Interaction of Enteroinvasive Pathogens with Neutrophils

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DESCRIPTION (provided by applicant): Shigella, Salmonella and Yersinia spp. infect millions of people worldwide, these related pathogens cause different diseases, many of which can be lethal. Neutrophils play a central role in host defense against invading organisms. Within hours, activated neutrophils migrate to the site of infection where they deploy their granule associated anti-microbial arsenal. We recently identified a key host defense, granule protein; neutrophil elastase (NE) that rapidly and specifically destroys virulence factors of Shigella, Salmonella and Yersinia. It is unclear how NE recognizes and interacts with pathogenic bacteria. We hypothesize that the exposure of surface-bound granule proteins, including NE, of activated neutrophils to bacterial virulence factors is a critical aspect of their defense function and propose the following aims: (1) To identify bioactive NE on the surface of activated neutrophils and determine whether bacterial virulence factors are targeted at the neutrophil surface. NE and other neutral proteases associate with the membrane of activated neutrophils. Since the biological consequences of the interaction of bacterial virulence factors with surface-bound granule proteins of activated neutrophils are unknown we will examine the role of bioactive NE and other proteases on intact neutrophils in the degradation of virulence factors. (2a) Identification of pathogen specific neutrophil granule proteins by "affinity" purification with target bacteria. Role of NE. We have previously observed that the association of NE to the outer envelope of Shigella was more effective with bacteria treated with a crude lysate of neutrophils than with equivalent concentrations of purified NE (unpublished). Based on these observations we predict that targeting of virulence factors by NE is augmented by the preferential binding of granule proteins to Lipopolysaccharide (LPS) of intact bacteria. (2b) To examine the role of LPS specific granule proteins BPI and hCAP18 on NE recruitment and specificity. Specific neutrophil granule proteins such as Bactericidal Permeability Increasing protein (BPI) and hCAP18 have a high affinity for the outer envelope of Gram-negative bacteria. We have previously observed (unpublished) increased binding of purified NE to Shigella in the presence of hCAP18 but not in its absence. We propose that these proteins could potentially "recruit" NE to the bacterial envelope resulting in increased NE specificity.