Clinical Persistence of Chlamydia trachomatis Sexually Transmitted Strains Involves Novel Mutations in the Functional αββα Tetramer of the Tryptophan Synthase Operon

Abstract

Clinical persistence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and β subunits that encode TrpA and TrpB, respectively, and form a functional αββα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αββα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism's tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis.

Keywords: Chlamydia trachomatis; indole; interferon gamma; sexually transmitted infections; trpA; tryptophan synthesis.

FIG 1

C. trachomatis clinical strains F I to IV (F I-IV) contain a trpA frameshift causing TrpA elongation. (A to C) Partial nucleotide sequences showing trpR (A), trpB (B), and trpA (C) polymorphisms of the four serial clinical strains F I-IV compared to 20 C. trachomatis (Ct) reference strains (A/HAR13, Ba/Apache2, C/TW3, D/UW3, Da/TW448, E/Bour, F/ICCal3, G/UW57, H/UW4, I/UW12, Ia/IU4168, J/UW36, Ja/UW92, K/UW31, L1/440, L₂/434, L₂a/TW396, L₂b/UCH-1/proctitis, L₂c, and L₃/404) including novel clinical F strains from the San Francisco Bay Area (n = 7) and all F strains previously sequenced and available from public databases (n = 59). A period in the sequence denotes homologous sequences that are not shown. Dashes denote nucleotide deletions at positions A408, T409, T410, and T528 for trpA of ocular strains and at position 758 for trpA of clinical strains F I-IV. Bold nucleotide letters denote substitution mutations, while bold amino acid letters denote nonsynonymous amino acid substitutions. Strains with homologous sequences are not shown: F/SotonF1-F4, F/Soton18-137, F/R4663-28312, F/STN15-22, F/UK35155-770010, F/SW4-5, F/SWFP, F/S1470-3948, F/NI1, F/NL30-36, F/Aus20, F/C55, F/It686-688, F/Sou9-100, F/Swab5, F/SwabB5, F/Fin106-219, and F/SF7-19. These strains had genes that were similar to the trpR gene of A/HAR13-L3/404, the trpB gene of D/UW-3-K/UW-31, and the trpA gene of F/IC-Cal3-I/UW-12.

FIG 2

Phylogeny of trpRBA for 20 Ct reference strains, clinical F I-IV strains and other publicly available C. trachomatis F strains clustered F I-IV as a subbranch of reference strain F/IC-Cal3. (A to C) trpR (A), trpB (B), and trpA (C) maximum likelihood trees were constructed using Tamura-3-parameters method with 10,000 bootstrap replicates of the F I-IV strains, seven novel clinical F strains collected from San Francisco Bay Area clinics, 59 Ct clinical F strains available from public databases, and the 20 reference strains of Ct. B/UW-3 contained no trpRBA operon and therefore was not included. Scale bar at the top and at each branch length correspond to sequence divergence scale and time. The numbers at branches indicate bootstrap score. Red dots, F I-IV strains; blue dots, San Francisco F strains; magenta dots, reference Ct strains; green dots, ocular Ct strains; maroon dots, LGV strains.

FIG 3

TrpA 3D predicted structures of clinical and reference C. trachomatis strains. (A to F) TrpA structures of Ct clinical strains F I-IV (cyan) (A), elongation of clinical strain F α254N and α255L aa surface area (yellow) and α-loop L6 (α-L6) of template 5tch (blue) and clinical strain F (cyan) (B), Ct urogenital reference strain F/IC-Cal3 (yellow) (C), α-L6 of template 5tch (blue) and F/IC-Cal3 (yellow) (D), Ct ocular reference strain A/HAR13 (green) (E), and α-L6 of template 5tch (blue) and A/HAR13 (green, truncated) (F). All structures were constructed using the template from the published crystal structures of the reconstructed putative last bacterial common ancestor (LBCA) tryptophan synthase 5ey5 (magenta) using MODELLER with visualization construction using Chimera (see Materials and Methods). Residues involved in both catalytic and subunit interaction are shown in orange. Dashed lines represent the tunnel and substrate binding site at the α subunit.

FIG 4

Comparative structure analyses of TrpB and TrpA for clinical strains F I-IV reveals mutations within the functional αββα tetrad complex. (A to C) TrpA-TrpB dimer structures of Ct clinical strain F (cyan) (A), Ct urogenital reference strain F/IC-Cal3 (yellow) (B), and Ct ocular reference strain A/HAR13 (green) (C). The TrpB structure is shown with the β-COMM domain in black. All TrpA and TrpB structures were constructed using the template from the published crystal structures of the reconstructed putative last bacterial common ancestor (LBCA) tryptophan synthase 5ey5 (magenta) using MODELLER with visualization construction using Chimera (see Materials and Methods). α-L6 residues are shown in blue, and residues involved in both catalytic and subunit interaction are shown in orange.

FIG 5

TrpA amino acid alignment and phylogeny of C. trachomatis clinical strains F I-IV, A/HAR13, F/IC-Cal3, and Protein Data Bank (PDB) templates. (A) For 3D model reconstructions, amino acid alignments of Ct clinical strains F/I-IV, F/IC-Cal3, and A/HAR13 with the PDB templates of LBCA 5ey5, M. tuberculosis 5tch, and S. Typhimirium 1qopA was performed using Clustal Omega (see Materials and Methods). Residues involved in both catalytic and subunit interaction (magenta), α-L6 residues α176-196 according to the 5tch sequence (cyan), clinical strain F mutation G253D and .254N and .255L aa elongation (yellow), gray, basic amino acids arginine and lysine in template sequences (gray), and acidic amino acid aspartic acid in clinical strains (green) are indicated. (B) Phylogenetic tree of Ct strains and PDB aa templates using neighbor joining with Jones-Taylor-Thornton (JTT) substitution method with 10,000 bootstrap replications using MEGA 7 (see Materials and Methods).

FIG 6

Recoverable infectivity of C. trachomatis clinical strains F I-IV was significantly lower than for F/IC-Cal3 following tryptophan starvation and rescue. Infected cells were incubated for 48 h in the presence of either complete DMEM medium (–IFN-γ), tryptophan-free DMEM medium treated with 5 ng/ml human IFN-γ (+IFN-γ), tryptophan-free DMEM medium treated with 5 ng/ml human IFN-γ supplemented with 50 μM Indole (+IFN-γ –Trp +Indole) or 10 mg/liter tryptophan (+IFN-γ –Trp +Trp). (A) Comparison between reference strain F/IC-Cal3 and clinical strains F I-IV. Mean recoverable IFUs/ml plus standard deviations (SD) (error bars) from three independent experiments. †, P = 0.0597; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (B) Inclusion morphology of F/IC-Cal3 under IFN-γ and indole treatment (+IFN-γ –Trp +Indole). Inclusions were stained with a FITC-conjugated Ct-specific LPS MAb (see Materials and Methods). White arrowheads indicate Ct inclusions. (C) Clinical strain F II under IFN-γ and indole treatment (+IFN-γ –Trp +Indole); inclusions were stained with a Ct-specific LPS-MAb at 48 hpi. The morphology of F I, III, and IV were similar to that of F II (data not shown). Arrowheads indicate Ct inclusions. (D) TEM of clinical strain F II and (E) reference strain F/IC-Cal3 under IFN-γ and indole treatment (+IFN-γ –Trp +Indole) at 48 hpi. White arrow, Elementary Body; Black arrow, Intermediate Body; white arrowhead, Reticulate Body; black arrowhead, Aberrant Body.

FIG 7

trpBA mRNA expression levels for clinical strains F I-IV were significantly reduced compared to F/IC-Cal3 under tryptophan depletion and rescue. At 48 h before infection, HeLa cells were treated with 5 ng/ml of human IFN-γ for 24 h in tryptophan-free media. Infected cells were incubated in the presence of either tryptophan-free DMEM (–Trp), tryptophan-free DMEM supplemented with 100 μM indole (–Trp +Indole), or tryptophan-free DMEM supplemented with 10 mg/liter tryptophan (–Trp +Trp). Infected cells were harvested at indicated times. RNA was reversed transcribed to cDNA, and expression levels were normalized to the Ct 16S rRNA and presented as means plus SD from three independent experiments. Data are shown for F I, although F II-IV had similar mRNA transcription patterns. There were no statistically significant differences at 48 hpi. *, P < 0.05.

FIG 8

In vitro intracellular tryptophan concentrations vary significantly for reference strain F/IC-Cal3 compared with clinical strains F I-IV. (A and B) Ct-infected HeLa cells were incubated with either tryptophan-free DMEM medium (−Trp) (A) or complete medium treated with IFN-γ (+IFN-γ) (B) (see Materials and Methods). The medium was supplemented with indole (−Trp +Indole or +IFN-γ +Indole) or tryptophan (−Trp +Trp or +IFN-γ +Trp) as indicated. Infected cells were harvested at 48 hpi, and intracellular tryptophan concentrations were measured using high-pressure liquid chromatography-linked tandem mass spectrometry (see Materials and Methods). Data are presented as the mean tryptophan concentration (in picomoles) normalized to Ct 16S rRNA plus SD based on two independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.