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.
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