Small RNAs regulating group A Streptococcus virulence

描述(申请人提供)：人类致病菌A组链球菌(GAS)可引起多种疾病，包括咽炎、坏死性筋膜炎和中毒性休克样综合征。这项拟议研究的长期目标是促进我们对控制细菌毒力的全球调控机制的理解。新发现的毒力相关调控机制将成为新型抗菌剂操纵的有吸引力的靶点，从而提高公众健康。小调控RNA(SRNAs)代表了GAS和相关病原体中基因调控的一个鲜为人知的区域。具体的假设是，sRNA代表着协调气体毒力的全球调控网络的主要组成部分。这一假设基于以下观察结果。首先，sRNA调节一些细菌病原体的毒力。其次，生物信息学分析表明，GAS编码56个推测的sRNA。第三，已经在GAS中实验验证了两个sRNA，这两个sRNA似乎都通过尚不清楚的机制来调节毒力因子的表达。我们的假设将通过以下具体目标进行检验： 1.验证sRNAs普遍存在于GAS中并在链球菌中保守的假设。将通过使用定制的Affymetrix微阵列来检测在一系列模拟人类感染的实验条件下转录的sRNA，包括体外和体内疾病模型，来研究sRNA是否代表GAS的主要调节机制。这一特定目标的完成将代表着首次对乳杆菌目病原体(包括链球菌和肠球菌)内的sRNA进行全基因组分析，并刺激对人类细菌病原体光谱的sRNA研究。 2.验证sRNAs调控气体毒力的假设。候选的sRNA将根据乳杆菌病原体之间的序列保守性以及体内生长过程中sRNA的表达模式进行分类。将为12个保守的sRNA构建等基因的sRNA突变株，并在体外和体外检测改变的毒力特性。毒力特征发生变化的菌株随后将在两种侵袭性疾病的小鼠模型中进行毒力减弱测试。完成这一具体目标将确定对气体感染的正常发展至关重要的sRNA，并为描绘这些调节因子发挥作用的途径提供动力，最终目标是通过使用新型抗菌剂来抑制这些途径。 与公共卫生相关：在美国，每年约有3000万例毒气咽炎病例。这项拟议的研究将为GAS和相关病原体的基因调控提供一个基本但尚未得到充分研究的领域，并为研究界提供sRNA位置和表达模式的详细蓝图，以促进进一步研究。通过将sRNA毒力调节途径的知识转化为基于新型抗菌剂对这些途径的抑制的新的治疗和/或预防制度的长期目标，可以加强公共卫生。

DESCRIPTION (provided by applicant): The human bacterial pathogen group A Streptococcus (GAS) causes a broad spectrum of diseases, including pharyngitis, necrotizing fasciitis, and a toxic-shock-like syndrome. The long-term aim of the proposed research is to advance our understanding of the global regulatory mechanisms controlling bacterial virulence. Newly identified virulence-related regulatory mechanisms would represent attractive targets for manipulation by novel antimicrobial agents, and thus enhance public health. Small regulatory RNAs (sRNAs) represent a poorly understood area of gene regulation in GAS and related pathogens. The specific hypothesis is that sRNAs represent a major constituent of the global regulatory networks coordinating GAS virulence. This hypothesis is based upon the following observations. First, sRNAs regulate virulence in some bacterial pathogens. Second, a bioinformatic analysis indicates that GAS encodes 56 putative sRNAs. Third, two sRNAs have been experimentally verified in GAS and both, through as-yet-unknown mechanisms, appear to regulate virulence factor expression. Our hypothesis will be tested by the following specific aims: 1. Test the hypothesis that sRNAs are prevalent in GAS and conserved across streptococcal species. Whether sRNAs represent a major mechanism of regulation in GAS will be investigated through use of a custom Affymetrix microarray to detect sRNAs transcribed under a series of experimental conditions mimicking human infections, including both ex vivo and in vivo disease models. Completion of this specific aim would represent the first genome-wide analysis of sRNAs within a pathogen from the order Lactobacillales, which includes the streptococci and enterococci, and stimulate sRNA research in a spectrum of human bacterial pathogens. 2. Test the hypothesis that sRNAs regulate GAS virulence. Candidate sRNAs will be triaged based upon sequence conservation across Lactobacillales pathogens, and also upon sRNA expression patterns during in vivo growth. Isogenic sRNA mutant strains will be constructed for 12 conserved sRNAs and assayed in vitro and ex vivo for altered virulence characteristics. Strains with altered virulence characteristics will subsequently be tested for attenuated virulence in two mouse models of invasive disease. Completion of this specific aim would identify sRNAs critical for normal progression of GAS infections, and provide impetus to delineate the pathways by which these regulators function, with the ultimate goal of inhibiting these pathways through use of novel antimicrobials. PUBLIC HEALTH RELEVANCE: Each year in the U.S. there are ~30 million cases of GAS pharyngitis. The proposed research would provide insight into a fundamental and yet understudied field of gene regulation in GAS and related pathogens, and provide the research community with a detailed blueprint of sRNA locations and expression patterns to stimulate further study. Public health may be enhanced through the long-term goal of translating knowledge of sRNA virulence-regulating pathways into new treatment and/or preventative regimes based upon the inhibition of these pathways by novel antimicrobial agents.