GAS Switch from Colonizing Bacterium to Invasive Pathogen

描述（由申请人提供）：GAS是一种卓越的革兰氏+细菌病原体，在人类宿主中引起广泛的疾病。虽然GAS遭遇的常见结果是无症状定植或自限性粘膜感染，但特定GAS菌株在其他健康个体中产生全身感染的倾向决定了其抵抗宿主先天免疫清除机制的能力，而宿主先天免疫清除机制通常起着防止传播超出上皮表面的作用。过去30年来，GAS M1T1血清型的克隆在全球范围内传播，是包括坏死性筋膜炎在内的侵袭性感染的主要原因。我们的实验室采用了多方面的方法来理解那些解释这种重要的宿主-病原体相互作用的不同结果的GAS和宿主因素，使用侵袭性M1T1 GAS克隆作为模型。我们的方法将候选毒力因子基因的精确、靶向诱变和异源表达与疾病发病机制的体外、离体和体内模型（包括WT、基因敲除和人转基因小鼠系）结合起来。我们假设GAS感染的结果是由这些GAS毒力因子对宿主先天免疫施加的选择压力的反应和调节所决定的。在本研究中，我们将定义在先天免疫选择下促进GAS M1T1菌株向侵袭性疾病表型转变的细菌毒力因子库。在Aim 1中，我们将在人源纤溶酶原小鼠中测试一组独特而广泛的等基因M1T1 GAS毒力因子突变体对中性粒细胞耐药、侵袭性表型转换和全身感染的影响，确定那些对全身毒力必要的先天免疫耐药因子。同时，我们将组成表达特定的毒力因子，以确定是否有任何足以促进疾病进展。在Aim 2的补充研究中，我们将使用药理学技术和敲除小鼠来确定宿主先天免疫防御的那些特定方面，这些方面对GAS M1T1施加选择性压力，有利于向侵袭性表型转变。在Aim 3中，我们将确定特异性M1T1 GAS毒力基因和侵袭性表型转移在上皮细胞相互作用和粘膜定植过程中对GAS适应度的贡献。以这种方式，我们将确定这种专性人类病原体在其整体生态的不同阶段所面临的竞争选择压力。最后，我们将评估我们的实验模型的稳健性（Nat Med 2007），获得噬菌体？编码DNase Sda1的M1T1Z在侵袭性M1T1感染的流行中是一个明显的哨兵，通过逃避中性粒细胞胞外陷阱促进对吞噬清除的抵抗。最后一个目标是通过探索实现的吗？M1T1Z的转导机制，噬菌体在美国和GAS高流行地区（澳大利亚北领地）不同M血清型菌株中的分布，以及在非m1t1菌株背景下研究噬菌体对疾病转换和侵袭性疾病的贡献。

DESCRIPTION (provided by applicant): GAS is a preeminent Gram+ bacterial pathogen causing a wide spectrum of diseases in the human host. While the common outcome of a GAS encounter is asymptomatic colonization or self-limited mucosal infection, the propensity of particular GAS strains to produce systemic infection in otherwise healthy individuals defines a capacity to resist host innate immune clearance mechanisms that normally function to prevent dissemination beyond epithelial surfaces. A clone of the GAS M1T1 serotype has spread globally over the last 30 years as the leading cause of invasive infections including necrotizing fasciitis. Our laboratory has adopted a multifaceted approach to understanding those GAS and host factors that explain the diverse outcomes of this important host-pathogen interaction, using the invasive M1T1 GAS clone as a model. Our methodology has coupled precise, targeted mutagenesis and heterologous expression of candidate virulence factor genes with in vitro, ex vivo and in vivo models of disease pathogenesis, including WT, knockout and human transgenic mouse lines. We hypothesize that the outcome of GAS infection is dictated by the action and regulation of these GAS virulence factors in response to selective pressures exerted by host innate immunity. In this proposal, we will define the repertoire of bacterial virulence factors that promote the shift of GAS M1T1 strains to an invasive disease phenotype in response to innate immune selection. In Aim 1, we will test a unique and extensive panel of isogenic M1T1 GAS virulence factor mutants for neutrophil resistance, invasive phenotype switching, and systemic infection in the humanized plasminogen mouse, defining those innate immune resistance factors necessary for systemic virulence. In parallel, we will constitutively express specific virulence factors to identify if any are sufficient to promote disease progression. In the complementary studies of Aim 2, we will use pharmacologic techniques and knockout mice to define those specific aspects of host innate immune defense that exert selective pressure on GAS M1T1 favoring the shift to invasive phenotype. In Aim 3, we will determine the contribution of specific M1T1 GAS virulence genes and invasive phenotype shifting on GAS fitness during epithelial cell interactions and mucosal colonization. In this fashion, we will identify the competing selective pressures faced by this obligate human pathogen during the different stages of its overall ecology. Finally, we will assess the robustness of our experimental model (Nat Med 2007) that acquisition of a phage ?M1T1Z encoding the DNase Sda1 was a sentinel evident in the epidemic of invasive M1T1 infection, promoting resistance to phagocytic clearance through evasion of neutrophil extracellular traps. The last Aim will be achieved by exploring ?M1T1Z transduction mechanisms, phage distribution in diverse M serotype strains in the U.S. and an area of high endemic GAS disease (the Australian Northern Territory), and studying the contribution of the phage to disease switching and invasive disease in non-M1T1 strain backgrounds. PUBLIC HEALTH RELEVANCE: Group A Streptococcus (GAS) is a bacteria that is a leading cause of infections in humans of all ages, from simple "strep throat" to life-threatening "flesh-eating" infections and shock. Serious disease is an unusual outcome, as most people can acquire the GAS bacterium in their throat or on their skin without developing symptoms. We are studying the ways in which the GAS bacteria shifts from an innocent member of our normal flora to an invasive pathogen, using molecular genetic techniques, assays of immune function, and mouse models of infection.