Abstract
Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Different strains are associated with ocular or urogenital infections, and a proposed mechanism that may explain this tissue tropism is the active tryptophan biosynthesis pathway encoded by the genomic trpRBA operon in urogenital strains. Here we describe genetic complementation studies that are essential to confirm the role of tryptophan synthase in the context of an ocular C. trachomatis genomic background. Ocular strain A2497 was transformed with the (urogenital) pSW2::GFP shuttle vector showing that there is no strain tropism barrier to this plasmid vector; moreover, transformation had no detrimental effect on the growth kinetics of A2497, which is important given the low transformation efficiency of C. trachomatis. A derivative of the pSW2::GFP vector was used to deliver the active tryptophan biosynthesis genes from a urogenital strain of C. trachomatis (Soton D1) to A2497 with the aim of complementing the truncated trpA gene common to most ocular strains. After confirmation of intact TrpA protein expression in the transformed A2497, the resulting transformants were cultivated in tryptophan-depleted medium with and without indole or tryptophan, showing that complementation of the truncated trpA gene by the intact and functional urogenital trpRBA operon was sufficient to bestow an indole rescuable phenotype upon A2497. This study proves that pSW2::GFP derived vectors do not conform to the cross-strain transformation barrier reported for other chlamydia shuttle vectors, suggesting these as a universal vector for transformation of all C. trachomatis strains. This vector promiscuity enabled us to test the indole rescue hypothesis by transforming ocular strain A2497 with the functional urogenital trpRBA operon, which complemented the non-functional tryptophan synthase. These data confirm that the trpRBA operon is necessary and sufficient for chlamydia to survive in tryptophan-limited environments such as the female urogenital tract.
Highlights
Chlamydia trachomatis is an obligate intracellular bacterium that is a major threat to human health
To ascertain whether pSW2::green fluorescent protein (GFP) had recombined with the native plasmid of A2497WT, whole-genomic DNA was extracted from the transformed A2497WT and cloned into E. coli DH5α
Whilst this shows that TrpA is an essential part of a properly functioning tryptophan synthase, the aggressive Lymphogranuloma venereum (LGV) serovars are more tolerant to tryptophan limitation than urogenital isolates (Caldwell et al, 2003), do not have an epithelial tropism and have no role in trachoma
Summary
Chlamydia trachomatis is an obligate intracellular bacterium that is a major threat to human health. This study focuses on the trachoma strains, which infect epithelial cells to cause two distinct diseases, namely the eye infection “trachoma” and the sexually transmitted infection “chlamydia.” The biological divide between ocular- and urogenital-associated strains is reflected by a clear phylogenetic separation, which occurred thousands of years ago (Hadfield et al, 2017). Whole genome sequencing has revealed that the trachoma biovar is divided into two distinct clades, T1 and T2 (Harris et al, 2012). T1 contains the common STI strains, whereas T2 contains two subclades comprising of the rarer STI strains and the ocular strains. The nesting of ocular strains within the T2 clade suggests that ocular C. trachomatis evolved from one common, urogenital ancestor (Harris et al, 2012)
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