Genetic Basis of Virulence of Community MRSA Clone USA300

描述(申请人提供)：金黄色葡萄球菌是一种主要的人类病原体。它可以引起迅速致命的感染。一种高毒力的耐甲氧西林金黄色葡萄球菌(MRSA)克隆USA300在美国的社区流行。耐甲氧西林金黄色葡萄球菌对所有与青霉素有关的抗生素都具有耐药性，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与USA300毒力有关的基因。USA300的出现与某些移动遗传元件的获得有关，我们怀疑这些遗传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测试中测试可疑毒力基因已被删除的USA300突变株，以确定与正常毒株相比，它们的毒力是否降低。中性粒细胞(PMN)是宿主抵抗金黄色葡萄球菌感染的第一道防线，是细菌清除的关键。将在体外检测PMN的功能，以确定毒力因素是否影响人PMN的趋化、吞噬或细菌杀灭。具体目标是：1)确定精氨酸分解代谢移动元件(ACME)中的毒力决定因素，该元件是USA300所独有的。ACME基因的缺失降低了毒力。三个可能的可疑毒力基因，arca，opp-3A和opp-3df，将被删除，并对每个突变体进行测试，以确定哪种影响毒力。2)确定非致命性暴露于β-内酰胺类抗生素诱导的SOS反应是否产生适应性突变，从而为MRSA提供生存优势。3)确定PVL基因的破坏是否减弱了兔肺炎模型的毒力。PVL(潘顿-瓦伦丁杀白素)通常被认为是USA300最重要的毒力因子，但这一点存在很大争议。兔作为一个物种对PVL敏感，肺可能是PVL的靶器官。这个模型的结果应该有助于解决关于PVL是否是主要毒力因素的争议。4)确定另外两个元件(SA3原噬菌体和SaPI5致病岛)内的基因是否编码毒力决定因素。相关性：在社区中出现具有毒力和抗药性的金黄色葡萄球菌菌株是一个公共问题，在范围和重要性上仅次于艾滋病。确定导致USA300菌株特殊毒力的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。与公共卫生相关的金黄色葡萄球菌是一种重要的人类病原体，其耐药菌株正在上升。有一种特殊的克隆，USA300，似乎特别致命。在USA300中有几个对毒力可能很重要的基因是独一无二的。通过敲除这些基因中的每一个并测试毒力的丧失，我们希望了解哪些基因编码毒力。一旦确定了这些基因，就可能开发出阻止其影响的策略。

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.