Chlamydia trachomatis virulence factor CT135 is stable in vivo but highly polymorphic in vitro

Abstract

Chlamydia trachomatis is an important human pathogen causing both ocular and sexually transmitted disease. Recently, we identified CT135 as an important virulence determinant in a mouse infection model. Results from CEL 1 digestion assays and sequencing analyses indicated that CT135 was much more polymorphic in high in vitro passage reference serovars than it was in clinical strains that had undergone limited passaging. Herein, we used targeted next-generation sequencing of the CT134-135 locus, from reference strains and clinical isolates, enabling accurate discovery of single nucleotide polymorphisms and other population genetic variations. Our results indicate that CT134 is stable in all C. trachomatis serovars examined. In contrast, CT135 is highly polymorphic in high-passaged reference ocular and non-LGV genital serovars, with the majority of the mutations resulting in gene disruption. In low-passaged ocular clinical isolates, CT135 was frequently disrupted, whereas in genital clinical isolates CT135 was intact in almost all instances. When a serovar K isolate, with an intact CT134 and CT135, was subjected to serial passage in vitro CT134 remained invariable, while numerous gene interrupting mutations rapidly accumulated in CT135. Collectively, our data indicate that, for genital serovars, CT135 is under strong positive selection in vivo, and negative selection in vitro.

Keywords: CT135; chlamydia trachomatis; deep sequencing; genomics; single nucleotide polymorphism; virulence factor.

Graphical Abstract Figure.

CT135 is a critical Chlamydia trachomatis virulence factor that undergoes rapid inactivation upon in vitro passage.

Figure 1.

SNPs in CT134 and CT135 from C. trachomatis reference serovars as determined by deep sequencing. Primers designed to span CT133 and CT136 were used with all C. trachomatis genomic DNA templates, in PCR reactions. The resulting amplicons were subjected to deep sequencing as random NextTera XT shotgun libraries applying the Illumina MiSeq platform. Reads were referenced mapped to C. trachomatis serovar D UW-3 chromosome sequence (Stephens et al. , NCBI NC_000117.1). SNP position are shown using the C. trachomatis D UW-3 genomic sequence numbering as the reference. CT134: 151671–152084 and CT135: 152143–153225. Not shown is a single intragenic region LGV serovar-specific SNP, a TA>T deletion, which occurs at position 152119. In all cases, high-quality SNPs indicated were supported by a Phred quality score of ≥3.

Figure 2.

SNPs in CT135 in serially passaged serovar K UW-31 as determined by deep sequencing. Serovar K, initial start passage number 40, was serially passed every 48 h in McCoy cells with DMEM-10 high glucose 10% FBS + 1 ug ml⁻¹ cycloheximide for nine additional passes. Genomic DNA was prepared from EBs from each passage and used for PCR and the resulting amplicons were subjected to deep sequencing and SNP analysis as described in the legend to Fig. 1. In all cases, high-quality SNPs indicated were supported by a Phred quality score of ≥3.