GAS Switch from Colonizing Bacterium to Invasive Pathogen

摘要 GAS是一种在人类宿主中引起广泛疾病的卓越的革兰氏+细菌病原体。 虽然GAS的常见结局是无症状的定植或自限性粘膜病变， 感染，特定GAS菌株在其他健康人群中产生全身感染的倾向 个体定义了抵抗宿主先天免疫清除机制的能力，该机制通常起作用， 防止扩散到上皮表面以外。GAS M1T1血清型的克隆已在全球传播 在过去的30年中，它是包括坏死性筋膜炎在内的侵袭性感染的主要原因。我们 一个实验室采用了多方面的方法来理解这些气体和主机因素，解释 这种重要的宿主-病原体相互作用的不同结果，使用侵袭性M1T1 GAS克隆作为一种 模型我们的方法将精确的靶向诱变和异源表达相结合， 候选毒力因子基因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。我们假设GAS感染的结果是 由这些GAS毒力因子响应选择压力的作用和调节决定 由宿主的先天免疫力产生在这个建议中，我们将定义细菌毒力因子的库 促进GAS M1T1菌株响应先天免疫应答而转变为侵袭性疾病表型 选择.在目标1中，我们将测试一组独特且广泛的同基因M1T1 GAS毒力因子 在人源化细胞中，中性粒细胞抗性、侵袭性表型转换和全身感染的突变体 纤溶酶原小鼠，定义那些先天性免疫抗性因子所必需的系统性毒力。在 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， 基因敲除小鼠来确定宿主先天免疫防御的那些特定方面， 对GAS M1T1的影响有利于向侵袭性表型的转变。在目标3中，我们将确定 特异性M1T1 GAS毒力基因和侵袭性表型转变对上皮细胞GAS适应性的影响 相互作用和粘膜定植。在这种方式下，我们将确定竞争的选择性压力， 在其整个生态的不同阶段，这种专性人类病原体所面临的挑战。最后我们将 评估我们的实验模型（Nat Med 2007）的鲁棒性，即噬菌体的获得？M1T1Z 编码DNA酶Sda1的基因在侵袭性M1T1感染的流行中是一个明显的哨兵， 通过逃避嗜中性粒细胞胞外陷阱对吞噬清除的抵抗。最后的目标将是 通过探索实现？M1T1Z转导机制，不同M血清型菌株中的噬菌体分布 在美国和地方性GAS疾病高发地区（澳大利亚北领地），并研究 噬菌体对非M1T1菌株背景中的疾病转换和侵袭性疾病的贡献。项目叙述 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致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他们的喉咙或皮肤上获得GAS细菌，而不会发展 症状我们正在研究GAS细菌从我们的无辜成员转移的方式。 正常植物群对入侵性病原体，使用分子遗传技术，免疫功能测定， 感染的小鼠模型。

Abstract 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. Project Narrative 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.