Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates

Abstract

A striking difference between genital and ocular clinical isolates of Chlamydia trachomatis is that only the former express a functional tryptophan synthase and therefore can synthesize tryptophan by indole salvage. Ocular isolates uniformly cannot use indole due to inactivating mutations within tryptophan synthase, indicating a selection against maintaining this enzyme in the ocular environment. Here, we demonstrate that this selection occurs in two steps. First, specific indole derivatives, produced by the human gut microbiome and present in serum, rapidly induce expression of C. trachomatis tryptophan synthase, even under conditions of tryptophan sufficiency. We demonstrate that these indole derivatives function by acting as de-repressors of C. trachomatis TrpR. Second, trp operon de-repression is profoundly deleterious when infected cells are in an indole-deficient environment, because in the absence of indole, tryptophan synthase deaminates serine to pyruvate and ammonia. We have used biochemical and genetic approaches to demonstrate that expression of wild-type tryptophan synthase is required for the bactericidal production of ammonia. Pertinently, although these indole derivatives de-repress the trpRBA operon of C. trachomatis strains with trpA or trpB mutations, no ammonia is produced, and no deleterious effects are observed. Our studies demonstrate that tryptophan synthase can catalyze the ammonia-generating β-elimination reaction within any live bacterium. Our results also likely explain previous observations demonstrating that the same indole derivatives inhibit the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prognosis of diseased conditions associated with bacterial dysbiosis.

Keywords: Chlamydia trachomatis; genital and ocular serovars; serine deamination; trp operon de-repression; tryptophan synthase β-elimination.

Fig. 1.

IAA and IPA de-repress the C. trachomatis trpRBA operon with deleterious effects. (A) IAA and IPA reduce C. trachomatis D/UW-3/CX IFUs recovered in a dose-dependent manner. HeLa cells were infected with D/UW-3/CX as described (Materials and Methods), following which indicated amounts of IAA or IPA were added. IFU recovery was evaluated at 42 h.p.i. Structures of IAA and IPA are shown as Insets within graphs. (B) IAA and IPA de-repress the C. trachomatis D/UW-3/CX trpRBA operon. RNA extracted at 24 h.p.i. from cells infected with D/UW-3/CX under control conditions or after IAA (100 μM) or IPA (200 μM) exposure. TrpRBA expression was evaluated by RT-qPCR for trpB. (C) IAA and IPA reduce the size of primary inclusions formed by D/UW-3/CX in HeLa cells. Inclusions were stained in cells fixed at 36 h.p.i. as described (Materials and Methods). Chlamydial inclusions are green, while DNA counterstained with Hoechst 33342 is blue. (Scale bar: 20 μm.) *P < 0.05, using the Wilcoxon rank sum test, and **P < 0.01, using the same test.

Fig. 2.

ILA is not deleterious to C. trachomatis D/UW-3/CX and does not de-repress the trpRBA operon. (A) D/UW-3/CX–infected HeLa cells were exposed to ILA as indicated, and IFU recovery was evaluated at 42 h.p.i. The structure of ILA is shown as an Inset. (B) ILA does not de-repress the D/UW-3/CX trpRBA operon. trpRBA expression was evaluated as described in Fig. 1. (C) Exposure to ILA does not affect the size of primary inclusions formed by D/UW-3/CX. Infected cells exposed to the indicated ILA concentration were fixed and stained at 36 h.p.i. as described in Fig. 1. (Scale bar: 20 μm.)

Fig. 3.

Ammonia is produced by C. trachomatis D/UW-3/CX–infected cells exposed to IPA and IAA, but not ILA. Cells were infected with D/UW-3/CX as described (Materials and Methods). Indicated concentrations of IPA, IAA, and ILA were added immediately postinfection. Ammonia levels in supernatants were evaluated at 24 h.p.i. as described (Materials and Methods). The white bars indicate ammonia levels detected in supernatants from uninfected cells under the specified condition. The gray bars indicate ammonia levels detected in supernatants from infected cells under the specified condition. **P < 0.01, using the Wilcoxon rank sum test.

Fig. 4.

Indole blocks the deleterious effect of IAA and IPA on C. trachomatis D/UW-3/CX. (A) Fifty micromolar indole along with the indicated concentration of IAA or IPA was added to D/UW-3/CX–infected HeLa cells. Inclusions were stained as described in Fig. 1. (B) IFU recovery was evaluated at 42 h.p.i. from cells infected with D/UW-3/CX and exposed to IAA and IPA alone (gray bars), or in combination with 50 μM indole (white bars). (C) Indole blocks the production of ammonia by HeLa cells infected with D/UW-3/CX and treated with IPA or IAA. Infected cells were treated with IAA (100 μM) or IPA (200 μM) alone, or together with 50 μM indole, following which supernatant ammonia levels were measured at 24 h.p.i. (D) Indole does not prevent de-repression of the D/UW-3/CX trpRBA operon by IAA and IPA. Infected HeLa cells were exposed to 50 μM indole alone (white bar), or in combination with 100 μM IAA or 200 μM IPA (gray bars). TrpRBA expression was evaluated as described. **P < 0.01, using the Wilcoxon rank sum test.

Fig. 5.

IAA and IPA do not affect C. trachomatis strains that lack tryptophan synthase activity. (A) HeLa cells were infected with a trpA mutant C. trachomatis serovar A strain (A2497), or a derivative of C. trachomatis D/UW-3/CX with an inactivating point mutation in trpB (D trpB mut). Infected cells were exposed to 100 μM IAA or 200 μM IPA for 42 h, following which IFU recovery was evaluated. (B) IAA and IPA do not affect primary inclusion formation by A2497 or D trpB mut. HeLa cells infected with the indicated strain were exposed to control media or IPA and IAA for 36 h following which chlamydial inclusions were stained. (Scale bar: 20 μm.) (C) IAA and IPA de-repress the trpRBA operon in A2497 and D trpB mut. HeLa cells, infected with A2497 or D trpB mut, were treated with 100 μM IAA or 200 μM IPA for 24 h, following which trpRBA expression was evaluated. (D) IPA and IAA do not induce ammonia production from HeLa cells infected with A2497 or D trpB mut. HeLa cells infected with A2497 or D trpB mut were treated with 100 μM IAA or 200 μM IPA for 24 h, following which ammonia levels in cell supernatants were evaluated. Control conditions are indicated by the white bar, while ammonia levels after IPA and IAA exposure are indicated by the gray bars. **P < 0.01, using the Wilcoxon rank sum test.

Fig. 6.

Physiological concentrations of IPA and IAA are deleterious to genital C. trachomatis under hypoxic (4% O₂) conditions. (A) D/UW-3/CX–infected HeLa cells were treated with 5 and 15 µM of IAA or IPA and incubated in at 4% O₂ as described in Materials and Methods. At 42 h.p.i., cells were harvested to measure IFUs released. (B) Physiological concentrations of IAA and IPA de-repress the D/UW-3/CX trpRBA operon. trpRBA expression was evaluated as described in Materials and Methods. (C) Ammonia is generated by D/UW-3/CX–infected cells incubated with physiological concentrations of IAA and IPA under hypoxic conditions. Ammonia levels were measured from supernatants obtained at 24 h.p.i. as described earlier. (D) Indole alleviates the deleterious effects of IAA and IPA on genital C. trachomatis growth. HeLa cells infected with genital C. trachomatis were exposed to 5 and 15 µM of IAA and IPA concurrently with 50 µM indole and incubated under hypoxic conditions. IFU release was measured at 42 h.p.i. *P < 0.05, using the Wilcoxon rank sum test, and **P < 0.01, using the same test.