Investigating important but overlooked streptococcal agents of disease in production animals.

我的研究计划的首要目标是促进知识的发展，以便更好地预防和控制传染病。介绍了动物病原菌--乳链球菌亚种的菌株多样性、致病机理及耐药性。等同种(SDE，猪败血症和关节炎的药剂)和乳链球菌亚种。乳房发育不良(SDD，一种牛乳房炎的病原体)鲜为人知。根据溶血活性和Lancefield抗原分组的差异，可以假设SDE和SDD分离株在基因上是不同的，它们的毒力武器库在含量和调节方面都不同，并且AMR基因含量存在亚种差异。在这里，我建议：1)调查动物SDE和SDD分离株的多样性和系统发育关系，2)利用宿主相关感染模型表征几个关键的SDE和SDD毒力因子和调节因子，3)阐明这两个亚种的AMR谱系。在目标1中，基于种群的分离株的基因组学将使人们能够彻底了解两个亚种的菌株多样性。将临床元数据覆盖到共享相似基因内容和/或突变星座的系统发育中，将有助于改进毒力预测。AIM 2将研究毒力因子和调节因子。毒力因子(即M蛋白、链激酶、丝氨酸蛋白酶CSPA、超抗原)将利用体外感染模型(即与上皮细胞的相互作用、吞噬细胞的摄取和杀伤、细胞增殖)和模拟猪脓毒症和牛乳房炎的小鼠感染模型进行突变和表征。将使用相同的方法结合转录组学来研究调节剂，如两个组分系统。来自Aim1的自然基因变异数据将被用来选择更多的菌株进行测试，从而使人们能够以多样性为导向，全面了解这两个亚种感染的发病机制。在目标3中，进一步挖掘基因组学数据并辅以表型易感性数据将确定SDE和SDD AMR谱系。通过测定携带抗性基因的移动遗传元件(MGES)，并与与SDE和SDD共享定植生态位的细菌物种进行比较，以及通过MGE切除试验和接合试验，将获得对AMR基因的获取和动员，以及SDE和SDD作为AMR储存库的潜力。因此，这项建议将通过1)揭示SDE和SDD种群结构，2)详细描述毒力因子的作用模式和调节，以及3)产生对其AMR的全面描述，从而促进对两种兽医病原体的了解。这些数据将促进动物生产系统中的感染控制工作，将有助于减少兽医环境中抗菌剂的使用，并最终将在减少经济损失的同时改善动物健康。

The overarching goal of my research program is to advance knowledge to enable better prevention and control of infectious diseases. The strain diversity, the pathogenesis of infection, and the antimicrobial resistance (AMR) of the animal pathogens Streptocococcus dysgalactiae subsp. equisimilis (SDE, an agent of septicemia and arthritis in swine) and S. dysgalactiae subsp. dysgalactiae (SDD, an agent of bovine mastitis) are poorly known. Based on differences in hemolytic activity and Lancefield antigen grouping, it can be hypothesized that SDE and SDD isolates are genetically diverse, that their virulence arsenals vary in both content and regulation, and that there are subspecies differences in terms of AMR gene content. Here, I propose to: Aim 1) investigate the diversity and phylogenetic relationships of animal SDE and SDD isolates, Aim 2) characterize several key SDE and SDD virulence factors and regulators using host-relevant infection models, and Aim 3) elucidate the AMR repertoire of both subspecies. In Aim 1, genomics of population-based isolates will permit a thorough understanding of strain diversity in both subspecies. Overlying of clinical metadata to phylogenies sharing similar gene content and/or constellation of mutations will allow for improved virulence prediction. Aim 2 will study virulence factors and regulators. Virulence factors (i.e., M protein, streptokinase, the serine protease CspA, superantigens) will be mutated and characterized using in vitro infection models (i.e., interactions with epithelial cells, uptake and killing by phagocytic cells, cellular proliferation) and mouse infection models mimicking porcine sepsis and bovine mastitis. Regulators such as two component systems will be studied using the same methods in combination with transcriptomics. Natural gene variation data from Aim1 will be leveraged to select further strains to be tested, thus enabling a diversity-driven, comprehensive understanding of the pathogenesis of infection of both subspecies. In Aim 3, further mining of the genomics data supplemented by phenotypic susceptibility data will determine the SDE and SDD AMR repertoire. Insights into acquisition and mobilization of AMR genes, and the potential of SDE and SDD to act as AMR reservoirs will be gained by determination of mobile genetic elements (MGEs) carrying resistance genes, and comparisons to bacterial species sharing colonization niches with SDE and SDD, as well as by MGE excision tests and conjugation assays. This proposal will thus advance knowledge on two veterinary pathogens by 1) unveiling the SDE and SDD population structure, 2) characterizing in detail the mode of action and regulation of virulence factors, and 3) generating a comprehensive portrait of their AMR. The data will facilitate infection control efforts in animal production systems, will help reduce antimicrobial usage in veterinary settings and, ultimately, will improve animal well-being while decreasing economic losses.