Molecular Basis Defining Human Chlamydia trachomatis Tissue Tropism

Abstract

Here we report the cloning and sequencing of a region of the chlamydiae chromosome termed the "plasticity zone" from all the human serovars of C. trachomatis containing the tryptophan biosynthesis genes. Our results show that this region contains orthologues of the tryptophan repressor as well as the ␣ and ␤ subunits of tryptophan synthase. Results from reverse transcription-PCR and Western blot analyses indicate that the trpBA genes are transcribed, and protein products are expressed. The TrpB sequences from all serovars are highly conserved. In comparison with other tryptophan synthase ␤ subunits, the chlamydial TrpB subunit retains all conserved amino acid residues required for ␤ reaction activity. In contrast, the chlamydial TrpA sequences display numerous mutations, which distinguish them from TrpA sequences of all other prokaryotes. All ocular serovars contain a deletion mutation resulting in a truncated TrpA protein, which lacks ␣ reaction activity. The TrpA protein from the genital serovars retains conserved amino acids required for catalysis but has mutated several active site residues involved in substrate binding. Complementation analysis in Escherchia coli strains, with defined mutations in tryptophan biosynthesis, and in vitro enzyme activity data, with cloned TrpB and TrpA proteins, indicate these mutations result in a TrpA protein that is unable to utilize indole glycerol 3-phosphate as substrate. In contrast, the chlamydial TrpB protein can carry out the ␤ reaction, which catalyzes the formation of tryptophan from indole and serine. The activity of the chlamydial Trp B protein differs from that of the well characterized E. coli and Salmonella TrpBs in displaying an absolute requirement for full-length TrpA. Taken together our data indicate that genital, but not ocular, serovars are capable of utilizing exogenous indole for the biosynthesis of tryptophan.

Chlamydial IFN-γ immune evasion is linked to host infection tropism

“…Infectivity of both human and mouse strains was rescued by the addition of exogenous tryptophan, but importantly, only the infectivity of human strains was rescued by exogenous indole (Fig. 3C), demonstrating the expression of a functional tryptophan synthase enzyme (17,18) by the human strains. Unlike HeLa cells, MECs treated with IFN-␥ produced iNOS mRNA and protein that resulted in NO production ( Fig.…”

Generation of targeted Chlamydia trachomatis null mutants

“…Paradoxically, genitotropic but not oculotropic strains have retained a functional trpBA operon (18)(19)(20)(21). The trpBA operon is negatively regulated and expresses the TrpBA tryptophan synthase that uses exogenous indole as substrate to synthesize tryptophan and escape the anti-microbial effect of IFN-γ-mediated tryptophan starvation (20,21). We used these phenotypic and genetic markers of the trpBA operon to characterize the CTD trpB− mutant.…”

“…The in vitro indole-rescue assays were performed as described previously (20,21). After indole rescue, chlamydiae-infected cells were harvested and titered on HeLa cell monolayers or analyzed by immunofluorescence after staining of fixed cells with anti-chlamydial antibody or DAPI for DNA.…”

“…Western blot analysis of TrpB and TrpA was performed as described previously (20,21). Anti-HSP-60 antibody was included in the analysis as a loading control.…”

Lower Levels of Cervicovaginal Tryptophan Are Associated With Natural Clearance of Chlamydia in Women

“…In response to tryptophan depletion, Ct survives by differentiating into an enlarged, non-infectious form (a process termed "persistence"), which results in a marked decrease in cellular metabolism and cellular division [7]. In addition, urogenital Ct strains encode a functional tryptophan synthase, which can synthesize tryptophan from extracellular precursor substrates, restoring tryptophan levels and permitting Ct survival in the presence of IFN--mediated immune pressure [8]. In vitro studies also demonstrate that indole is a precursor that can be used for tryptophan synthesis [8].…”