Data_Sheet_3_Genetic Transformation of a C. trachomatis Ocular Isolate With the Functional Tryptophan Synthase Operon Confers an Indole-Rescuable Phenotype.PDF

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.

沙眼衣原体（Chlamydia trachomatis）是引发可预防性失明的首要病原体，亦是最为常见的细菌性性传播感染病原。不同菌株分别与眼部或泌尿生殖道感染相关，目前已提出的可解释该组织嗜性的潜在机制，是泌尿生殖道菌株基因组中由trpRBA操纵子（trpRBA operon）编码的活性色氨酸生物合成通路。本研究描述了必要的基因互补实验，以确认色氨酸合酶在沙眼衣原体眼部菌株基因组背景下的功能作用。研究人员将（源自泌尿生殖道菌株的）pSW2::GFP穿梭载体转化至眼部菌株A2497中，证实该质粒载体不存在菌株嗜性障碍；此外，转化过程未对A2497的生长动力学产生不利影响——鉴于沙眼衣原体的转化效率极低，这一点至关重要。研究人员使用pSW2::GFP载体的衍生载体，将源自泌尿生殖道沙眼衣原体菌株Soton D1的活性色氨酸生物合成基因递送至A2497中，旨在互补大多数眼部菌株共有的截短trpA基因。在确认转化后的A2497可完整表达TrpA蛋白后，研究人员将所得转化株置于添加或不添加吲哚/色氨酸的色氨酸缺乏培养基中培养，结果显示，通过完整且具有功能的泌尿生殖道trpRBA操纵子互补截短trpA基因，足以使A2497获得可被吲哚挽救的表型。本研究证实，源自pSW2::GFP的载体并未遵循其他衣原体穿梭载体所报道的跨菌株转化障碍，提示该载体可作为通用载体用于所有沙眼衣原体菌株的转化。这种载体的广谱适用性使我们得以验证吲哚挽救假说：将携带功能性泌尿生殖道trpRBA操纵子的载体转化至眼部菌株A2497中，以互补其失活的色氨酸合酶。上述数据证实，trpRBA操纵子对于衣原体在色氨酸受限环境（如女性泌尿生殖道）中存活是必需且足够的。