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The obligate intracellular pathogen Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections and blindness globally. To date, Ct urogenital strains are considered tryptophan prototrophs, utilizing indole for tryptophan synthesis within a closed-conformation tetramer comprised of two α (TrpA)- and two β (TrpB)-subunits. In contrast, ocular strains are auxotrophs due to mutations in TrpA, relying on host tryptophan pools for survival. It has been speculated that there is strong selective pressure for urogenital strains to maintain a functional operon. Here, we performed genetic, phylogenetic, and novel functional modeling analyses of 595 geographically diverse Ct ocular, urethral, vaginal, and rectal strains with complete operon sequences. We found that ocular and urogenital, but not lymphogranuloma venereum, TrpA-coding sequences were under positive selection. However, vaginal and urethral strains exhibited greater nucleotide diversity and a higher ratio of nonsynonymous to synonymous substitutions [Pi(a)/Pi(s)] than ocular strains, suggesting a more rapid evolution of beneficial mutations. We also identified nonsynonymous amino acid changes for an ocular isolate with a urogenital backbone in the intergenic region between TrpR and TrpB at the exact binding site for YtgR-the only known iron-dependent transcription factor in Chlamydia-indicating that selective pressure has disabled the response to fluctuating iron levels. In silico effects on protein stability, ligand-binding affinity, and tryptophan repressor (TrpR) affinity for single-stranded DNA (ssDNA) measured by calculating free energy changes (ΔΔG) between Ct reference and mutant tryptophan operon proteins were also analyzed. We found that tryptophan synthase function was likely suboptimal compared to other bacterial tryptophan prototrophs and that a diversity of urogenital strain mutations rendered the synthase nonfunctional or inefficient. The novel mutations identified here affected active sites in an orthosteric manner but also hindered α- and β-subunit allosteric interactions from distant sites, reducing efficiency of the tryptophan synthase. Importantly, strains with mutant proteins were inclined toward energy conservation by exhibiting an altered affinity for their respective ligands compared to reference strains, indicating greater fitness. This is not surprising as l-tryptophan is one of the most energetically costly amino acids to synthesize. Mutations in the tryptophan repressor gene (trpR) among urogenital strains were similarly detrimental to function. Our findings indicate that urogenital strains are evolving more rapidly than previously recognized with mutations that impact tryptophan operon function in a manner that is energetically beneficial, providing a novel host-pathogen evolutionary mechanism for intracellular survival.IMPORTANCE Chlamydia trachomatis (Ct) is a major global public health concern causing sexually transmitted and ocular infections affecting over 130 million and 260 million people, respectively. Sequelae include infertility, preterm birth, ectopic pregnancy, and blindness. Ct relies on available host tryptophan pools and/or substrates to synthesize tryptophan to survive. Urogenital strains synthesize tryptophan from indole using their intact tryptophan synthase (TS). Ocular strains contain a trpA frameshift mutation that encodes a truncated TrpA with loss of TS function. We found that TS function is likely suboptimal compared to other tryptophan prototrophs and that urogenital stains contain diverse mutations that render TS nonfunctional/inefficient, evolve more rapidly than previously recognized, and impact operon function in a manner that is energetically beneficial, providing an alternative host-pathogen evolutionary mechanism for intracellular survival. Our research has broad scientific appeal since our approach can be applied to other bacteria that may explain evolution/survival in host-pathogen interactions.

Chlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L2/434 and outbreak strain L2b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L2c, isolated from an MSM with severe hemorrhagic proctitis. L2c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L2c was a recombinant of L2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation., IMPORTANCE Lymphogranuloma venereum (LGV) is a prevalent and debilitating sexually transmitted disease in developing countries, although there are significant ongoing outbreaks in Australia, Europe, and the United States among men who have sex with men (MSM). Relatively little is known about LGV virulence factors, and only two LGV genomes have been sequenced to date. We isolated an LGV strain from an MSM with severe hemorrhagic proctitis that was morphologically unique in tissue culture compared with other LGV strains. Bioinformatic and statistical analyses identified the strain as a recombinant of L2 and D strains with highly conserved clustered regions of genetic exchange. The unique culture morphology and, more importantly, disease phenotype could be traced to the genes involved in recombination. The findings have implications for bacterial species evolution and, in the case of ongoing LGV outbreaks, suggest that recombination is a mechanism for strain emergence that results in significant disease pathology.