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Ammonia Generation by Tryptophan Synthase Drives a Key Genetic Difference Between Genital and Ocular Chlamydia Trachomatis Isolates

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Ammonia Generation by Tryptophan Synthase Drives a Key Genetic Difference Between Genital and Ocular Chlamydia Trachomatis Isolates Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates Shardulendra P. Sherchanda and Ashok Aiyara,1 aDepartment of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center New Orleans, New Orleans, LA 70112 Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved April 19, 2019 (received for review December 19, 2018) A striking difference between genital and ocular clinical isolates of the microbiome at the site of infection produces indole, permitting Chlamydia trachomatis is that only the former express a functional chlamydial tryptophan synthase to generate tryptophan and thus tryptophan synthase and therefore can synthesize tryptophan by circumvent host-imposed tryptophan starvation (3, 4, 6, 7). The indole salvage. Ocular isolates uniformly cannot use indole due to composition of the cervicovaginal microbiome indicates the pres- inactivating mutations within tryptophan synthase, indicating a ence of indole-producing genera during dysbiotic conditions, pro- selection against maintaining this enzyme in the ocular environ- viding a strong reason for genital isolates to maintain an active ment. Here, we demonstrate that this selection occurs in two tryptophan synthase (6, 7). In contrast, factors that drive the loss of steps. First, specific indole derivatives, produced by the human gut tryptophan synthase in the majority of oculotropic strains are un- microbiome and present in serum, rapidly induce expression of C. tra- clear and the focus of this report (5). chomatis tryptophan synthase, even under conditions of tryptophan Tryptophan synthase has two α-subunits, encoded by trpA, and sufficiency. We demonstrate that these indole derivatives function by two β-subunits, encoded by trpB (11, 12). Comprehensive studies acting as de-repressors of C. trachomatis TrpR. Second, trp operon de- by Caldwell et al. (3), evaluating the tryptophan synthase status repression is profoundly deleterious when infected cells are in an indole- of a large number of clinical genital and ocular isolates, revealed deficient environment, because in the absence of indole, tryptophan that no clinical ocular isolates encoded an active tryptophan synthase deaminates serine to pyruvate and ammonia. We have used synthase. With rare exception, this failure arose from point biochemical and genetic approaches to demonstrate that expression mutations in either trpA or trpB (3). In most bacteria, tryptophan of wild-type tryptophan synthase is required for the bactericidal synthase catalyzes two reactions termed α and β. In the α reac- production of ammonia. Pertinently, although these indole deriva- tion, which is catalyzed solely by TrpA (11, 12), indole-3-glycerol tives de-repress the trpRBA operon of C. trachomatis strains with phosphate undergoes a retro-aldol cleavage to release indole and trpA or trpB mutations, no ammonia is produced, and no deleterious glyceraldehyde-3-phosphate (G3P) (13). Indole is channeled to effects are observed. Our studies demonstrate that tryptophan syn- TrpB by TrpA (11, 14), to undergo a β-replacement reaction with thase can catalyze the ammonia-generating β-elimination reaction serine to generate tryptophan (11, 13). In addition to β-replacement, within any live bacterium. Our results also likely explain previous in vitro studies using tryptophan synthase from Escherichia coli observations demonstrating that the same indole derivatives inhibit demonstrated that, in the absence of indole, TrpB deaminates the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prog- Significance nosis of diseased conditions associated with bacterial dysbiosis. Chlamydia trachomatis is a human bacterial pathogen that Chlamydia trachomatis | genital and ocular serovars | tryptophan synthase causes distinct pathologies upon infecting ocular and urogen- β-elimination | serine deamination | trp operon de-repression ital compartments. Previous studies have shown that all uro- genital strains can express tryptophan synthase, an enzyme he bacterium Chlamydia trachomatis is an obligate intracel- they use to synthesize tryptophan by salvaging indole pro- Tlular pathogen that causes urogenital and ocular infections of duced by other bacterial species in the infection microenvi- humans. Distinct serovars of C. trachomatis, classified based on ronment. In stark contrast, all ocular strains of Chlamydia their outer membrane proteins, are associated with infections at trachomatis lack tryptophan synthase, typically because of these sites. Ocular infections are associated with serovars A–C, inactivating point mutations. Here, we suggest why ocular while urogenital infections are associated with serovars D–K. strains lose tryptophan synthase activity; activation of this Genetic and sequence studies indicate that the genomes of enzyme in an indole-deficient environment, like the eye, re- oculotropic and genitotropic strains are >99% identical with two sults in the deleterious production of ammonia. By identifying striking differences (1). Urogenital strains encode an intact cy- the mechanism that underlies this effect, our findings provide totoxin with a GTPase-inactivating domain (CT166) (2) and, in strategies to target infections by Chlamydia and other bacteria. addition, encode a functional tryptophan synthase that provides them the capacity to synthesize tryptophan via indole salvage (3, Author contributions: S.P.S. and A.A. designed research, performed research, contributed 4). The observation that urogenital C. trachomatis isolates ex- new reagents/analytic tools, analyzed data, and wrote the paper. press a functional tryptophan synthase extends to genital serovar The authors declare no conflict of interest. B isolates, indicating a strong selective pressure to maintain the This article is a PNAS Direct Submission. capacity to salvage indole in the urogenital environment (3, 5). Published under the PNAS license. C. trachomatis is a tryptophan auxotroph and cannot synthesize Data deposition: Predicted structures for TrpR and tryptophan synthase from C. tracho- tryptophan de novo. The capacity of urogenital isolates to syn- matis D/UW-3/CX have been deposited in the Zenodo data repository, https://doi.org/10. thesize tryptophan via indole salvage is proposed to reveal an 5281/zenodo.2662318. intricate interplay between C. trachomatis, the IFN-γ–induced See Commentary on page 12136. host enzyme indoleamine 2,3-dioxygenase (IDO1), and the 1To whom correspondence should be addressed. Email: [email protected]. microbiome at the site of infection (3, 4, 6, 7). When induced, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IDO1 degrades tryptophan to kynurenine, thereby starving 1073/pnas.1821652116/-/DCSupplemental. Chlamydia (8–10). The antibacterial effect of IFN-γ is hampered if Published online May 16, 2019. 12468–12477 | PNAS | June 18, 2019 | vol. 116 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1821652116 Downloaded by guest on September 27, 2021 serine via β-elimination to generate ammonia and pyruvate (SI results clearly indicate that expression of trpBA in the absence of Appendix,Fig.S1)(15–17). The ammonia product of this alternative indole is severely deleterious to C. trachomatis. reaction can be directly antibacterial if it saponifies bacterial lipids Our studies reveal that the ammonia-generating β-elimination SEE COMMENTARY (18–20). Alternatively, as first shown for Helicobacter pylori,am- reaction can occur when tryptophan synthase is expressed within monia kills infected human cells that express the NMDA receptor a live bacterium. Finally, the widely reported antiinflammatory (21). Given the negative implications of the ammonia-generating properties associated with these indole derivatives may result β-elimination reaction, it is unsurprising that it is biochemically from the bacterial burden-reducing consequence of inducing curtailed in two ways. First, β-elimination proceeds at a slower rate tryptophan synthase activity in an indole-poor environment. than the tryptophan-generating β-replacement reaction (22, 23). Thus, it is not favored if indole is present. Second, β-elimination is Results suppressed by allosteric interactions within tryptophan synthase. Indole-3-Propionic Acid and Indole-β-Acrylic Acid Are Predicted to De- Biochemical studies using Salmonella typhimurium tryptophan syn- repress the C. trachomatis trpRBA Operon. The C. trachomatis thase indicate that the G3P product of the α reaction remains trpRBA operon is expressed in conditions where tryptophan is bound to TrpA forcing an interaction between loop 6 of TrpA with limiting (33, 34), presumably because the TrpR apo-repressor is no helix 6 of TrpB (23–27). This allosteric change further decreases the longer associated with its corepressor, tryptophan (33, 34). Al- rate of β-elimination. Confirming this, mutations placed in S. though C. trachomatis TrpR has diverged from its E. coli homolog typhimurium tryptophan synthase that disrupt the TrpA/TrpB allo- (SI Appendix,Fig.S2A), we sought to test whether it could be de- steric interaction permit β-elimination to proceed (23–27). Relevant repressed by the same indole derivatives as E. coli TrpR. For E. to C. trachomatis tryptophan synthase, studies indicate that the coli TrpR, indole-3-propionic acid (IPA) and indole-β-acrylic acid TrpA/TrpB allosteric interaction is not
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