Role of precipitous virulence in invasiveness of community-acquired methicillin-r

描述（由申请人提供）：我们的长期目标是更深入地了解细菌适应性和毒力在发病机制中是如何交叉的。本提案的重点是葡萄球菌病毒在社区获得性耐甲氧西林金黄色葡萄球菌（CA-MRSA）引起的感染中的作用。虽然CA-MRSA遭遇的结果通常是无症状的定植，但CA-MRSA菌株产生侵袭性感染的倾向定义了抵抗宿主先天免疫清除机制的能力。CA-MRSA因素负责这一重要的宿主-病原体相互作用的不同结果尚不清楚。agr基因座是一种全球性的毒力基因表达调控因子，由于其在动物模型中侵袭性的关键作用而受到广泛关注。我们的初步数据表明，CA-MRSA在人体内的侵袭伴随着agr活性增强的变异的富集，这支持了这一观点，并表明疾病的早期阶段伴随着细菌的变化，从而产生侵袭性疾病表型。这些变异的DNA测序显示agr位点没有改变，表明“上游”agr调控有助于表型。替代sigma因子sigB的功能障碍被确定为表型的常见原因，但我们对临床分离株的初步调查表明，其他未知基因也发生了改变。关键的是，变异在体外具有适应性优势，这表明独立于宿主因素的选择压力可以部分解释它们的出现。这些考虑产生了以下两部分假设，本建议基于此：1)通过基因突变，金黄色葡萄球菌经常在感染中产生一组毒力不同的克隆；2)CA-MRSA菌株从共生表型向侵袭性疾病表型的转变与体内对增强毒力的突变的选择有关。为了验证这一假设，我们将1)确定临床CA-MRSA皮肤感染和侵袭性疾病期间宿主内agr功能上升的频率，2)表征这些上升的遗传基础，以及3)在体外评估具有上升的菌株的相对毒力和适应性

DESCRIPTION (provided by applicant): Our long-term goal is a deeper understanding of how bacterial fitness and virulence intersect in pathogenesis. The present proposal focuses on the role of the Staphylococcal virulon in infections caused by community- acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA). While the outcome of a CA-MRSA encounter is usually asymptomatic colonization, the propensity of CA-MRSA strains to produce invasive infection defines a capacity to resist host innate immune clearance mechanisms. The CA-MRSA factors responsible for the diverse outcomes of this important host-pathogen interaction are unknown. The agr locus, a global regulator of virulence gene expression, has received much attention due to its critical role in invasiveness in animal models. Our preliminary data suggesting that invasion of CA-MRSA in humans is accompanied by enrichment for variants with enhanced agr activity supports this idea and indicates that the early stages of disease are accompanied by bacterial changes that give rise to an invasive-disease phenotype. DNA sequencing of such variants revealed that the agr locus was unaltered, indicating that "upstream" agr regulation contributes to the phenotype. Dysfunction of the alternative sigma factor sigB was identified as a frequent cause of the phenotype, but our preliminary survey of clinical isolates indicates the occurrence of alterations in other, unknown genes. Critically, variants had a fitness advantage in vitro, suggesting that selective pressures independent of host factors can account in part for their emergence. These considerations give rise to the following two-part hypothesis, upon which the present proposal is based: 1) through mutation of genes, S. aureus frequently generates within an infection a group of clones differing in their virulence, and 2) the shift of CA-MRSA strains from a commensal to an invasive-disease phenotype is associated with selection in vivo for mutations that enhance virulence. To test the hypothesis, we will 1) determine the frequency of within-host upshifts in agr function during clinical CA-MRSA skin infection and invasive disease, 2) characterize the genetic basis of these upshifts, and 3) evaluate the relative virulence and fitness of strains with upshifts both in vitro and in vivo using serial passage and competition tests. Field studies will focus on community subjects, where the barrier to infection is higher than in hospitalized patients for whom disruption of barrier functions by disease and clinical intervention permit S. aureus strains that lack full virulence to cause infection. The proposed work will impact critical questions in infectious disease research, such as the best use of therapeutic modalities that are being developed to target agr and virulence, as well as more basic yet closely intertwined problems, such as the ways in which S. aureus transitions from an innocent member of our normal flora to an invasive, life-threatening pathogen. The work will have broad implications for forestalling such events.