's leading cause of infectious preventable blindness. The chlamydial 7.5-kb plasmid and chromosomal gene CT135 have been shown to be important virulence factors in both nonhuman primate and mouse infection models. Chlamydia trachomatis plasmid-deficient urogenital isolates and a predicted CT135 null mutant have been evaluated independently in the female mouse genital tract model and both have been shown to reduce infectivity and virulence. However, these attenuating phenotypes have not been evaluated collectively in the murine model. Here, we test the infectivity of C. trachomatis serovar D strains in the mouse model that are plasmid-deficient, CT135 disrupted, or possess a combination of these attenuating genotypes. We find that the presence of the plasmid results in infections with higher infectious burdens, whereas CT135 facilitates a more protracted or chronic infection. Not unexpectedly, a combination of these genetic deficiencies resulted in a strain with enhanced infection attenuation characteristics.

Human enteroendocrine cell responses to infection with Chlamydia trachomatis: a microarray study.

BackgroundEnteroendocrine cells (EEC) are highly specialized cells producing signalling molecules vital to the normal functions of the gut. Recently, we showed altered protein distribution in Chlamydia infected EEC in vitro. The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model.MethodsTwo human EEC lines: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected using cultured C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. We used microarray analysis (Affymetrix GeneChip®) for studying changes in gene expression at different stages of infection.ResultsTwenty-four hours after active and persistent infection, 66 and 411 genes in LCC-18 and 68 and 170 genes in CNDT-2 cells, respectively showed mean expression ratios >2-fold compared to non-infected cells. These genes encoded factors regulating apoptosis, cell differentiation, transcription regulation, cytokine activity, amine biosynthesis and vesicular transport. We found significant differences in gene transcription levels between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) and in 5 genes related to endocrine function (GABARAPL1, GRIP1, DRD2, SYT5 and SYT7).ConclusionsInfected EEC cells exhibit cell-type specific patterns related to vesicular transport, secretion and neurotransmitters. EEC play a pivotal role in regulation of gut motility and an impairment of enteroendocrine function can contribute to motility disorders.

Transformation of Sexually Transmitted Infection-Causing Serovars of Chlamydia trachomatis Using Blasticidin for Selection

Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis.

Plasmid-mediated transformation tropism of chlamydial biovars

Chlamydia trachomatis and C.muridarum are human and mouse pathogens, respectively, which show high conservation of gene order and content. Both species contain a common 7.5-kb plasmid that is an important virulence factor. Recently described transformation systems have been used to characterize C.trachomatis L2 plasmid gene functions; however, similar studies have not been reported for C.trachomatis ocular tropic serovar A or the mouse strain, C.muridarum. Here, we have conducted genetic experiments with C.trachomatis serovar A and C.muridarum and report the following: (1) successful transformation of C.muridarum and C.trachomatis serovar A is restricted to a shuttle vector with a C.muridarum or C.trachomatis serovar A plasmid backbone, respectively; (2) transformation of plasmid-deficient C.muridarum with the C.muridarum-based shuttle vector complement glycogen accumulation and inclusion morphology; and (3) C.muridarum plasmid-encoded Pgp4 is a regulator of chromosomal (glgA) and plasmid (pgp3) virulence genes. In summary, our findings show a previously unrecognized and unexpected role for the chlamydial plasmid in its transformation tropism and confirm the plasmids regulatory role of virulence genes in C.muridarum.

A Case of Late-Stage Lymphogranuloma Venereum in a Woman in Europe

Lymphogranuloma venereum is caused by Chlamydia trachomatis serovars L1-L3. The third stage of the disease leads to chronic progressive lymphangitis. The first European case of elephantiasic enlargement of the female genitalia caused by C. trachomatis L2 serovar in decades is reported.

Genotyping of Chlamydia trachomatis from endocervical specimens in Shiraz, Iran

Summary The objective of this study was to determine the prevalence and distribution of Chlamydia trachomatis genotypes in Shiraz, Iran. Two hundred twelve cervical swab samples were collected from women attending Shahid Motahari Polyclinic in Shiraz, Iran. The endocervical specimens were screened for C. trachomatis by plasmid PCR. Genotyping was performed in C. trachomatis-positive samples by nested PCR amplification and sequencing of 571 fragment encompassing VD1 and VD2 of omp1 gene. The overall prevalence rate of C. trachomatis in endocervical specimens determined by plasmid nested PCR was 8%. The deduced serovars found, in descending order of prevalence, were F (46.6%), E (33.3%), and D (13.3%), and serovar G was found in a single sample. Sequence mutation analysis by BLAST search against GenBank reference sequences identified 4 genetic variants. This study can be considered a contribution to increasing knowledge on C. trachomatis genotype distribution and sequence variations within each genotype in Shiraz. Further studies are needed to better define molecular epidemiology of C. trachomatis serovars and to investigate its genotype variations in Iran.

Chloramphenicol acetyltransferase as a selection marker for chlamydial transformation

BackgroundChlamydia is a common bacterial pathogen responsible for many diseases. Methods for transforming this important organism using a β-lactamase as a selection marker have been developed very recently. However, the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules do not permit transformation experiments with β-lactamase gene-containing vectors for certain human chlamydial pathogens. Therefore, a different selection marker is urgently needed for transformation of those chlamydiae.ResultsAfter transformation of plasmid-free Chlamydia trachomatis with pGFP:SW2, which carries a β-lactamase and a chloramphenicol acetyltransferase gene fused to a green fluorescence protein gene, transformants were obtained by selection with either ampicillin or chloramphenicol. Stable chloramphenicol-resistant, but ampicillin-sensitive, transformants were obtained using a pGFP:SW2 derivative without the β-lactamase. All transformants expressed green fluorescence protein and had glycogen synthesis activity restored.ConclusionsChloramphenicol resistance may be used as a selection marker for genetic experiments in Chlamydia. This eliminates the requirement for the use of β-lactamase, of which dissemination to some C. trachomatis serovars may jeopardize clinical treatment of chlamydial infections in pregnant women. Chloramphenicol acetyltransferase may also serve as a useful secondary selection marker for genetic analyses in β-lactamase-transformed chlamydial strains.

Correction: Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

The following should be included as the first sentence of the abstract: “The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. The authors have confirmed the identity of the C. trachomatis serovar E strain used in this study by PCR and sequencing of the ompA gene.” The sentence at the bottom of page 10 should be corrected. It currently reads, “Thus, at least two different serovars of C. trachomatis appear to be capable of co-opting the FGF2 pathway to facilitate bacterial spread.” We would correct it to “Thus, at least two different species of Chlamydia appear to be capable of co-opting the FGF2 pathway to facilitate bacterial spread.”

Correction: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

The following should be included as the first sentence of the abstract: “The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. The authors have confirmed the identity of the C. trachomatis serovar D strain used in this study by PCR and sequencing of the ompA gene.”

Correction: RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry

The following should be included as the first sentence of the abstract: “The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP.”

Chlamydia trachomatis GlgA Is Secreted into Host Cell Cytoplasm

Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.

P3.296 Prevalence of Chlamydial Infections Within Eight South African Provinces (2006–2011)

BackgroundThe microbiological surveillance was undertaken in eight provinces of South Africa during 2006 –2011 to determine the aetiology of the male urethritis syndrome (MUS), vaginal discharge syndrome (VDS) and genital...