Exploration of chlamydial type III secretion system reconstitution in Escherichia coli

BACKGROUND: Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli. RESULTS: We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli. CONCLUSIONS: We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity

Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability

Lactobacilli inactivate Chlamydia trachomatis through lactic acid but not H2O2.

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H2O2, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H2O2 concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H2O2 production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H2O2, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection

Acquisition and synthesis of folates by obligate intracellular bacteria of the genus

We undertook studies focused on folate acquisition by Chlamydia trachomatis L2, Chlamydia psittaci 6BC, and C. psittaci francis. Results from in situ studies, using wild-type host cells, confirmed that C. trachomatis L2 and C. psittaci 6BC are sensitive to sulfonamides whereas C. psittaci francis is resistant. In addition C. trachomatis L2 and C. psittaci francis were inhibited by methotrexate in situ whereas C. psittaci 6BC was not. In contrast to C. trachomatis, neither C. psittaci strain was affected by trimethoprim. Surprisingly our results indicate that all three strains are capable of efficient growth in folate-depleted host cells. When growing in folate-depleted cells C. psittaci francis becomes sensitive to sulfonamide. The ability of all three strains to carry out de novo folate synthesis was demonstrated by following the incorporation of exogenous 13HJpABA into intracellular folates and by detecting dihydropteroate synthase activity in reticulate body crude extract. Dihydrofolate reductase activity was also detected in reticulate body extract. In aggregate the results indicate that C. trachomatis L2, C. psittaci francis, and C. psittaci 6BC can all synthesize folates de novo, however, strains differ in their ability to transport preformed folates directly from the host cell. (J. Clin. Invest. 1992.90:1803-1811.) Key words: parasite * dihydropteroate synthase * dihydrofolate reductase * sulfonamide * methotrexat