Comparative analysis of the plasmid sequences has revealed the most conserved regions …

• Abstract
• Background
• Results and Discussion
• Conclusion
• Methods
• Acknowledgements
• References
• Table 1
• Table 2
• Table 3
• Figures

Chlamydia trachomatis is the most common cause of non specific urethritis in the industrialised world, and the major infectious cause of preventable blindness (trachoma) in the third world [1,2]. C. trachomatis can be divided into at least 15 serovars or serotypes, distinguished by ompA sequencing [3], which are associated with different disease pathologies [4]. Serotypes A, B, Ba and C are generally associated with blinding trachoma and serotypes D to K cause non-disseminating sexually transmitted infections. These 12 serotypes are all naturally restricted to infection of genital or ocular epithelial cells and have not been observed to be invasive [5]. By contrast, serotypes L1, L2 and L3 cause a rare invasive and systemic sexually transmitted infection normally found in the tropics, known as lymphogranuloma venereum (LGV) [6,7].

In October 2006 a new variant of C. trachomatis was described in Sweden that evaded several of the then current commercial molecular diagnostic tests for detecting the microorganism, which were based on the presence of specific plasmid sequences [8]. A deletion of 377 bp of plasmid DNA, in the region used for nucleic acid amplification tests (NAATs), is responsible for the negative diagnoses [9]. All new variant strains of C. trachomatis belong to serotype E [10]. Failure to detect the plasmid and hence treat those infected with the new variant has led to a significant increase in cases, and in some Swedish counties 20 – 64% of current infections are caused by this strain of C. trachomatis [10].

Strains of C. trachomatis have a highly conserved small genome of approximately 1 Mb and harbour a plasmid of approximately 7 kb [11]. Some plasmid-free isolates of C. trachomatis have been described, but these are exceedingly rare and the only viable clinical isolates described that are plasmid free belong to serotypes L2, D and E [12-14]. The C. trachomatis plasmids sequenced to date each contain eight predicted coding sequences (CDSs), along with a set of four 22 bp repeats which is understood to be the origin of replication [15,16]. Characterization of plasmid functions and of chlamydial genes in general has been greatly impeded by the lack of a genetic system for studying C. trachomatis [17] and so little is known about the function of these CDSs beyond in silico predictions of function for 5 CDSs and the observation that the CDS previously designated ORF5 encodes a protein of 28 kDa (pgp3) when expressed in Escherichia coli [18,19]. However, the role of pgp3 in the chlamydial developmental cycle and the overall biological function of the plasmid remains unknown.

Since the study of C. trachomatis genes is hampered by the lack of any molecular tools with which to manipulate strains, the emergence of the new variant strain and naturally occurring mutants provides an opportunity to investigate the evolution of the plasmids and to determine whether the evolutionary pathway of these plasmids matches that of the chromosome. In this study the nature and extent of chromosome and plasmid variation in C. trachomatis has been investigated. To extend the current dataset, two isolates of the previously unsequenced serotype B were completely sequenced, as were four plasmids from Swedish strains belonging to serotypes E and F, including that of the new variant strain. The plasmid is useful as a target for diagnostic testing because it is relatively stable and hence more resistant to nuclease damage than the genome, and present at up to ten copies per genome [20]. Thus a detailed knowledge of the sequence conservation and the stability of chlamydial plasmids will be critically informative in determining whether the plasmid is a reliable target for future diagnostic tests.