INTESTINAL M CELLS: INTERACTION WITH ENTERIC PATHOGENS

Children'S Hospital Boston, Boston MA

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Abstract

DESCRIPTION: This project will examine in detail the interactions of enteric microorganisms with epithelial M cells in the intestine, and the early events following M cell transport of pathogens and live vaccines into organized mucosal lymphoid tissues. We will focus on a virus and two bacteria whose interactions with M cells differ dramatically: reovirus type 1, Vibrio cholerae and Salmonella typhimurium. Specific aim 1 seeks to identify M cell surface components that serve as receptors for attachment and transport of these microorganisms. Specific lectins and glycosidase digestion will be used to test the hypothesis that initial recognition events are mediated by specific oligosaccharide epitopes displayed only on M cell apical membranes. Epithelial isolation, cell surface biotinylation, and Western blot overlay methods will be applied to identify the glycosylated or non-glycosylated M cell membrane components that serve as receptors. The role of microbial particle size in limiting access of microorganisms to receptors on M cell apical surfaces will be defined using adhesin-coated inert particles of defined sizes. Whether redistribution or loss of M cell surface components is a prerequisite for close interaction and phagocytosis of V. Cholerae, or membrane ruffling and macropinocytosis of S. typhimurium by M cells, will be investigated by enzyme digestion and EM immunocytochemical analysis at progressive stages of microbial uptake. Specific aim II will focus on V. cholerae as a model for defining the bacterial factors that allow efficient, non-destructive interaction with M cells and antigen-presenting cells: desirable attributes of safe, effective vaccine strains. V. Cholerae mutants lacking defined surface components or enzymes will be tested for alterations in M cell adherence and uptake. The fate of selective V. cholerae candidate vaccine strains in intra-epithelial and subepithelial antigen-presenting cells after M cell transport will be defined and compared. The mucosal and systemic immune responses to the V. cholerae mutants that differ in M cell transport and/or subepithelial cell localization will be analyzed. Finally, the effects of pre-existing specific IgA antibodies on interactions of V. cholerae vaccine strains with M cells and subepithelial cells, and on subsequent mucosal immune responses, will be analyzed. These studies will enhance our understanding of how M cells "select" pathogens for mucosal immune surveillance, how pathogens exploit the M cell transport pathway to cause disease, and how live vaccines and vaccine vectors may be efficiently targeted to the mucosal immune system.