The Nature and Extent of Plasmid Variation in Chlamydia trachomatis.

Charlotte A Jones,Nicholas R Thomson,Colette E O’Neill,James Hadfield,Peter Marsh,Ian N Clarke,David W Cleary

doi:10.3390/microorganisms8030373

Abstract

Chlamydia trachomatis is an obligate intracellular pathogen of humans, causing both the sexually transmitted infection, chlamydia, and the most common cause of infectious blindness, trachoma. The majority of sequenced C. trachomatis clinical isolates carry a 7.5-Kb plasmid, and it is becoming increasingly evident that this is a key determinant of pathogenicity. The discovery of the Swedish New Variant and the more recent Finnish variant highlight the importance of understanding the natural extent of variation in the plasmid. In this study we analysed 524 plasmid sequences from publicly available whole-genome sequence data. Single nucleotide polymorphisms (SNP) in each of the eight coding sequences (CDS) were identified and analysed. There were 224 base positions out of a total 7550 bp that carried a SNP, which equates to a SNP rate of 2.97%, nearly three times what was previously calculated. After normalising for CDS size, CDS8 had the highest SNP rate at 3.97% (i.e., number of SNPs per total number of nucleotides), whilst CDS6 had the lowest at 1.94%. CDS5 had the highest total number of SNPs across the 524 sequences analysed (2267 SNPs), whereas CDS6 had the least SNPs with only 85 SNPs. Calculation of the genetic distances identified CDS6 as the least variable gene at the nucleotide level (d = 0.001), and CDS5 as the most variable (d = 0.007); however, at the amino acid level CDS2 was the least variable (d = 0.001), whilst CDS5 remained the most variable (d = 0.013). This study describes the largest in-depth analysis of the C. trachomatis plasmid to date, through the analysis of plasmid sequence data mined from whole genome sequences spanning 50 years and from a worldwide distribution, providing insights into the nature and extent of existing variation within the plasmid as well as guidance for the design of future diagnostic assays. This is crucial at a time when single-target diagnostic assays are failing to detect natural mutants, putting those infected at risk of a serious long-term and life-changing illness.

Highlights

Chlamydia trachomatis is a long-established cause of infection in humans, with mentions of the ocular form of the disease dating back to ancient Egyptian times [1]
The plasmid consists of eight coding sequences (CDS) and two small antisense RNA sequences [6]. sRNA-2 is antisense to CDS2 and the most abundantly expressed feature of the plasmid, with up to 100-times the expression level of any plasmid-encoded CDS; sRNA-7 is antisense to CDS7 and whilst having relatively high expression levels when compared to the CDS features [7], this was later found to be 12-fold lower than that seen for sRNA-2 [8]
This study describes the largest in-depth analysis of the C. trachomatis plasmid to date, mined from whole genome sequences spanning fifty years and from a worldwide distribution

Summary

Introduction

Chlamydia trachomatis is a long-established cause of infection in humans, with mentions of the ocular form of the disease dating back to ancient Egyptian times [1]. Iterons are critical for control of the plasmid copy number [14] due to the “hand-cuffing” mechanism in which separate plasmid molecules are coupled via the ATP-dependent DNA helicase Rep protein, resulting in steric hindrance and prevention of subsequent rounds of replication [15]. There are usually four repeat sequences in the plasmid of C. trachomatis strain SW5, only three repeats were identified with no apparent effect on plasmid copy number [16]. This suggests that three copies are sufficient for plasmid replication, or that a different mechanism of copy number control exists in this species

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