RESEARCH ARTICLE Seth-­Smith et al., Microbial Genomics 2021;7:000599 DOI 10.1099/mgen.0.000599 OPEN OPEN DATA ACCESS Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains Helena M. B. Seth-­Smith1,2,3,*, Angèle Bénard4‡, Sylvia M. Bruisten5,6, Bart Versteeg5,7, Björn Herrmann8, Jen Kok9,10, Ian Carter10, Olivia Peuchant11, Cécile Bébéar11, David A. Lewis9,12, Teresa Puerta13, Darja Keše14, Eszter Balla15, Hana Zákoucká16, Filip Rob17, Servaas A. Morré18,19, Bertille de Barbeyrac11, Juan Carlos Galán20, Henry J. C. de Vries5,6, Nicholas R. Thomson4,7, Daniel Goldenberger1† and Adrian Egli1,2† 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 overrep- resented 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. DATA SUMMARY antimicrobial treatment and, if not detected and treated, may be associated with serious clinical complications [2, 3]. All genome data have been submitted to the European Nucle- otide Archive (ENA) under project number PRJEB19884 OmpA-­serotyping was the traditional method for typing (https://www.​ncbi.​nlm.​nih.​gov/​bioproject/​PRJEB19884). C. trachomatis strains, now superseded by ompA-­genotyping. Data for ompA variants have been submitted under project The ompA-­genotypes A–C are associated with ocular infec- PRJEB37762 with the accession numbers given in Table S1 tions (trachoma), D–K with urogenital infections, and L1– (available in the online version of this article). L3 with LGV. OmpA is the major outer membrane protein (also known as MOMP), a porin and highly immunogenic surface-­exposed antigenic determinant [4, 5]. Within OmpA, INTRODUCTION four variable domains (VDs) have been described, which are Chlamydia trachomatis is the most common agent of bacte- proposed to be surface-­exposed and therefore subject to rial sexually transmissible infection (STI) globally, with an immune selection [4, 6]. No crystal structure of chlamydial estimated 127 million new cases of chlamydia infection in OmpA exists as yet. 2016 worldwide [1]. Lymphogranuloma venereum (LGV) is Among MSM infected with LGV C. trachomatis, L2 was the an invasive disease causing ulcerative anogenital infection, dominant circulating OmpA-­serotype until the early 2000s extending often to regional lymph nodes, and is commonly [7], when ompA-­genotype L2b was found to be responsible associated with men who have sex with men (MSM) in many for an outbreak in the Netherlands associated with severe high-­income countries. A diagnosis of LGV chlamydia is proctitis [8], which spread globally [9]. Retrospectively, it of clinical importance, as the infection requires prolonged was found that ompA-­genotype L2b isolates were circulating 000599 © 2021 The Authors This is an open-­access article distributed under the terms of the Creative Commons Attribution NonCommercial License. 1 Seth-­Smith et al., Microbial Genomics 2021;7:000599 in the USA as early as 1979–1985 [8, 10]. The difference in the ompA gene between L2 and L2b is an SNP causing Impact Statement the N162S amino acid substitution within VD2. On the Lymphogranoma venereum (LGV) is a destructive and whole genome level, the reference strains ompA-­genotypes serious sexually transmitted infection (STI) caused by L2/434/Bu and L2b/UCH-1 are differentiated by 573 SNPs across their 1.04 Mb genome [11], and have almost identical a more aggressive and invasive variant of chlamydia. It gene content. The highest variation between the genomes is affects mainly men who have sex with men. Over the last found within tarp, encoding a translocated actin recruiting 20 years, one LGV variant has dominated the diagnosed phosphoprotein, and pmpH, encoding an immunogenic auto-­ cases reported globally. We have investigated the recent transporting polymorphic membrane protein, with possible evolution of this variant, and find that it seems to be adhesin function. Isolates within ompA-­genotype L2b differ continuing to adapt to its hosts and environment. This is from each other by up to 20 SNPs [12], reflecting a relatively particularly apparent in the ompA gene, commonly used recent common ancestor. to categorize or type strains of chlamydia. Many muta- tions in ompA, including novel ones which have not been Laboratory diagnosis of LGV as opposed to urogenital strains seen before, have now been identified. In particular, a relies on in-­house molecular tests [13–16] or can be based on single mutation in ompA has resulted in ompA-­genotype epidemiological and clinical findings [3]. While differences in LGV ompA-­genotypes do not affect treatment recommenda- L2 in this clade, which generally refers to a strain with tions, they are of epidemiological importance in continuing to a different genomic structure. The variant that we have survey the dynamics and evolution of LGV in MSM popula- studied may now be the dominant circulating clone, tions. However, in most laboratories, LGV typing is still not which presents a challenge for typing. The changes in the performed, despite IUSTI guidelines [17]. ompA gene may aid the bacteria to induce reinfections, by avoiding host immunity. Further surveillance of this In order to diagnostically distinguish ompA-­genotype L2b disease is warranted. from other LGVs, a real-­time PCR (RT-­PCR) was previously developed, based on a characteristic 9 bp insertion found in the pmpH gene of ompA-­genotype L2b isolates relative to ompA-­genotype L2 [18], referred to here as pmpH-­genotype ompA-­genotype L2 is making a resurgence [19–24] and that L2b. Recent work has shown that the pmpH-­genotype L2b additional diversity in LGV ompA-­genotypes is present in is no longer specifically associated with ompA-­genotype circulating strains (Table S1) [10, 20, 22–27]. It is important to L2b, since it is also present in strains carrying ompA-­ note that strains and their genomes are currently categorized genotype L2 [19]. Several further publications indicate that by their ompA-­genotype, despite increasing data suggesting that ompA-­genotype does not reflect the rest of the genomic Received 05 November 2020; Accepted 04 May 2021; Published 29 June 2021 Author affiliations: 1Clinical Bacteriology & Mycology, University Hospital Basel, University of Basel, Switzerland; 2Applied Microbiology Research, Department of Biomedicine, University of Basel, Basel, Switzerland; 3SIB Swiss Institute of Bioinformatics, Basel, Switzerland; 4Wellcome Trust Sanger Institute, Cambridge, UK; 5Department of Infectious Diseases, GGD Public Health Service of Amsterdam, Amsterdam, The Netherlands; 6Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity (AII), Location Academic Medical Centre, Amsterdam, The Netherlands; 7 Clinical Research Department, London School of Hygiene and Tropical Medicine, London, UK; 8Section of Clinical Bacteriology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; 9Marie Bashir Institute for Infectious Diseases and Biosecurity & Westmead Clinical School, University of Sydney, Sydney, New South Wales, Australia; 10Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia; 11CHU Bordeaux, Department of Bacteriology, French National Reference Center for bacterial STIs, Bordeaux, France; 12Western Sydney Sexual Health Centre, Western Sydney Local Health District, Parramatta, New South Wales, Australia; 13Unidad de ITS/VIH, Centro Sanitario Sandoval, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain; 14University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Ljubljana, Slovenia; 15Bacterial STI Reference Laboratory, National Public Health Center (former National Center for Epidemiology), Budapest, Hungary; 16National Reference Laboratory for Diagnostics of Syphilis and Chlamydia Infections, National Institute of Public Health, Srobarova 48, 100 42, Prague 10, Czech Republic; 17Department of Dermatovenereology, Second Faculty of Medicine, Charles University and Hospital Bulovka, Budinova 2, 180 81, Prague 8, Czech Republic; 18Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, VU University Medical Center Amsterdam, Amsterdam, The Netherlands; 19 Institute for Public Health Genomics (IPHG), Department of Genetics and Cell Biology, Research Institute GROW, University of Maastricht, Maastricht, The Netherlands; 20Servicio de Microbiología, Hospital Universitario Ramón y Cajal. Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain. CIBER en Epidemiología y Salud Pública (CIBERESP). *Correspondence: Helena M. B. Seth-­Smith, ​helena.​seth-​smith@​usb.​ch Keywords: whole genome sequencing; selective pressure; outer membrane protein; sexually transmitted infections; homosexuality; surveillance; molecular epidemiology; evolution; LGV. Abbreviations: CDS, coding sequence; LGV, lymphogranuloma venereum; MLST, mulit locus sequence typing; MLVA, multi locus variable-­number tandem-­repeat analysis; MOMP, major outer membrane protein; MSM, men who have sex with men; SNP, Single Nucleotide Polymorphism; STI, sexually transmitted infection; VD, variable domain; WGS, whole genome sequencing. ‡Present address: Healthcare Systems Research Group, VHIR, Universitat Autònoma de Barcelona, Passeig de la Vall d’Hebron 119-129, 08035 Barcelona, Spain. †These authors contributed equally to this work Data statement: All supporting data, code and protocols have been provided within the article or through supplementary data files. Six supplementary tables and four supplementary figures are available with the online version of this article. 2 Seth-­Smith et al., Microbial Genomics 2021;7:000599 backbone [28–30]. Throughout this paper, we try to distin- [34], multiplexed (96-­plex) samples were sequenced using guish between ompA-­genotype (defined by ompA sequence), Illumina Hiseq V4 (Illumina) with 75 bp paired-­end reads. pmpH-­genotype (defined by the 9 bp insertion in L2b strains) and genomic backbone (to date generally associated with the Sequence analysis ompA-­genotype, for example in the first published reference Resulting data were mapped using bwa [35] against refer- strains). ence genomes L2/434/Bu (EMBL accession AM884176) Recombination is a driver of C. trachomatis evolution and L2b/UCH-1 (EMBL accession AM884177) using a [9, 12, 31]; clinical isolates with recombinations between minimum read identity threshold of 0.9 (minimum 90 % LGV and urogenital strains have been described [28, 30, 32], mapping identity of the read to a reference) to avoid including a recent L2b-­D/Da hybrid strain identified in mapping of contaminating reads. Bcftools was used for Portugal, which resulted from a recombination across ompA variant calling, with the following options: minimum in an L2b genomic background [30]. Therefore, a plausible number of reads matching SNP=8, minimum number of hypothesis for this observation of these pmpH–ompA reads matching SNP per strand=3, SNP/mapping quality discrepancies is that recombination between the genomes of ratio cutoff=0.8, minimum base call quality to call a ompA-­genotype L2b and ompA-­genotype L2 has occurred. To SNP=50, minimum mapping quality to call a SNP=20. investigate the pmpH–ompA discrepancy phenomenon and After extracting mapping parameters and finding that L2b/ to determine the current genome dynamics of LGV strains at UCH-1 was the better reference for this study, this was the high resolution, we used ompA- and pmpH-­genotyping along reference used in all subsequent analyses. Samples meeting with whole genome sequencing (WGS) of clinical samples coverage criteria (over 15× mean coverage) are detailed in sampled between 2011 and 2017 across Europe and Australia. Table S3: 53 provided insufficient coverage, probably due to sample quality and chlamydial load. All called SNPs were checked manually using BAM files visualized in Artemis METHODS [36]. The SNP phylogeny was generated using RAxML v8.2.8 AVX and GTRGAMMA model with 100 bootstraps. Read Sample collection data were similarly mapped against the plasmid sequence Samples yielding positive amplification for LGV targets, in from L2b/UCH-2 (EMBL accession NC_020956). Indel particular those of pmpH-­genotype L2b, were assembled by investigation was beyond the scope of this phylogenetic our consortium, providing a collection as comprehensive study. Alignments of the ompA gene sequences and amino and international as possible (Table S2). Our dataset was acid sequences were performed within Seaview [37] using selected to explore the particular phenomenon of pmpH-­ Clustal Omega with default settings. The ompA phylogeny genotype L2b linked to other ompA-­phenotypes and, as was generated in Seaview from a muscle alignment of such, is not a random survey of circulating strains. Samples ompA gene sequences, using PhyML and the GTR model were named after the country of origin (AU, Australia; and default settings with 100 bootstraps. Sequences of the CZ, Czechia; CH, Switzerland; ES, Spain; FR, France; HU, pmpH gene were confirmed by extracting the gene sequence Hungary; NL, Netherlands; SL, Slovenia; SW, Sweden), from Unicycler v0.4.8 [38] assemblies using Artemis [36] sample number and ompA-­genotype. Samples positive for and aligning against the reference L2/434/Bu and L2b/ pmpH-­genotype L2b and ompA-­genotype L2 were labelled UCH-1 pmpH gene sequences within Seaview using Clustal ‘L2new’. Detailed diagnostic methods in the various source Omega with default settings. countries are provided in the supplementary data. pmpH-­genotyping was performed as previously described Recombination and time tree analysis [18]. pmpH-­genotype L2b was defined as possessing the 9 Read data from the wider LGV clade were analysed as bp insertion TCTAGTAGT at position 1884. The ompA gene above. Gubbins v2.4.1 [39] was run on 69 genomes from the was amplified using the primer pair P1 : 5′-​ATGA​AAAA​ L2b clade, providing 63 genomes with sufficient data. Linear ACTC​T TGA​AATCGG-3′ and OMP2 : 5′-​ACTG​TAAC​ root-­to-­tip analysis was performed in TempEst v1.5.1 [40] TGCG​TATT​TGTCTG-3′ [33], and capillary-­sequenced using the calculated root based on outgroups. BactDating using primer OMP2. Traces were compiled and manually v1.0.11 [41] was run in R Studio v1.2.5033 [42] with R inspected in CLC Genomics Workbench (version 9.5.3, v3.6.2 [43] on the resulting dataset. All models were run, Qiagen). each with 1 million iterations, and compared: arc (additive relaxed clock) was found to be the best model [44]. Whole genome sequencing SureSelect target capture was used as previously described Antigenicity prediction [12] on a selection of 95 isolates including selected samples The Immune Epitope Database (​IEDB.​org), B cell epitope from a previous study on LGV diversity [19]. The Sure- prediction using either Kolaskar and Tongaonkar Anti- Select baits were custom designed around known variable genicity and Bepipred Linear Epitope Prediction) was regions from all sequenced ompA-­genotypes [9]. Sequence used to model antigenicity of omopA epitopes. Results are capture (48-­plex) was performed according to the manu- given as an antigenicity score, with higher values indicating facturer’s protocols (Agilent). After library preparation higher antigenicity (http://​tools.​iedb.​org/​bcell/​help/). 3 Seth-­Smith et al., Microbial Genomics 2021;7:000599 RESULTS exception of Hungary. The phenomenon of ompA-­genotype Typing shows that pmpH-genotype L2b is L2 and pmpH-­genotype L2b (‘L2new’) is represented by 26 associated with several ompA genotypes genomes, and ompA-­genotype L2b by three. Of the 12 L2/ L2b ompA-­variants identified above, eight are represented A total of 389 LGV-­positive samples from eight countries by at least one sample with a complete genome sequence. from 2011 to 2017 (Spain n=97, Netherlands n=67, Switzer- The ompA-­genotypes without genome data are: L2bv6, land n=64, Australia n=53, Sweden n=37, Hungary n=31, L2bv8, L2bv10 and L2bv11. All sequenced genomes show Czechia n=30, Slovenia n=10) were obtained. Of these, 321 much higher identity to the genome of L2b/UCH1 (differing (83 %) gave positive and interpretable results for pmpH-­ by one to eight SNPs) than that of L2/434/Bu (differing by genotyping and ompA-­sequencing (Table S2: Spain n=86, ≥245 SNPs). The presence of the 9 bp insertion in the pmpH Netherlands n=63, Switzerland n=50, Australia n=38, Sweden gene was confirmed in 37 of the 42 samples: the assemblies n=34, Hungary n=28, Czechia n=20, Slovenia n=2). All the of five samples (ES89_L2bv7, ES96_L2v3, FR1378_L2new, samples were obtained from men and, where sexual orienta- NLB19_L2new and AU39_L2Bv1) were too fragmented over tion was provided (Netherlands, Hungary and Slovenia), all the pmpH locus to analyse, owing to low sequence coverage were from MSM. The mean age of the patients, where data in these samples. were available (n=313), was 37.9 years (range 19–72 years). Each genome shows one to eight SNP differences compared All the LGV samples included in this study were found to be to the genome of L2b/UCH1 from 2006, and all derive from pmpH-­genotype L2b (n=321). However, ompA-­sequencing this L2b genomic backbone (Fig. 3). The phylogeny describes revealed ompA-­genotype L2b (n=83; 26 %) and ompA-­ the ‘L2new’ samples in this study, with ompA-­genotype L2 genotype L2 (n=180; 56 %) as well as sequences which vary and pmpH-­genotype L2b, having arisen through an initial from these by up to 2 bp: L2b (n=45; nine variant types) and SNP reversion at genome position 59 342 (G485A in ompA; L2 (n=7; three variant types) (Table S1). We also identified OmpA S162N), and then undergoing further mutation. Thus, ompA-­genotype L1 variants (n=4), a D variant (n=1) and the full genome phylogeny (Fig. 3) does not agree with the a single mixed L2/L2b infection (n=1). As our focus is on ompA gene phylogeny (Fig. 1), as the ‘L2new’ genomes are a ompA-­ genotypes L2/L2b, these latter samples were not further development from the L2b genomic backbone, and do analysed further, except to note that the Swiss D variant ompA not represent the genome of the reference L2/434/Bu. sequence matches that of the L2b-­D/Da hybrid (MN094864) [30] across base pairs 193–1021. The diversity of the ompA-­ As both recombination and mutation are potential sources variants sampled is shown in Fig. 1. The phylogeny cannot be of genome variation, we aimed to investigate which mech- fully resolved due to homoplasy (the same mutation in two anism was responsible for these SNPs. The distribution separate branches of the phylogeny), and one base reversion of SNPs across the genome is shown in Fig. S2. With the at position G485A causing the S162N amino acid change. exception of the ompA gene with nine SNPs, there are five Notably, all the nucleotide substitutions (n=13; as well as three loci with two SNPs within 1 kb, and the rest are spread further SNPs from previous studies) lead to predicted amino along the genome. Recombination analysis on a dataset acid changes in OmpA (Table S1). including these and other available genomes from the L2b clade [12] resulted in a collection of 63 LGV genomes, between which no recombinations were identified (data Large ompA diversity in our sample collection not shown). To investigate whether the SNPs identified Typing data from our whole collection show a decrease in above have been seen before in LGV genomes, we analysed ompA-­genotype L2b samples while rates of ompA-­genotype in detail these 59 genomes, which altogether possess 1894 L2 increased from 2010 to 2015 (Fig. 2). The trends vary SNPs relative to L2b/UCH1. Of the 66 SNPs identified in per country, with data from parts of Europe showing strains this study, five have been seen (separately) in previously carrying ompA-­genotype L2 increasing from 2011 (France, sequenced LGV strains: four within ompA and one causing Switzerland), being dominant in several countries since 2011 a Met-­Val change at codon 12 within CTLon_0845/rbsU, (Spain, Sweden, Hungary), or maintaining a stable propor- encoding a sigma regulatory family protein-­PP2C phos- tion (Netherlands) (Fig. S1). Of the further ompA-­genotypes phatase, which was seen previously in two UK strains (assigning new genotypes with every SNP difference), many (Table S4). In all cases, no SNPs which fall adjacent to (n=10) are specific to single countries (Fig. 1). these were identified in the genomes in this study, as would be expected to co-­t ransfer during recombination: this is also the case at the ompA locus, with the closest WGS analysis of pmpH-genotype L2b and ompA SNP found over 13 kb away, suggesting that the ompA variants SNPs in these genomes did not occur as part of a larger A selection of 95 samples, chosen from those with lower recombination. The rate of SNP accumulation, with one Ct values (Table S2) to represent the widest geographical, to eight SNPs over 10 years (2006–2016), broadly concurs temporal and ompA-­ genotype diversity, underwent with the mutation rate calculated for the LGV lineage of sequence capture and WGS. Genome data for further 2.15×10−7 SNPs per site per year (0.23 SNPs per genome analysis were obtained from 42 samples (44 %), repre- per year) [12]. As such, our data suggest that these SNPs senting at least one genome from each country, with the arose by mutation. 4 Seth-­Smith et al., Microbial Genomics 2021;7:000599 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. A comparison of these data with previously published genome The isolates at the root of the L2b clade are from the USA sequences from the L2b clade [12] shows the context of in the 1980s, and the earliest isolate from the branch this ongoing evolution (Fig. 4). This phylogenetic tree also representing the L2b outbreak is from the USA in 2001 highlights the wide presence of ompA-­genotype L2 across (SF156531), which is genomically identical to the refer- the phylogeny, showing that this feature is not monophyletic ence ompA-­type L2b isolate L2b/UCH1, among others. (Fig. 4). We attempted to date this clade, including the USA 1980s isolates onwards. A first root-­to-­tip regression analysis 5 Seth-­Smith et al., Microbial Genomics 2021;7:000599 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. shows that the genomes have a temporal signal, but the Nine of the 66 SNPs (14 %) are located within a single gene, mutation rate of 0.44 SNPs per genome per year does not ompA, all of which are non-­synonymous (Table S4). This agree with previously published data (Fig. S4). Bayesian gene comprises 1185 bp of the 1 038 863 bp genome (0.1 %), analysis of the data using BactDating gives a mutation rate showing that SNPs are clearly enriched in this gene. Of the of 0.213 SNPs per genome per year (95 % CI: 0.147–0.283, nine SNPs in the ompA gene, six are novel compared to rate equivalent to 2.1×10−7 SNPs per site per year) and a putative date for the ancestor of ompA-­genotype L2b clade sequenced LGV genomes [12] (Table S4). The three that are of 1935 CE (common era) (95 % CI: 1903–1959) (Fig. S4). seen elsewhere in the phylogeny also often exist in combi- nation with other ompA SNPs, and in some cases these combinations have also been seen in previously sequenced The genomes, particularly ompA, are under genomes. Two of the ompA SNPs (58 830 and 59 312, causing selective pressure amino acid changes S333G and K172T) are homoplasic, Across the phylogeny of the 42 genomes from this study, a found in two locations in our phylogeny. Together, these total of 66 SNPs were identified (Fig. 3): six (9 %) are inter- genic, 15 [23 %; 25 % of SNPs found within coding sequences findings suggest either independent mutations in separate (CDSs)] are synonymous, and 45 (68 %; 75 % of SNPs found lineages, or possible recombination, although no adjacent within CDSs) are non-­synonymous, one of which causes a SNPs were identified, which would be a signature of recom- premature stop codon in incA (CTLon_0370) (Table S4). bination (see above). 6 Seth-­Smith et al., Microbial Genomics 2021;7:000599 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]. The 48 CDSs affected by mutations identified across the [48]), ST1 (Dean scheme [49]) and ST58 (Uppsala scheme phylogeny include 33 with at least one non-­synonymous muta- [46, 47]). No SNPs were identified within the plasmid. tion. A comparison of these CDSs with the 34 identified as having a possible role in pathoadaptation [45] find four in common: Antigenic impact of OmpA substitutions CTLon_0243/CTL0247 (ChlaDub1), CTLon_0247/CTL0251 (pmpH), CTLon0370/CTL0374 (incA) and CTLon0845/ All the 13 SNPs identified in the samples in our study (WGS CTL0851 (rbsU) (Table S4). Note that tarp (translocated actin and genotyping) within ompA cause amino acid changes, each recruiting phosphoprotein) gene repeats were not considered of which is located on the external face in the variable domains in this analysis as the mapping and assembly of this region is (VD1, VD2 and VD4), as previously modelled [6] (Fig. 5). The known to be problematic. S162N substitution changes the ompA-­genotype from L2b to None of these SNPs are located in any of the multilocus sequence L2, and vice versa. The majority of substitutions are from polar typing (MLST)/multilocus variable-­number tandem-­repeat amino acids to polar or hydrophobic residues, in agreement analysis (MLVA) genes from any of the schemes [46–49], and with previous findings [50, 51]. With the K172T substitution, a as such all these 42 samples are from ST44 (Pannekoek scheme charged amino acid is replaced by a polar one. 7 Seth-­Smith et al., Microbial Genomics 2021;7:000599 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. The substitutions in OmpA are all located in regions found to be DISCUSSION antigenic in C. trachomatis ompA-­genotype L2 in rabbits [51]. Investigations into circulating C. trachomatis genotypes are These regions contain B cell epitopes as defined in the trachoma very useful in an epidemiological context. In this study, we biovar of C. trachomatis [50, 52]. Modelling of OmpA epitope antigenicity indicates that the L2b version of OmpA with the show that the evolution of the LGV outbreak L2b-­lineage serine at position 162 has marginally higher antigenicity (scores between 2006 and 2016 has resulted particularly in diversity of 0.939 and 0.589 respectively with B cell epitope prediction in the immunogenic OmpA protein. Our main finding is the using either Kolaskar and Tongaonkar Antigenicity or Bepipred reversion of this L2b genomic backbone to ompA-­genotype Linear Epitope Predictions) than L2 with the glutamine at posi- L2, an event which appears to have occurred recently, between tion 162 (0.906 and 0.529) (Tables S5 and S6). 2006 and 2011, at least once. This confounds traditional 8 Seth-­Smith et al., Microbial Genomics 2021;7:000599 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. typing, as the L2b genomic backbone is now linked to the L2 [30]: from ompA evidence, this variant may also be present ompA-­genotype. The original hypothesis of recombination in Switzerland. This high ompA variability is reflected in between these lineages appears to be unlikely, based on the our study, where seven novel ompA-­genotypes have been phylogeny, low SNP numbers, lack of identified recombina- identified. It is a key observation that all the 13 mutations in tions within this clade and absence of co-­transferred adjacent ompA identified in this study cause amino acid changes in the SNPs. A more likely scenario is that the ompA gene is under variable domains of OmpA, and thus may affect the epitopes strong selective pressure on specific immunogenic residues, targeted by the hosts’ immune system. Many of the ompA-­ such that chance mutations in these residues are selected genotypes are specific to single countries, whereas others are for in the circulating strains. It has previously been shown found throughout our international collection. that ompA is under positive selection [53, 54]; this has been hypothesized to be a way to promote immune evasion and The ompA-­ genotype L2b strain was responsible for an enable repeat infections [9]. Among LGV strains, there is high outbreak among MSM starting in the early 2000s, thought variability of ompA [45], partially as a result of high levels to have arisen in the USA [2] and rapidly spread globally. We of recombination. A probable recombination event between have dated the ancestor of the L2b clade to 1935, in agreement ompA-­genotype D/Da and the L2b genomic backbone has with Hadfield et al. [12], although it is important to bear in been recently reported as a seemingly successful combination mind that genomic data from outbreaks may confound the 9 Seth-­Smith et al., Microbial Genomics 2021;7:000599 time tree algorithm [55]. The clonality of the data can also ompA-­genotype distinction is, with isolates sequenced mean that phylogenies are difficult to accurately calculate. We here with the L2b genomic backbone showing reversion found this L2b genomic backbone present across the many of the ompA mutation to L2 (G485A). Further confusion countries we sampled, despite changes in the ompA-­genotype, arose with the definition of ‘hypervirulent L2c’ [28], which argues that this is a successful and expanding lineage, which has an ompA-­genotype L2 and a chimeric genome diversifying over time. It is possible that this success is due between L2 and D genomic backbones. The ‘L2c’ nomen- to increased virulence encoded by the ompA-­genotype L2b clature in this case refers to the genomic context, which is genome compared to other LGV lineages, although there are unprecedented and has led to problems in the literature, few functional differences predicted between the genomes with strains carrying ompA-­genotype L2 believed to be of ompA-­genotype L2/434/Bu and ompA-­genotype L2b/ the ‘hypervirulent L2c’ [29, 61, 62], whereas they may UCH-1 [11]. Another possibility is that this represents a have represented cases of the ‘L2new’ described here. clonal expansion of an epidemiologically present strain From a clinical microbiology and phylogenetic point of among high-­risk groups with international sexual networks, view, the ompA-­genotyping concept is misleading, as it fuelled by changes in sexual practice over the past decades. refers to a useful typing target which does not necessarily The observed combination of the L2b genomic backbone with equate to ancestral lineage. In the cases described here, the L2 ompA appears to be expanding successfully, perhaps the ompA-­genotype L2b backbone is better described selected to improve infection chances in certain populations by the pmpH-­genotype rather than the ompA-­genotype, with some pre-­existing immunity to ompA-­genotype L2b. which confounds attempts at following epidemiology The L2 variant of OmpA is predicted to be slightly less with single or even dual targets. We argue that there is antigenic than that of L2b, which may be a selective advan- an urgent requirement for a new nomenclature. As most tage to a naïve host, or perhaps this variant succeeds as it nomenclatures are devised around existing typing tools, a favours reinfection. This may explain the wide presence of genomovar nomenclature based on the whole genome may this ompA-­genotype across the LGV phylogeny. Exploration be too complex. The Uppsala database http://​mlstdb.​bmc.​ of this, and whether the immunological impact of human uu.​s e/ [46, 63] already has an internal ompA nomenclature immunodeficiency virus (HIV) co-­infection could affect as well as the five-­t arget MLST scheme, and it would be immune recognition and selective pressure would be a interesting to see how combinations of these would allow fascinating area for further study. Future studies should discrimination across the whole LGV dataset. investigate the fitness, virulence, clinical outcomes and C. trachomatis has provided us with yet another diagnostic re-­infection rates in patients infected with LGV. conundrum [64–69], elucidated by genomics to show how the Our study found that 75 % of the SNPs in protein coding ompA-­genotype L2b outbreak continues to spread globally, regions across the whole genome phylogeny are non-­ diversifying its ompA gene. The impact and implications of synonymous. While this is similar to the expected maximum this need to be investigated. of 23 % synonymous mutations based on C. trachomatis codon usage [45], it suggests that the mutated sites in these isolates are under positive selection, supporting previous Funding information J.C.G. was supported by the Instituto de Salud Carlos III (Plan Estatal de observations in the L2b clade of 90.2 % of the SNPs in I+D+ i 2013–2016), Grant PI16-01242. CDSs being non-­synonymous [45]. Four of the 33 CDSs identified as having non-­synonymous mutations in our Acknowledgements Many thanks to Dr Victoria Feher and Professor Rommie Amaro for the study were also identified as having an overrepresentation original figure of the modelled OmpA, annotated in Fig. 5. Thanks to of non-­synonymous mutations in a previous study on LGV Dr Alfredo Mari, University of Basel, for help with Fig. S1. Thanks to [45], suggesting that there is indeed a pressure on specific Prof. Xavier Didelot for assistance with BactDating. We thank Elisabeth targets in this lineage. Three of these genes are probably Schultheiss for excellent technical assistance in ompA sequencing. Many thanks also to Dr Simon Harris for help with pipeline clarifica- involved in interaction with or evasion of the immune tions, and Dr Madlen Stange and Dr Fanny Wegner for help with selec- system: ChlaDub1 [56], pmpH [57] and incA [58]. Whether tion analysis. this potential patho-­adaptation is responsible for increased Author contributions virulence or increased numbers of asymptomatic cases [59] Conceptualization and methodology: H.M.B.S-­ S., D.G., A.E. Formal is not clear, and would be useful to test in future studies. analysis: H.M.B.S-­S., A.B. Resources: S.M.B., B.V., B.H., J.K., I.C., O.P., C.B., T.P., D.K., E.B., H.Z., F.R., B.d.B., J.C.G., N.R.T. Supervision: D.G., M.B., The nomenclature used for C. trachomatis lineages S.A.M., D.A.L., J.C.G., H.J.C.d.V., N.R.T. Writing – original draft preparation: is ever more confusing. The strain and genome in C. H.M.B.S-­S. Writing – review and editing: all authors. trachomatis are currently referred to by the genotype of Conflicts of interest a single gene (ompA). It is increasingly clear that ompA-­ The authors declare that there are no conflicts of interest genotype does not reflect the genomic heritage of strains [16, 30, 60]. 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