Interaction Of Pathogenic Bacteria With Human Phagocytic

项目目标和目的：有两个具体的项目，1）研究人类吞噬细胞对病原菌的反应机制，2）鉴定和表征病原菌逃避或破坏正常吞噬细胞反应从而导致疾病的特定机制。研究成果和发现：项目1：人类的微生物感染通常伴有急性炎症反应。对抗感染的人类白色血细胞（中性粒细胞或PMN）的正常周转由程序性细胞死亡（凋亡）介导。PMN凋亡对于正常周转和炎症的消退是重要的，因为它防止了如果细胞破裂并溢出其有毒的抗微生物成分则会发生的对健康组织的损伤。1)我们发现了一个遗传程序，在人类中性粒细胞的吞噬作用与细胞凋亡，使用先进的微阵列技术筛选数千个人类基因。我们发现了100多个与程序性细胞死亡相关的基因。关键凋亡基因和相关细胞通路的识别将有助于未来的研究，旨在了解细胞凋亡的过程。2)在随后的微阵列研究中，我们发现凋亡的中性粒细胞在基因表达水平上调节多种代谢途径，从而证明了人类吞噬细胞的基因表达与生物学功能之间的直接相关性。我们假设，全球性的基因表达变化吞噬后，包括在人类中性粒细胞的凋亡分化程序。凋亡分化程序代表转录调节的PMN成熟或造血分化的最后阶段，其通过吞噬作用显著加速。3)利用微阵列技术和流式细胞术，我们发现人中性粒细胞凋亡在基因表达水平下调促炎能力。135个编码促炎因子、信号转导介质、粘附分子和其他促进炎症反应的蛋白质的基因在PMN凋亡过程中下调。这三项研究为解决人类PMN激活后炎症的分子事件提供了新的见解。项目二：A组链球菌（GAS）能成功地逃避中性粒细胞的吞噬和杀伤，引起人类感染，如咽炎、蜂窝织炎和坏死性筋膜炎（食肉综合征），这些感染是全球高发病率和死亡率的原因。我们继续阐明GAS逃避PMN介导的杀伤的机制。1)在与James Musser的合作中，我们发现Sic（链球菌补体抑制剂）抑制PMN吞噬作用，以促进GAS存活和死亡。2)我们进一步阐明了链球菌Mac蛋白（人CD 11b的同源物）阻断PMN吞噬作用的机制。3)最重要的是，我们使用GAS DNA微阵列发现了GAS用于逃避PMN吞噬和杀伤的遗传程序。在这些研究中，我们确定了349个GAS基因，在吞噬细胞与人中性粒细胞的相互作用过程中差异调节。我们发现11种新的GAS分泌蛋白在PMN吞噬过程中上调，并发现GAS中以前未表征的双组分基因调控系统有助于免疫逃避，以促进GAS存活并导致人类疾病。

Project Goals and Objectives: There are two specific projects which 1) investigate human phagocyte response mechanisms to pathogenic bacteria, and 2) identify and characterize specific mechanisms used by pathogenic bacteria to evade or subvert normal phagocyte responses and therefore cause disease. Research Accomplishments and Discoveries: PROJECT 1: Microbial infections in humans are generally accompanied by an acute inflammatory response. Normal turnover of human white blood cells that fight infection (neutrophils or PMNs) is mediated by programmed cell death (apoptosis). PMN apoptosis is important for normal turnover and resolution of inflammation because it prevents damage to healthy tissues that would otherwise occur if cells were to rupture and spill their toxic anti-microbial components. 1) We discovered a genetic program that links phagocytosis in human PMNs with apoptosis using cutting edge microarray technology to screen thousands of human genes. We identified more than one hundred genes relevant to programmed cell death. Identification of key apoptosis genes and those involved in associated cellular pathways will facilitate future research directed toward understanding the process of apoptosis. 2) In subsequent microarray studies, we discovered that apoptotic PMNs regulate multiple metabolic pathways at the level of gene expression, thereby demonstrating a direct correlation between gene expression in human phagocytes and biological function. We hypothesize that global changes in gene expression following phagocytosis comprise an apoptosis differentiation program in human PMNs. The apoptosis differentiation program represents a final stage of transcriptionally regulated PMN maturation or hematopoietic differentiation, which is accelerated significantly by phagocytosis. 3) Using microarray technology and flow cytometry, we discovered that apoptosis in human PMNs down-regulates pro-inflammatory capacity at the level of gene expression. One hundred thirty-five genes encoding pro-inflammatory factors, signal transduction mediators, adhesion molecules, and other proteins that facilitate the inflammatory response were down-regulated during PMN apoptosis. These three studies provide new insight into the molecular events that resolve inflammation following PMN activation in humans. PROJECT 2: Group A Streptococcus (GAS) successfully evades PMN phagocytosis and killing to cause human infections such as pharyngitis, cellulitis, and necrotizing fasciitis (flesh-eating syndrome).These infections are responsible for high morbidity and mortality globally. We continue to elucidate the mechanisms used by GAS to evade PMN-mediated killing. 1) In collaboration with James Musser, we discovered that Sic (streptococcal inhibitor of complement) inhibits PMN phagocytosis to promote GAS survival and diesease. 2) We have eludidated further the mechanmism for blocking PMN phagocytosis by streptoccocal Mac protein, a homologue of human CD11b. 3) Most significantly, we discovered a genetic program used by GAS to evade PMN phagocytosis and killing using a GAS DNA microarray. In these studies, we identified 349 GAS genes that were differentially regulated during phagocytic interaction with human PMNs. We found that 11 novel GAS secreted proteins were up-regulated during PMN phagocytosis, and discovered that a previously uncharacterized two-component gene regulatory system in GAS facilitates immune evasion to promote GAS survvival and cause disease in humans.