Trachomatis L2 Research Articles

Chlamydia trachomatis serovar L2 infection model using human lymphoid Jurkat cells

Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C. trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C. trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C. trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis L2 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement.

Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis

A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MβCD) and cholesterol-loading MβCD complexed cholesterol (chol-MβCD). The invasion was attenuated by MβCD-treatment while chol-MβCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells.

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.

Identification and Characterization of the Chlamydia trachomatis L2 S -Adenosylmethionine Transporter

ABSTRACTMethylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-l-[methyl-14C]methionine with an apparent Km of 5.9 µM and a Vmax of 32 pmol min−1 mg−1. Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.

HIV-1 does not significantly influence Chlamydia trachomatis serovar L2 replication in vitro

Individuals with lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis serovar L2, are commonly co-infected with human immunodeficiency virus type 1 (HIV-1), for reasons that remain unknown. One hypothesis is that a biological synergy exists between the two pathogens. We tested this by characterising for the first time in vitro C. trachomatis L2 replication in the presence of HIV-1. The human epithelial cell-line, MAGI P4R5 was infected with C. trachomatis L2 and HIV-1 (MN strain). Co-infected cultures contained fewer and larger chlamydial inclusions, but the inclusions did not contain morphologically aberrant organisms. C. trachomatis remained infectious in the presence of HIV-1 and showed neither an alteration in genome accumulation, nor in the acumulation of ompA, euo or unprocessed 16S rRNA transcripts. However, omcB was slightly elevated. Taken together, these data indicate that HIV-1 co-infection did not significantly alter C. trachomatis replication and the association between HIV-1 and LGV is likely due to other factors that require further investigation. The fewer, larger inclusions observed in co-infected cultures probably result from the fusion of multiple inclusions in HIV-1 induced syncytia and indicate that C. trachomatis-host-cell interactions continue to function, despite considerable host-cell re-modelling.

Transcriptome Analysis Indicates an Enhanced Activation of Adaptive and Innate Immunity byChlamydia‐Infected Murine Epithelial Cells Treated with Interferon γ

Interferon γ (IFN‐γ) is the major cytokine involved in the elimination of Chlamydia infection. Despite its importance, the combined effect of Chlamydia infection and IFN‐γ on the gene expression of murine epithelial cells has only partially been described. The DNA chip method was used to evaluate the impact of IFN‐γ and both the human strain Chlamydia trachomatis L2 infection and the murine strain Chlamydia muridarum infection on the transcriptome of murine epithelial cells. The gene expression analysis revealed that IFN‐γ had an enhancing effect on both the up‐regulation and down‐regulation of the epithelial gene expression. The influenced gene functional classes included cytokine and chemokine expression, antigen presentation, apoptosis, and genes involved in basic metabolic processes such as fatty acid oxidation. We also detected the up‐regulation of various genes that could be directly antichlamydial, such as members of the p47 GTPase family, inducible nitric oxide synthase, and monokine induced by IFN‐γ (MIG). As a functional validation of DNA chip data, we measured the antichlamydial effect of MIG on the extracellular form of Chlamydia. Our results show that IFN‐γ is a key cytokine that primes epithelial cells to activate adaptive and innate immunity and to express antichlamydial effector genes both intracellularly and extracellularly.

Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseriagonorrhoeae

Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis (serovar L2), Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 μM towards C. trachomatis L2. Three INPs had a MIC <12.5 μM against N. gonorrhoeae. Inhibition was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection ( P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.

Immunization with the attenuated plasmidless Chlamydia trachomatis L2(25667R) strain provides partial protection in a murine model of female genitourinary tract infection

Here we report on the safety, immunogenicity, and vaccine efficacy of the naturally occurring plasmid-free attenuated Chlamydia trachomatis L2-25667R (L2R) strain in a murine infection model. Intravaginal immunization induced both chlamydial specific serum antibody and systemic CD4+ Th1 biased immune responses but failed to induce local IgA antibodies. Immunization induced no pathological changes in the urogenital tract. Protective immunity was evaluated by vaginal challenge with a natural occurring non-attenuated plasmid positive C. trachomatis urogenital strain (serovar D). Vaccinated mice were not protected from colonization/infection but exhibited a reduction in infectious burden at early time periods (1–2 weeks) post-challenge. Partial protective immunity did not protect against inflammatory disease. Thus, intravaginal vaccination with the live-attenuated L2R stain is safe, induces a systemic antibody and CD4+ Th1 biased immune response, but its protective efficacy is limited to reducing chlamydial burden at early time periods post-infection.

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

BackgroundChlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.ResultsWe demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry.ConclusionINPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.

Macrophages are significant producers of IL-1beta during C. muridarum genital tract infection and IL-1beta secretion is dependent on chlamydial viability but not growth (133.3)

Abstract Recent findings have implicated secretion of the proinflammatory cytokine interleukin-1β as a crucial player in female genital tract pathology during chlamydial infection. However, the major cellular source of IL-1β during an in vivo infection and mechanistic details of its release have yet to be determined. In the present study, flow cytometry was used to isolate highly enriched cell populations from the genital tracts of C. muridarum infected mice at the peak of IL-1β secretion. Purified F4/80+ macrophages and Ly-6G+ neutrophils, but not CD45- epithelial cells, expressed high levels of both IL-1β mRNA and protein. Macrophages infected with C. muridarum in vitro secrete low amounts of IL-1β, however they can be induced to secrete high amounts when prestimulated with E. coli LPS prior to infection. Prestimulation with purified LPS from C trachomatis L2 was also sufficient to amplify IL-1β secretion, suggesting that prestimulation by chlamydial ligands could account for the high levels of IL-1β observed in vivo. Using LPS prestimulated macrophages as a model system, it was further determined that IL-1β secretion was dependent on activation of the protease caspase-1 in a manner that required both potassium efflux and the activity of serine proteases. Additionally, chlamydial-induced IL-1β secretion was blocked when chlamydiae were inactivated by UV irradiation or heat treatment but occurred normally during treatment with chloramphenicol, rifampin, and INP0007, illustrating that only bacterial viability and not bacterial growth is necessary for this response. This study was supported by NIH grant #AI067678.

Manifestations and management of lymphogranuloma venereum

This review was prompted by a sustained outbreak of lymphogranuloma venereum that has been observed among men who have sex with men (MSM) worldwide since 2004. Recent developments in the epidemiology, diagnosis and management of the infection are summarized. Between the early 1980s and 2003, lymphogranuloma venereum was rarely seen in the developed world. In 2003, a cluster of cases was seen in the Netherlands occurring mostly in HIV-positive MSM with high levels of sexual risk. With the assistance of novel molecular diagnostic techniques, more than a thousand cases of Chlamydia trachomatis L2 serovar disease have now been reported in MSM worldwide. Almost all have presented with rectal infection, usually manifesting as severe proctitis, with ulcer adenopathy syndrome seldom seen. Oral doxycycline remains the recommended treatment and has proven effective in the recent outbreak. Conflicting data exist regarding the prevalence of asymptomatic infection, and our understanding of the exact modes of transmission remains incomplete. Lymphogranuloma venereum appears to have reestablished endemicity among MSM populations in many industrialized nations. In the relative absence of recent publications from its traditional endemic regions it can be assumed that these populations remain afflicted by the infection as well.

Analysis of proteins inChlamydia trachomatisL2 outer membrane complex, COMC

The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary amino groups of in vivo generated proteolytic cleavage sites facilitated identification of such sites in known outer membrane proteins (MOMPs). Our results further support a proposed prediction of the topology of the MOMPs. Furthermore, a previously unknown MOMP, CTL0626 (Ct372), was assigned as an MOMP with a carbohydrate-selective porin (OprB) family motif, and the presence of CTL0626 was confirmed using antibodies raised against the protein.

The chlamydial functional homolog of KsgA confers kasugamycin sensitivity to Chlamydia trachomatis and impacts bacterial fitness

BackgroundrRNA adenine dimethyltransferases, represented by the Escherichia coli KsgA protein, are highly conserved phylogenetically and are generally not essential for growth. They are responsible for the post-transcriptional transfer of two methyl groups to two universally conserved adenosines located near the 3'end of the small subunit rRNA and participate in ribosome maturation. All sequenced genomes of Chlamydia reveal a ksgA homolog in each species, including C. trachomatis. Yet absence of a S-adenosyl-methionine synthetase in Chlamydia, the conserved enzyme involved in the synthesis of the methyl donor S-adenosyl-L-methionine, raises a doubt concerning the activity of the KsgA homolog in these organisms.ResultsLack of the dimethylated adenosines following ksgA inactivation confers resistance to kasugamycin (KSM) in E. coli. Expression of the C. trachomatis L2 KsgA ortholog restored KSM sensitivity to the E. coli ksgA mutant, suggesting that the chlamydial KsgA homolog has specific rRNA dimethylase activity. C. trachomatis growth was sensitive to KSM and we were able to isolate a KSM resistant mutant of C. trachomatis containing a frameshift mutation in ksgA, which led to the formation of a shorter protein with no activity. Growth of the C. trachomatis ksgA mutant was negatively affected in cell culture highlighting the importance of the methylase in the development of these obligate intracellular and as yet genetically intractable pathogens.ConclusionThe presence of a functional rRNA dimethylase enzyme belonging to the KsgA family in Chlamydia presents an excellent chemotherapeutic target with real potential. It also confirms the existence of S-adenosyl-methionine - dependent methylation reactions in Chlamydia raising the question of how these organisms acquire this cofactor.

New Chlamydia trachomatis L2 Strains Identified in a Recent Outbreak of Lymphogranuloma Venereum in Vienna, Austria

Since 2003, an ongoing outbreak of lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis biovar L2b, has been reported among men who have sex with men. Twenty-four samples positive for C. trachomatis were analyzed for specific biovars and genovariants by genotyping of the variable segment (VS) 4, VS2 and VS1 regions of the outer membrane protein (omp) A. In addition we assessed the patients' sociodemographic background and clinical signs and symptoms. Twenty-four men who have sex with men presented with either anorectal or inguinal symptoms and tested positive for C. trachomatis DNA. Of these, the L2 genotype accounted for 15 patients, with a high coinfection rate with HIV (73.3%) and other sexually transmitted infections (53.4%). Analysis of the VS1, VS2, and VS4 regions of the ompA gene revealed the variant L2b in 8 patients. In 4 patients, 3 new L2 sequences were identified with nucleotide changes in the VS1, VS2, and VS4 region, respectively, defining new strains designated L2c, d, e. This outbreak of LGV represents the further spread of C. trachomatis L2 infection. Sequence analysis of ompA regions shows heterogeneity of L2 variants, suggesting more than 1 source of the LGV infections diagnosed in Vienna.

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

To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified ∼226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRβ may function as a receptor, as inhibition of PDGFRβ by RNA interference or by PDGFRβ neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRβ or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRβ and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.

Lymphogranuloma venereum: a review of literature.

Lymphogranuloma venereum (LGV) is a systemic STD caused by Chlamydia trachomatis serotypes L1, L2 and L3. The disease is endemic in parts of Africa, Asia, South America and the Caribbean but rare in Western countries where the disease occurs mainly in sporadic form. Large outbreaks occurred recently in Europe and America mostly among men who have sex with men (MSM). The clinical course of the disease is stratified into primary, secondary and late stages but the presentation may be atypical particularly when coexisting with HIV and may result in diagnostic confusion. We present here a review of literature on LGV.African Journal of Clinical and Experimental Microbiology Vol. 9 (1) 2008: pp. 53-58

Effect of host fatty acid-binding protein and fatty acid uptake on growth of Chlamydia trachomatis L2

Chlamydia trachomatis is an obligate intracellular bacterium and acquires both building blocks and energy from host cells for growth. The fatty acid-binding protein (FABP) plays an important role in uptake of long-chain fatty acids (LCFA) and energy metabolism by eukaryotic cells. The roles of FABP and LCFA in chlamydial infection were evaluated. Infection of liver cells with chlamydial organisms promoted fatty acid uptake by the infected cells, suggesting that LCFA may benefit chlamydial growth. Introduction of FABP into the liver cells not only enhanced fatty acid uptake, but also increased chlamydial intravacuolar replication and maturation. The FABP-enhanced chlamydial intracellular growth was dependent on the host cell uptake of fatty acids. These results have demonstrated that C. trachomatis can productively infect liver cells and utilize FABP-transported LCFA for its own biosynthesis.

P1855 Heterogeneity of Chlamydia trachomatis L2 strains involved in the current outbreak of Lymphogranuloma venereum

P1855 Heterogeneity of Chlamydia trachomatis L2 strains involved in the current outbreak of Lymphogranuloma venereum

The effect of penicillin on Chlamydia trachomatis DNA replication

Chlamydia trachomatis L2 was used to infect BGMK cells at an m.o.i. of 1.0, and the developmental cycle was followed by transmission electron microscopy and quantitative PCR (QPCR) for both chromosomal and plasmid DNA. Samples were taken at sequential 6 h time points. Subsequent analysis by QPCR showed that there was an initial slow replication period (0-18 h), followed by a rapid phase (18-36 h) coinciding with exponential division when the DNA doubling time was 4.6 h. Chromosomal DNA was amplified 100-200-fold corresponding to 7-8 generations for the complete developmental cycle. Penicillin (10 and 100 units ml(-1)) was added to cultures at 20 h post-infection (p.i.). This blocked binary fission and also prevented reticulate body (RB) to elementary body transition. However, exposure to penicillin did not prevent chromosomal or plasmid DNA replication. After a short lag period, following the addition of penicillin, chlamydial chromosomal DNA replication resumed at the same rate as in control C. trachomatis-infected cells. C. trachomatis-infected host cells exposed to penicillin did not lyse, but instead harboured large, aberrant RBs in massive inclusions that completely filled the cell cytoplasm. In these RBs, the DNA continued to replicate well beyond the end of the normal developmental cycle. At 60 h p.i. each aberrant RB contained a minimum of 16 chromosomal copies.

Ulcerative proctitis in men who have sex with men: an emerging outbreak

Lymphogranuloma venereum (LGV) is a sexually transmitted infection that is caused by Chlamydia trachomatis types L1, L2, and L3.1 Although endemic in some tropical countries, lymphogranuloma venereum is usually uncommon...