Investigating virulence functions of mastitis-associated Extraintestinal pathogenic Escherichia coli relevant to human disease

摘要/项目摘要 肠外大肠杆菌（ExPEC）是一种生活在哺乳动物肠道内的细菌，可导致生命- 当它们感染其他组织时会威胁疾病。这组病原体造成的社会负担是巨大的 而且还在增长，部分原因是抗生素耐药性增加和缺乏疫苗接种选择。尿脓毒症， 肺炎和新生儿脑膜炎是由这些菌株引起的人类疾病。我们的长期目标是 了解在动物和人类中传播的ExPEC发病机制的分子机制。许多 动物易受这些病原体的感染，是人类感染的宿主。中央 基于大量的初步数据，本申请的假设是牛的一些乳房炎菌株 来源的病毒也有可能在包括人类在内的多种宿主中引起疾病。这是一个新奇的发现， 基于证据，包括与人类ExPEC谱系的遗传相似性，Galleria中的毒力 梅隆菌感染、对人血清的耐药性以及引起败血症和尿路感染的能力 小鼠这项工作的目的是表征毒力因子，使一些乳腺炎相关菌株 感染多个宿主并引起肠外疾病乳腺炎菌株M12的全基因组筛选 毒力因子导致发现了编码组3荚膜的荚膜生物合成基因簇。 第3组胶囊存在于许多ExPEC菌株中，但其作用尚未明确。未包封 突变株不能感染小鼠的脾或肾。在我们的研究中， 收集还编码组3胶囊，这可能有助于这些细菌避免毒力 嗜中性粒细胞吞噬作用中性粒细胞对于防御许多细菌病原体至关重要 包括ExPEC，但我们并不完全理解ExPEC抵抗被吞噬的机制， 被这些细胞杀死。本提案的目标将通过三个具体目标实现：（1） 估计乳腺炎相关E.在已建立的人类模型中， ExPEC感染。我们将利用新的DNA条形码策略来测量多个 在这些实验中。(2)描述在乳腺炎相关菌株中发现的第3组胶囊的作用 与人类疾病相关的条件。我们将通过以下方式测试这些胶囊是否能提高对杀戮的抵抗力： 中性粒细胞或血清和实验感染期间。(3)找出所有的基因， M12第3组胶囊的产生，包括胶囊位点内部和外部的那些。以来 构建胶囊是一个关键的毒力功能，这可以确定新疗法的靶点。这项建议 是创新的，因为它是基于新的概念，乳腺炎相关菌株是潜在的人类 病原体这是重要的，因为它将提供更好的理解ExPEC的分子基础 在多种宿主中的毒力，以及在不同环境中发现的ExPEC之间的关系。这些 这些发现可能为预防这些细菌引起的疾病和改善公共卫生提供新的方法。

Abstract/Project Summary Extraintestinal Escherichia coli (ExPEC) are bacteria that live in the intestines of mammals and cause life- threatening illnesses when they infect other tissues. The societal burden due to this group of pathogens is vast and growing, in part because of increasing antibiotic resistance and a lack of vaccination options. Urosepsis, pneumonia and neonatal meningitis are human diseases caused by these strains. Our long-term goal is to understand the molecular mechanisms of pathogenesis of ExPEC that circulate in animals and humans. Many animals are susceptible to these pathogens and represent reservoirs for human infection. The central hypothesis of this application, based on substantial preliminary data, is that some mastitis strains of bovine origin also have the potential to cause disease in multiple hosts including humans. This is a novel finding, based on evidence that includes genetic similarities with human ExPEC lineages, virulence in Galleria mellonella infections, resistance to human serum, and the ability to cause sepsis and urinary tract infections in mice. The objective of this work is to characterize virulence factors that allow some mastitis-associated strains to infect multiple hosts and cause extraintestinal disease. A genome-wide screen for mastitis strain M12 virulence factors led to the discovery of a cluster of capsule biosynthesis genes encoding a Group 3 capsule. Group 3 capsules are present in many ExPEC strains, but their roles are not well defined. An unencapsulated mutant strain was unable to infect spleens or kidneys of mice. Additional mastitis-associated strains in our collection also encode Group 3 capsules, which may promote virulence by helping these bacteria avoid neutrophil phagocytosis. Neutrophils are critically important for defense against many bacterial pathogens including ExPEC, but we do not fully comprehend the mechanisms whereby ExPEC resist being engulfed or killed by these cells. The objectives of this proposal will be accomplished with three specific aims: (1) Estimate the fraction of mastitis-associated E. coli that can cause disease in established models of human ExPEC infection. We will utilize novel DNA barcoding strategies to measure competitive fitness of multiple strains in these experiments. (2) Characterize the role of Group 3 capsules found in mastitis-associated strains in conditions relevant to human disease. We will test whether these capsules promote resistance to killing by neutrophils or serum and during experimental infections. (3) Identify all of the genes that are needed for production of the M12 Group 3 capsule, including those both inside and outside the capsule locus. Since building the capsule is a critical virulence function, this could identify targets for new therapies. This proposal is innovative because it is based on the novel concept that mastitis-associated strains are potential human pathogens. It is significant because it will provide greater understanding of the molecular basis for ExPEC virulence in multiple hosts, and the relationships between ExPEC found in different environments. These findings may inform new ways to prevent disease caused by these bacteria and improve public health.