Generation of deletion mutants in any chlamydial species: Quick and easy!

摘要 衣原体属的成员是革兰氏阴性细菌，其也引起各种急性 作为人类的慢性疾病。虽然披衣者与他们的典型的 细菌的生活方式，如细胞内发育周期和偏好，主要 这些人类病原体采用复杂的物种特异性策略， 导致组织向性和疾病表现的异常多样性。C. 沙眼感染人泌尿生殖道的粘膜表面或柱状上皮细胞 的眼睛。衣原体介导的生殖器感染是细菌性性病的主要原因 在美国，2018年向CDC报告了1，758，668例感染。C.沙眼衣原体 分离株分为15种主要血清变异体。侵入性较小的分离物， 粘膜层感染，包括眼血清型A、B、Ba、C，而血清型D至K 引起性传播疾病。性病性淋巴肉芽肿（LGV）血清型，L1，L2， 和L3，瞬时感染上皮细胞，然后侵入粘膜下层感染巨噬细胞， 这促进了细菌向局部淋巴结的传播。遗传 操纵C。沙眼，L2，在过去的十年中得到了广泛的发展， 有助于阐明这些病原体如何与宿主相互作用， 引起疾病。然而，为了发现和理解衣原体的机制- 每种衣原体都具有特异性的宿主嗜性和致病性， 将分子遗传学技术扩展到所有衣原体物种和菌株。我们提出 这里，为了优化一种独特、最近开发的通过等位基因产生无效突变体的方法， 通过同源重组交换，使用pUC 19穿梭载体，这将是适用的 衣原体属的任何成员。我们打算利用这种方法生产 C. pmpI和pmpD缺失突变体;沙眼，血清型L2、D和B。而且 pmpI和pmpD将在C中被敲除。mudirarum，因为这种啮齿动物衣原体物种是 通常用于研究体内衣原体发病机理的研究。表型 研究中使用了BpmpI和BpmpD衣原体及其WT对应物，并补充 菌株将在各种人以及鼠细胞培养物中进行，以鉴定 可能的物种特异性和宿主特异性相互作用。

Abstract Members of the genus Chlamydia are Gram-negative bacteria that cause various acute as well as chronic diseases in humans. Although chlamydiae share numerous aspects of their typical bacterial life style, such as intracellular developmental cycle and the preference to primarily invade epithelial cells, these human pathogens employ sophisticated species-specific strategies contributing to an extraordinary diversity in tissue tropism and disease manifestation. C. trachomatis infects mucosal surfaces of the human urogenital tract or columnar epithelial cells of the eye. Chlamydia-mediated genital infections represent the leading cause of bacterial STDs in the United States, with 1, 758,668 infections reported to the CDC in 2018. C. trachomatis isolates are divided into 15 major serovariants. The less invasive isolates, which cause infections of mucosal layers, include ocular serovars A, B, Ba, C, while serovars D through K cause sexually transmitted diseases. The lymphogranuloma venereum (LGV) serovars, L1, L2, and L3, transiently infect epithelial cells but then invade the submucosae to infect macrophages, which facilitates the dissemination of the bacteria to the regional lymph nodes. Genetic manipulation of C. trachomatis, L2, which has been extensively developed in the last decade, significantly helped to elucidate how these pathogens interact with their hosts and possibly, cause diseases. However, in order to discover and understand the mechanism(s) for chlamydia- specific host tropism and pathogenesis outcomes with each chlamydial species, it is imperative to expend molecular genetics technologies to all chlamydial species and strains. We propose here, to optimize a unique, recently developed approach for generation of null mutants by allelic exchange via homologous recombination, using pUC19 shuttle vector, that would be applicable to any member of the genus Chlamydia. We intend to utilize this approach in production of deletion mutants in pmpI and pmpD in C. trachomatis, serovars L2, D, and B. Moreover, the pmpI and pmpD will be knocked out in C. mudirarum since this rodent chlamydial species is typically employed in studies investigating chlamydial pathogenesis in vivo. Phenotypical studies with ∆pmpI and ∆pmpD chlamydiae with their WT counterparts and complemented strains will be conducted in various human as well as murine cells cultures in order to identify possible species-specific and host-specific interactions.