Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

本研究设定了三项核心目标：一是将基于质粒的转化实验方案（plasmid-based transformation protocol）拓展应用于属于沙眼生物型（trachoma biovar）的沙眼衣原体（Chlamydia trachomatis）临床分离株；二是提供原理验证，证实可对选定的质粒基因进行基因敲除（同时保留复制型质粒）；三是探究质粒的功能可塑性。本研究对新近开发的、针对沙眼衣原体LGV（Lymphogranuloma venereum，淋巴肉芽肿）分离株的基于质粒的转化方案进行了优化改造，并对一株源自瑞典的无质粒生殖道沙眼衣原体分离株（SWFP-）完成了遗传转化。针对该非LGV型沙眼衣原体宿主的转化操作需引入离心步骤（centrifugation step），但由于宿主本身不含天然质粒，因此无需对转化子（transformants）进行噬斑纯化（plaque purification）。转化子可表达绿色荧光蛋白（GFP）、表现出青霉素抗性，且碘染结果呈阳性，证实其可积累糖原（glycogen）。本次使用的转化用质粒未与宿主染色体（host chromosome）发生重组。我们构建了携带质粒编码序列5（CDS5）缺失突变的pGFP::SW2衍生载体。CDS5编码pgp3蛋白——一种从衣原体包涵体（inclusion）分泌至宿主细胞质（cell cytoplasm）的分泌蛋白。我们使用该衍生质粒（pCDS5KO）对沙眼衣原体SWFP-进行转化，实验证实pgp3对于质粒功能而言并非必需。本研究首次证实可对衣原体质粒的特定片段进行选择性缺失改造，这是实现质粒功能详细分子解析的关键第一步。我们将3.6 kb的β-半乳糖苷酶（β-galactosidase）表达盒插入CDS5的缺失位点，构建得到重组质粒placZ-CDS5KO。该转化子同样表现为青霉素抗性、可表达GFP且糖原碘染呈阳性；此外，转化子可稳定表达β-半乳糖苷酶，表明lacZ表达盒（lacZ cassette）在沙眼衣原体中可正常发挥功能。我们开发了一种可通过X-gal染色（X-gal staining）可视化观察单个衣原体包涵体的实验方法。能够在衣原体包涵体内表达有活性的β-半乳糖苷酶是一项重要技术进展，该方法可实现简便、快速的直接检测衣原体感染性的实验，无需依赖噬斑计数、荧光检测或抗体染色。