GAS Switch from Colonizing Bacterium to Invasive Pathogen

描述（由申请人提供）：GAS是一种在人类宿主中引起广泛疾病的卓越革兰氏阳性细菌病原体。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定义了抵抗宿主先天免疫清除机制的能力，该机制通常用于防止上皮表面以外的传播。在过去的30年里，GAS M1 T1血清型的克隆已经在全球范围内传播，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那些GAS和宿主因素，这些因素解释了这种重要的宿主-病原体相互作用的不同结果，使用侵入性M1 T1 GAS克隆作为模型。我们的方法已经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、敲除和人转基因小鼠系）相结合。我们假设GAS感染的结果是由这些GAS毒力因子的作用和调节决定的，这些毒力因子响应于宿主先天免疫施加的选择性压力。在这个提议中，我们将定义细菌毒力因子的库，这些因子促进GAS M1 T1菌株响应先天免疫选择而转变为侵袭性疾病表型。在目标1中，我们将测试一个独特的和广泛的面板的等基因M1 T1 GAS毒力因子突变体的中性粒细胞耐药性，侵袭性表型转换，和全身感染的人源化纤溶酶原小鼠，定义那些先天免疫耐药因子所需的全身毒力。同时，我们将组成型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。在目标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 M1 T1施加选择性压力，有利于向侵袭性表型转变。在目标3中，我们将确定特定的M1 T1 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期间对GAS适应性的贡献。以这种方式，我们将确定竞争的选择压力所面临的专性人类病原体在其整体生态的不同阶段。最后，我们将评估我们的实验模型（自然医学2007年），收购噬菌体？编码DNA酶Sda 1的M1 T1 Z在侵袭性M1 T1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。最后一个目标将通过探索来实现。M1 T1 Z转导机制，美国和高地方性GAS疾病地区（澳大利亚北领地）不同M血清型菌株中的噬菌体分布，并研究噬菌体对非M1 T1菌株背景中疾病转换和侵袭性疾病的贡献。 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“链球菌性咽喉炎”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获得GAS细菌而不会出现症状。我们正在研究GAS细菌如何从我们正常植物群的无辜成员转变为入侵性病原体，使用分子遗传技术，免疫功能测定和小鼠感染模型。

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.