Genetic Basis of Virulence of Community MRSA Clone USA300

描述（由申请方提供）：金黄色葡萄球菌是一种主要的人类病原体。它可以迅速引起致命的感染。甲氧西林抗性S.金黄色葡萄球菌（MRSA），USA 300，在美国社区流行。MRSA对所有与青霉素化学相关的抗生素都有耐药性，因此这种克隆的出现是一个严重的公共卫生问题。我们的研究目标是确定USA 300的毒力基因。USA 300的出现与获得某些移动的遗传元件有关，我们怀疑这些元件携带影响毒力的基因。为了验证这一假设，将在兔感染模型和体外试验中检测疑似毒力基因缺失的USA 300突变体，以确定其毒力是否低于正常菌株。中性粒细胞（polymorphisms neutrophil，PMNs）是宿主防御沙门氏菌的第一道防线。金黄色葡萄球菌感染，并对细菌清除至关重要。将在体外测定PMN功能，以确定毒力因子是否影响人PMN趋化性、吞噬作用或细菌杀伤。具体目标是：1）确定USA 300特有的精氨酸分解代谢移动的元件（ACME）中的毒力决定因子。ACME的缺失降低了毒力。将删除三个可能的可疑毒力基因arcA、opp-3A和opp-3DF，并对每个突变体进行检测，以确定哪些影响毒力。2)确定非致死性暴露于β-内酰胺抗生素诱导的SOS反应是否产生适应性突变，为MRSA提供生存优势。3)确定PVL基因的破坏是否减弱兔肺炎模型中的毒力。PVL（Panton-Valentine leukocidin）被认为是USA 300中最重要的毒力因子，但这一点存在很大争议。家兔对PVL敏感，肺可能是靶器官。从这个模型的结果应该有助于解决的争议，PVL是否是一个主要的毒力因子。4)为了确定是否在其他两个元件（Sa 3原噬菌体和SaPI 5致病岛）的基因编码的毒力决定因子。相关性：沙门氏菌毒性、耐药性菌株的出现。金黄色葡萄球菌在社区中是一个范围和重要性仅次于艾滋病的公共问题。确定USA 300菌株的特殊毒力基因是开发治疗社区相关MRSA菌株引起的严重感染的新药和方法的关键第一步。公共卫生相关性金黄色葡萄球菌（一种重要的人类病原体）的抗生素耐药性菌株正在上升。一个特别的克隆，USA 300，似乎特别致命。几个可能对毒力重要的基因是USA 300中唯一存在的。通过敲除这些基因中的每一个并测试毒性的丧失，我们希望了解哪些基因编码毒性。一旦这些基因被识别出来，就可能开发出阻断其影响的策略。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a major human pathogen. It can cause rapidly fatal infections. A highly virulent clone of methicillin resistant S. aureus (MRSA), USA300, is epidemic in communities in the United States. MRSA is resistant to all antibiotics chemically related to penicillin, thus emergence of this clone is a serious public health issue. Our research objective is to identify genes that contribute to virulence of USA300. Emergence of USA300 is associated with acquisition of certain mobile genetic elements, which we suspect carry genes that impact virulence. To test this hypothesis, mutants of USA300 in which suspected virulence genes have been deleted will be tested in rabbit infection models and in vitro assays to determine if their virulence is reduced compared to the normal strain. Polymorphonuclear neutrophils (PMNs) are the first line of innate host defense against S. aureus infection and are critical for bacterial clearance. PMN function will be assayed in vitro to determine whether virulence factors affect human PMN chemotaxis, phagocytosis, or bacterial killing. Specific aims are: 1) To identify virulence determinants in the arginine catabolic mobile element (ACME), which is unique to USA300. Deletion of ACME reduces virulence. Three "likely suspect virulence genes, arcA, opp-3A and opp-3DF, will be deleted and each mutant tested to determine which impact virulence. 2) To determine whether SOS response induced by non-lethal exposure to beta-lactam antibiotic generates adaptive mutations offering a survival advantage to MRSA. 3) To determine if disruption of PVL genes attenuates virulence in a rabbit pneumonia model. PVL (Panton-Valentine leukocidin) is generally assumed to be the most important virulence factor in USA300, but this is highly controversial. The rabbit as a species is sensitive to PVL and lung may be target organ. Results from this model should help resolve the controversy as to whether PVL is a major virulence factor. 4) To determine whether genes within two other elements, (Sa3 prophage and SaPI5 pathogenicity island, encode virulence determinants. Relevance: Emergence of virulent, drug-resistant strains of S. aureus in the community is a public problem second only to AIDS in scope and importance. Defining genes contributing to the special virulence of USA300 strains is a critical first step for developing new drugs and approaches for treatment of severe infections caused by community-associated MRSA strains. PUBLIC HEALTH RELEVANCE Antibiotic resistant strains of Staphylococcus aureus, an important human pathogen, are on the rise. One particular clone, USA300, seems to be especially virulent. Several genes that could be important for virulence are uniquely present in USA300. By knocking each of these genes out and testing for loss of virulence, we hope to learn which genes code for virulence. Once these genes are identified, strategies to block their effects may be developed.