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

在任何衣原体物种中产生缺失突变体：快速而简单！

• 批准号: 10495216
• 负责人: Katerina Wolf
• 金额: $ 19.13万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495216
• 关键词: Acute; Algorithms; Alleles; Antibiotics; Bacteria; Bacterial Adhesins; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Chlamydia; Chlamydia Infections; Chlamydia genome; Chlamydia muridarum; Chlamydia trachomatis; Chlamydiaceae; Chronic Disease; Cloning; Columnar Epithelium; Complement; Development; Disease; Environment; Epithelial Cells; Ethyl Methanesulfonate; Excision; Eye; Family; Future; Gene Silencing; Generations; Genes; Genetic; Genital; Genitalia; Genome; Gram-Negative Bacteria; Human; Infection; Invaded; Knock-out; Life Style; Lymphogranuloma Venereum; Mediating; Methods; Molecular Genetics; Mucous Membrane; Mus; Mutagenesis; Nutrient; Open Reading Frames; Organ; Organism; Outcome; Pathogenesis; Phenotype; Plasmids; Play; Production; Replication Origin; Reporting; Rodent; Role; Sexually Transmitted Diseases; Shuttle Vectors; Site; Stains; Surface; System; Tandem Repeat Sequences; Tropism; United States; Untranslated RNA; draining lymph node; genetic manipulation; genetic technology; genital infection; homologous recombination; human pathogen; immunogenic; in vivo; macrophage; member; mutant; novel; pathogen; preference; pressure; promoter; suicide vector; tissue tropism; urogenital tract; vector

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.

抽象的 衣原体属的成员是革兰氏阴性细菌，也会引起各种急性疾病 尽管衣原体在许多方面与其典型疾病有共同之处。 细菌的生活方式，例如细胞内发育周期和主要偏好 侵入上皮细胞，这些人类病原体采用复杂的物种特异性策略 导致组织趋向性和疾病表现的异常多样性 C. 沙眼衣原体感染人类泌尿生殖道粘膜表面或柱状上皮细胞 衣原体介导的生殖器感染是细菌性 STD 的主要原因。 在美国，2018 年向 CDC 报告了 1, 758,668 例感染病例。 沙眼衣原体 分离株分为 15 个主要血清变种，其引起的侵袭性较小。 粘膜层感染，包括眼部血清型 A、B、Ba、C，以及血清型 D 至 K 引起性传播疾病。性病淋巴肉芽肿 (LGV) 血清型 L1、L2、 和L3，短暂感染上皮细胞，但随后侵入粘膜下层感染巨噬细胞， 这有利于细菌传播到区域淋巴结。 沙眼衣原体 L2 的操纵，在过去十年中得到了广泛的开发， 极大地帮助阐明这些病原体如何与其宿主相互作用，并且可能， 然而，为了发现和了解衣原体的机制- 每种衣原体物种的特定宿主嗜性和发病机制结果，势在必行 我们建议将分子遗传学技术扩展到所有衣原体物种和菌株。 在这里，优化一种独特的、最近开发的通过等位基因产生无效突变体的方法 通过同源重组进行交换，使用 pUC19 穿梭载体，这将是适用的 我们打算利用这种方法来生产衣原体。 沙眼衣原体 L2、D 和 B 血清型中 pmpI 和 pmpD 的缺失突变体。 pmpI 和 pmpD 将在 C. mudirarum 中被淘汰，因为这种啮齿动物衣原体物种是 通常用于调查衣原体体内发病机制的研究。 ΔpmpI 和 ΔpmpD 衣原体研究及其 WT 学分和补充 菌株将在各种人类和小鼠细胞培养物中进行，以鉴定 物种可能的特定和宿主特定的相互作用。