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

我的研究计划的首要目标是推进知识，使更好地预防和控制传染病。动物致病菌失乳链球菌（Streptocococcus dysgalactiae subsp）的菌株多样性、感染发病机制及耐药性（AMR）。猪脓毒杆菌（SDE，猪败血症和关节炎的一种病原体）和不良乳杆菌亚种。泌乳不良（SDD，牛乳腺炎的一种诱因）鲜为人知。根据溶血活性和兰斯菲尔德抗原分组的差异，可以推测SDE和SDD分离株具有遗传多样性，其毒力库在含量和调控上都存在差异，AMR基因含量存在亚种差异。在此，我建议：目标1)研究动物SDE和SDD分离株的多样性和系统发育关系；目标2)利用宿主相关感染模型表征SDE和SDD的几个关键毒力因子和调节因子；目标3)阐明两个亚种的AMR库。在目标1中，基于种群的分离株的基因组学将允许对两个亚种的菌株多样性进行彻底的了解。将临床元数据与系统发育重叠，共享相似的基因内容和/或突变群，将有助于改进毒力预测。目标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、超级抗原）将发生突变，并利用体外感染模型（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腺炎的小鼠感染模型进行表征。将使用与转录组学相结合的相同方法研究双组分系统等调节因子。来自Aim1的自然基因变异数据将被用于选择进一步的菌株进行测试，从而使多样性驱动，全面了解两个亚种感染的发病机制。在Aim 3中，进一步挖掘基因组数据，辅以表型易感性数据，将确定SDE和SDD AMR库。通过测定携带抗性基因的移动遗传元件（MGEs），比较与SDE和SDD共享定植生态位的细菌物种，以及MGE切除试验和偶联试验，将深入了解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，该提案将通过以下方式推进对两种兽医病原体的认识：1)揭示SDE和SDD的种群结构，2)详细描述毒力因子的作用模式和调控，以及3)生成其AMR的全面描述。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微生物药物的使用，并最终改善动物福利，同时减少经济损失。

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.