Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains

Abstract

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

Keywords: LGV; evolution; homosexuality; molecular epidemiology; outer membrane protein; selective pressure; sexually transmitted infections; surveillance; whole genome sequencing.

Fig. 1.

Diversity of ompA by sequence clustering. ompA sequences of the cohort tested were compared with sequences from NCBI (accession numbers given). A PhyML tree was created from a muscle alignment of ompA gene sequences in Seaview using the GTR model and default settings, and using ompA-genotype L2 as the root. Full-length ompA sequences were extracted from whole genome sequences and used to complement the PCR data where possible (see further below). The numbers of samples in our study carrying these variants are shown on the right. Distributions are given by country, that with the most samples of this ompA-genotype given first: AU, Australia; CZ, Czechia; CH, Switzerland; ES, Spain; HU, Hungary; NL, Netherlands; SW, Sweden. SNP numbers on branches refer to bases in the reference gene from AM884176, L2/434/Bu; all are non-synonymous. Homoplasic SNPs (those found twice in the tree) are coloured green and blue (bold) respectively. The SNP distinguishing ompA-genotype L2 from L2b is shown in red (bold), as is the ‘revertant’ SNP in sample E91_L2h (according to this phylogeny). L2f_EU676181_128C/07 is identical to the L2/434/Bu reference. Not all sequences are full length: in particular, L2c_EF460796, L2d_EF460797 and L2bv4_KU518892_CV544 do not cover all the assigned SNPs. L2e_EF460798 was excluded from the analysis as it comprises only 150 bp and covers a part of the ompA gene which does not align with many other sequences: it carries a single SNP, C954T. Bootstrap values are low (0–61 %), reflecting the low sequence diversity and presence of homoplasies.

Fig. 2.

Distribution of ompA-genotypes temporally from our combined cohort. The SNPs within these ompA-genotypes are shown in Table S1. The data are broken down by country in Fig. S1.

Fig. 3.

Phylogeny of whole genome sequences of 42 sequenced LGV isolates. The reference strain L2b/UCH1_AM884177 was used as the root of the tree, based on additional analysis using outgroups SF41806 and SF46445. Columns to the right of the phylogeny indicate ompA-genotype, country and year of sampling for each sample. The evolution of ompA-genotype L2 from the ompA-genotype L2b backbone is clearly shown, with the star representing the SNP causing this ompA-genotype change. All samples were pmpH-genotype L2b. The same phylogeny with SNP locations on branches is given in Fig. S3. The figure was generated using Phandango [70].

Fig. 4.

Phylogeny of whole genome sequences available for LGV-clade samples. The phylogeny is based on reference strain L2b/UCH1_AM884177 (italicized), including 59 additional LGV genomes for context [12]. Sequences from this study are in bold. Columns to the right of the phylogeny indicate ompA-genotype, country and year of sampling for each sample [12]. The tree is rooted according to the full species phylogeny [12]. This phylogeny agrees largely with that in Fig. 3, with the exception of the location of NLB25, differently located due to the homoplasy at position 59 312 (Fig. S3). Bootstraps of 100 replicates are shown on key branches; the bootstrap to the branch with the ompA L2 SNP is 46. Bar, number of substitutions per site.

Fig. 5.

Location of altered amino acids within OmpA. This is from the model developed in [6], based on the C. trachomatis serovar C ompA sequence accession number DQ116399 and associated amino acid numbering. Amino acid substitutions differing from those in ompA-genotype L2b are given next to the position of the amino acid according to L2b/UCH-1 numbering: all are located in variable domains VD1, VD2 and VD4.