Global Genetic Analysis of the Mga Virulence Regulon

描述（由申请人提供）：A群链球菌（GAS）具有在许多宿主组织中存活的能力。重要的是要更好地了解这种病原体能够适应和持续存在于宿主不同部位的机制。GAS细胞利用多种调控途径来感知宿主环境；其中一个途径是Mga毒力调控。在这个应用中，我们希望通过生化和遗传学研究来确定Mga作为毒力调节因子的作用以及它在细胞生理学中的作用。菌株5448-AN，其基因组序列可供我们获取，将作为侵入性和全球传播的M1T1 GAS克隆的代表。为了鉴定对Mga调控重要的基因，我们将使用M1T1 5448- AN基因组中的Pmga-gusA报告等位基因。该菌株将进行饱和转座子诱变，以便使用新开发的基于水手的转座子（OSKAR）建立突变文库，该转座子已在GAS中优化为有效的随机诱变。通过鉴定Mga调控所需的基因，我们可以开始组装基因对Mga转录的直接和间接相互作用。我们还将使用创新的转座子突变体深度测序（Tn-seq）基因组方法，以确定mga基因的遗传相互作用。这将使我们更好地了解Mga毒力调节因子如何促进发病机制和细胞生理学。这将通过使用OSKAR来完成，在野生型和mga 5448-AN背景中创建饱和突变文库。我们将确定用于富培养基（THY）生长的基因的适合度。为了更好地描述我们的研究结果，我们将选择一些与mga相互作用有关的基因进行进一步分析，以确定它们在GAS毒力中的作用。本研究提出的总体目标是定义Mga调控网络的全部范围，以及它如何利用全球遗传分析将临床相关M1T1 GAS的毒力和细胞生理学的不同方面联系起来。这些研究的成功完成将有助于解决GAS发病机制中重要的调控网络之间的复杂性和相互作用。

DESCRIPTION (provided by applicant): The Group A Streptococcus (GAS) has the ability to survive in many host tissues. It is important to have a better understanding of the mechanisms by which this pathogen can adapt and persist at the different sites within the host. The GAS cell utilizes many regulatory pathways in order to sense the host environment; one such pathway is the Mga virulence regulon. In this application, we hope to identify the role Mga plays as a virulence regulator as well as its role in cellular physiology through biochemical and genetic studies. The strain 5448-AN, whose genome sequence is available to us, will be used as a representative of the invasive and globally disseminated M1T1 GAS clone. In order to identify genes that are important for Mga regulation, we will use a Pmga-gusA reporter allele within the M1T1 5448- AN genome. This strain will be subjected to saturating transposon mutagenesis in order to create a mutant library using a newly developed mariner-based transposon (OSKAR), which has been optimized for efficient random mutagenesis within GAS. By identifying genes required for Mga regulation, we can begin to assemble the direct and indirect interactions of genes on the transcription of mga. We will also use the innovative genomic methodology of deep sequencing of transposon mutants (Tn-seq) in order to identify genetic interactions of the mga gene. This will provide us with a better understanding of how the Mga virulence regulator contributes to both pathogenesis and cellular physiology. This will be accomplished by using OSKAR, to create saturated mutant libraries in both wild type and mga 5448-AN backgrounds. We will determine the fitness of genes that are utilized for growth in rich medium (THY). In order to better characterize our findings, a selected number of genes shown to be involved in interacting with mga will be chosen for further analysis to identify their role in GAS virulence. The overarching goal of the work proposed in this fellowship is to define the full extent of the Mga regulatory network and how it links virulence and different aspects of cell physiology in the clinically relevant M1T1 GAS using global genetic analyses. The successful completion of these studies will help to resolve the complexity and interactions between regulatory networks important for GAS pathogenesis. PUBLIC HEALTH RELEVANCE: GAS is a Gram-positive obligate human pathogen responsible for causing a broad spectrum of diseases including benign infections (pharyngitis, impetigo), to invasive diseases (necrotizing fasciitis, streptococcal toxic shock syndrome), as well as post immune sequelae (glomerulonephritis and acute rheumatic fever). It is important to have a better understanding of the mechanisms by which this pathogen can adapt and persist at different host tissues. This application focuses on resolving the complexity behind the Mga regulatory networks associated with GAS pathogenesis.