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，也会引起各种急性 作为人类的慢性病。尽管衣原体在许多方面都与其典型的 细菌的生活方式，如细胞内发育周期和偏爱主要 入侵上皮细胞，这些人类病原体采用复杂的物种特异性策略 有助于组织取向和疾病表现的非凡多样性。C。 沙眼衣原体感染人泌尿生殖道粘膜表面或柱状上皮细胞 眼睛的。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年向疾控中心报告的感染人数为1758,668人。沙眼衣原体 分离株分为15个主要血清变异体。侵袭性越小的菌株，就会导致 粘膜层感染，包括眼部A型、B型、B型、C型，以及D型至K型 引发性传播疾病。淋巴肉芽肿(LGV)血清型L1、L2、 和L3，瞬时感染上皮细胞，然后侵入粘膜下感染巨噬细胞， 这有助于细菌传播到区域淋巴结处。遗传 操作沙眼衣原体，L2，这在过去十年中得到了广泛的发展， 极大地帮助阐明了这些病原体如何与宿主相互作用，并可能， 引发疾病。然而，为了发现和了解衣原体的发病机制(S)- 针对每个衣原体物种的特定宿主趋向性和致病结局，这是势在必行的 将分子遗传学技术推广到所有衣原体物种和菌株。我们建议 在这里，为了优化一种独特的、最近开发的通过等位基因产生零突变体的方法 使用pUC19穿梭载体，通过同源重组进行交换，这将是适用的 对衣原体属的任何成员。我们打算将这种方法用于生产 沙眼衣原体、血清型L2、D和B中pmpI和pmpD的缺失突变 在木霉中，pmpI和pmpD将被淘汰，因为这种啮齿动物的衣原体物种是 通常用于研究衣原体在体内的致病机制。表型 ∆pmpI和∆pmpD衣原体及其WT对应物的研究和补充 菌株将在各种人类和小鼠细胞培养中进行，以便鉴定 可能的物种特定和宿主特定的相互作用。

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.