Microbial Response to Neutrophil Phagocytosis

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DESCRIPTION (provided by applicant): Neutrophils are a key component of host defense against bacterial and fungal pathogens. When neutrophils are severely diminished in number or function, the host becomes highly susceptible to serious infection. Neutrophils typically ingest and kill bacteria within a short space of time. In order to gain a better understanding of the bacterial response to phagocytosis, genomic arrays will be used to assess changes in mRNA abundance for Escherichia coli cells ingested by neutrophils. We expect to learn how ingested bacteria prioritize responses to stresses generated in the phagocyte and whether these responses enhance microbial survival. The effects of neutrophils deficient in antimicrobial systems mediated by the phagocyte NADPH oxidase (CGD, chronic granulomatous disease) or the enzyme myeloperoxidase will be compared with normal neutrophils. Initial studies indicate that normal neutrophils, but not CGD neutrophils, evoke an anti-oxidant response mediated by the bacterial transcription factor, OxyR. Disruption of the oxyR gene generated a bacterial strain that was 10-fold more susceptible to killing by normal neutrophils. The oxyR strain also appears to be markedly attenuated for virulence in a mouse model of ascending pyelonephritis. This proposal seeks to characterize oxyR effects more fully, and to explore the effects of selected other major transcriptional responses both to neutrophil phagocytosis and to isolated antimicrobial model systems. It also seeks to implement methodology that will emphasize detection of phagocytosis-induced changes for mRNA transcripts present in a pathogenic E. coli strain but not in a laboratory-adapted strain. The driving hypothesis for the latter goal being that: pathogen-specific transcripts contribute substantially to the pathogenic phenotype. Expression profiling of phagocytosed bacteria appears to provide useful information about conditions in the phagocytic vacuole and about important bacterial defenses mounted in response to this hostile environment.