GAS Switch from Colonizing Bacterium to Invasive Pathogen

描述(申请人提供)：GAS是一种主要的革兰氏+细菌病原体，在人类宿主中引起广泛的疾病。虽然气体接触的常见结果是无症状的定植或自限性黏膜感染，但在其他健康的人中，特定气体菌株产生系统性感染的倾向定义了抵抗宿主先天性免疫清除机制的能力，该机制通常起到防止扩散到上皮表面以外的作用。在过去的30年里，GAS M1T1血清型的克隆已经在全球传播，成为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这些气体和宿主因素，这些因素解释了这一重要的宿主-病原体相互作用的不同结果，并以侵袭性M1T1气体克隆为模型。我们的方法将候选毒力因子基因的精确、定向突变和异源表达与疾病发病的体外、体外和体内模型相结合，包括WT、基因敲除和人类转基因小鼠品系。我们推测，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性压力的作用和调节决定的。在这项提案中，我们将定义促进GAS M1T1菌株向侵袭性疾病表型转变的细菌毒力因子库，以响应先天免疫选择。在目标1中，我们将测试一组独特的和广泛的等基因M1T1 GAS毒力因子突变体，以检测人源化纤溶酶原小鼠的中性粒细胞抵抗、侵袭性表型转换和全身感染，确定那些对全身毒力所必需的先天免疫抵抗因子。同时，我们将结构性地表达特定的毒力因子，以确定是否有足以促进疾病进展的因子。在AIM 2的补充研究中，我们将使用药理学技术和基因敲除小鼠来定义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的转变。在目标3中，我们将确定特定的M1T1 GAS毒力基因和侵袭性表型转变在上皮细胞相互作用和粘膜定植过程中对GAS适合性的贡献。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(NAT Med 2007)的稳健性，即获得编码DNase Sda1的噬菌体M1T1Z是侵袭性M1T1感染流行的明显哨兵，通过逃避中性粒细胞胞外陷阱促进对吞噬细胞清除的抵抗。最后一个目标将通过探索M1T1Z的转导机制，噬菌体在美国不同M血清型毒株中的分布，以及在非M1T1毒株背景下研究噬菌体在疾病转换和侵袭性疾病中的贡献来实现。 公共卫生相关性：A组链球菌(GAS)是一种细菌，是所有年龄段人类感染的主要原因，从简单的“链球菌喉咙”到危及生命的“食肉”感染和休克。严重的疾病是一种不寻常的结果，因为大多数人可以在喉咙或皮肤上感染这种气体细菌，而不会出现症状。我们正在使用分子基因技术、免疫功能分析和小鼠感染模型，研究气体细菌如何从我们正常菌群中的无辜成员转变为入侵病原体。

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.