Antibiotic Resistance and Virulence Gene Transfer in Environmental Pathogens

描述(申请人提供)：食源性疾病的严重程度和持续时间受质粒编码的毒力因子和抗生素耐药基因的影响。环境病原体，如大肠埃希氏菌、沙门氏菌和志贺氏菌，从受污染的灌溉和施肥系统传播到食物链，已被证明同时具有这两种质粒编码元件。感染了来自受污染食品的毒力或抗药性微生物的患者面临严重的治疗选择有限或显着延长的治疗方案。我的长期目标是增加我们对环境病原体之间的接合质粒转移的理解，因为它与抗生素耐药基因和毒力因子的传播有关。接合质粒转移是这些元素在致病和非致病微生物菌株之间传播的方法。典型的F(育性)质粒和大量的R(抗性)质粒的转移是由一种质粒特异性的Tral酶介导的。我们已经确定了有效的(EC50和KI值在纳摩尔范围内)抑制F质粒转移和Tral介导的质粒切割。由于F和R质粒Tral酶的序列同源性高达98%，我推测类似的小分子将是R质粒转移的有效抑制剂。这项建议的具体目的是：第一，确定F质粒Tral酶的有效抑制剂；第二，比较F质粒Tral酶和R质粒Tral酶的结构；第三，确定针对R质粒Tral酶的抑制剂。我将结合分子生物学和生物化学来阐明Tral抑制的结构基础，并鉴定编码在F和R质粒上的Tral酶的新抑制剂。这是解决普遍和日益严重的抗生素耐药性问题的必要的第一步。

DESCRIPTION (provided by applicant): The severity and duration of food-borne diseases are influenced by plasmid-encoded virulence factors and antibiotic resistance genes. Environmental pathogens such as Escherchia coli, Salmonella, and Shigella, which are transmitted to the food chain from polluted irrigation and fertilization systems, have been shown to possess both of these plasmid-encoded elements. Patients infected with either virulent or antibiotic resistant microorganisms from contaminated food face severely limited treatment options or significantly prolonged therapeutic regimens. My long term objectives are to increase our understanding of conjugative plasmid transfer between environmental pathogens as it relates to the spread of antibiotic resistance genes and virulence factors. Conjugative plasmid transfer is the method by which these elements are spread among pathogenic and non-pathogenic microbial strains. Transfer of the prototypical F (fertility) plasmid and the numerous R (resistance) plasmids is mediated by a plasmid-specific Tral enzyme. We have identified potent (EC50 and Ki values in the nanomolar range) inhibitors of F plasmid transfer and Tral-mediated plasmid cleavage. Because F and R plasmid Tral enzymes share up to 98% sequence identity, I hypothesize that similar small molecules will be effective inhibitors of R plasmid transfer. The specific aims of this proposal are to: first, identify potent inhibitors of the F plasmid Tral enzyme; second, compare the structure of the Tral enzyme from the F plasmid to those of R plasmid Tral enzymes; third, identify inhibitors targeting the R plasmid Tral enzymes. I will use a combination of molecular biology and biochemistry to elucidate the structural basis of Tral inhibition and to identify new inhibitors of Tral enzymes encoded on the both F and R plasmids. This is a necessary first step to address the widespread and growing problem of antibiotic resistance.