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Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host-pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host-pathogen niche revolves around a restricted repertoire of host species-specific IFN-␥-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-␥ induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-␥ induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (Iigp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-␥-mediated immunity.immunity ͉ virulence factors ͉ pathogenesis ͉ defense ͉ coevolution

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.

Gamma interferon (IFN-␥

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H 4 folate are products of the reaction. Production of H 4 folate rather than H 2 folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA ؊ E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH 2 H 4 folate and follow an ordered sequential mechanism, CTThyX follows a pingpong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and sitedirected mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.Chlamydiae are obligate eubacterial parasites that cause a variety of diseases in humans, birds, and numerous animals (1, 2). Of the four recognized chlamydial species, two, Chlamydia pneumoniae and Chlamydia trachomatis, are established pathogens of humans. Human chlamydial infections are a leading cause of sexually transmitted disease, blindness, and respiratory disease (3). One of the reasons for chlamydiae's success as a pathogen is no doubt linked to its highly specialized biphasic growth cycle (4). The elementary body (EB) 1 is the metabolically inert, osmotically stable extracellular form that is capable of initiating infection by attaching to and entering the host cell. Within a few hours, the internalized EB differentiates into the metabolically active, osmotically fragile reticulate body (RB), which divides by binary fission. About 20 h later, the process of RB differentiation back to EB begins.Over time, chlamydiae have evolved an intimate metabolic relationship with their host. Early studies on chlamydial metabolism indicated that they were auxotrophic for amino acids, nucleotides, and many other components of intermediary metabolism that most free living bacteria are capable of synthesizing themselves (4, 5). Studies from our laboratory have shown that chlamydiae are capable of transporting NTPs but not dNTPs directly from the host cell (6, 7). In addition, we isolated a mutant C. trachomatis isolate resistant to the cytotoxic effects of hydroxyurea, a r...

SummaryWe previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase ( trpA and trpB ). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene ( trpR ). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae-infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan-depleted medium or in the presence of IFN-g . Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5-fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis . The growth-inhibitory properties of 5-fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN-g g g g is via the induction of the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase.

SummaryWe previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase ( trpBA ) and the tryptophan repressor ( trpR ). Here we employ primer extension analysis to identify the transcriptional origins of both trpR and trpBA , allowing for the identification of the putative operator sequences for both trpR and trpBA . Moreover we demonstrate that native recombinant chlamydial TrpR binds to the predicted operator sequence upstream of trpR . A restriction endonuclease protection assay was designed and used to demonstrate that 5-fluorotryptophan was the only tryptophan analogue capable of activating binding of native recombinant chlamydial TrpR to its operator. Additionally, 5-fluorotryptophan was the only analogue that repressed expression of trpBA at a level analogous to L -tryptophan itself. Based on these findings, a mutant selection protocol was designed and a C. trachomatis isolate containing a frameshift mutation in trpR was isolated. This chlamydial mutant synthesizes a truncated TrpR protein that cannot regulate expression of trpBA and trpR in response to changes in tryptophan levels. These findings provide the first genetic proof that TrpR acts as a negative regulator of transcription in C. trachomatis .