Genomic insights into local scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages
Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovar A-C, is the leading infectious cause of blindness worldwide. Its impact is substantial, with approximately 1.9 million individuals suffering from visual impairment. Africa bears the highest burden, accounting for over 86% of global trachoma cases. We investigated Ct serovar A (SvA) and serovar B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swab samples across nine Gambian villages during a six-month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted Whole-Genome Sequencing (WGS) on a limited number (n=43) of Ct positive samples. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had a highest identity to Ct strain A/HAR13 (NC_007429) and 30 had a highest identity to Ct strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains; Ct A/HAR13 and Ct B/Jali20 indicated a single nucleotide polymorphism accumulation rate of 2.4 {xi} 10^-5/site/year for the Gambian SvA and 1.3 {xi} 10^-5/site/year for SvB variants (p<0.0001). Variant calling resulted in a total of 1,371 single nucleotide variants (SNVs) with a frequency > 25% in SvA sequences, and 438 SNVs in SvB sequences, from which 740 (62.8%), and 241 (66.4%) were non-synonymous, respectively. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/ repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP), and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies. Impact StatementChlamydia trachomatis (Ct) is a globally significant pathogen. It is the leading infectious cause of blindness--a disease called trachoma. In addition, Ct causes the majority of bacterial sexually transmitted infections. Current control measures for trachoma are based on the "SAFE" strategy; Surgery for trichiasis (S), Antibiotics (A), Facial cleanliness (F) and Environmental improvement (E). Whilst this strategy has achieved remarkable success, the target date for the global elimination of blinding trachoma as a public health problem has been pushed back from 2020 to 2030. Prior studies demonstrated evidence indicating variations in infection loads and severity among different ocular Ct serovars. However, there remains a significant knowledge gap regarding the specific genes and mechanisms responsible for these variations. We generated genetic data from two main serovars of Ct that infect human eyes: Serovar A (SvA) and Serovar B (SvB) variants collected from four villages in two different administrative regions on opposing sides of the river Gambia to elucidate (i) the factors driving the diversification of ocular Ct strains; (ii) disparities in mutation frequency/accumulation profiles; (iii) selective pressures between serovar A and B; and (iv) the dynamics of mutation accumulation within the Gambian ocular Ct positive population over a short timeframe. Our findings suggest a different variation accumulation pattern in SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.