Michael J. Mahan

In vivo expression technology for selection of bacterial genes specifically induced in host tissues

We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The system is based on complementation of an attenuating auxotrophic mutation by gene fusion, and it is designed to be of use in a wide variety of pathogenic organisms. In Salmonella typhimurium, we have successfully used the system to identify a number of genes that are induced in BALB/c mice, and that, when mutated, confer a virulence defect. The IVET system has several applications in the area of vaccine and antimicrobial drug development. The technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live attenuated vaccines. In addition, the identification of promoters that are optimally expressed in animal tissues provides a means of establishing in vivo regulated expression of heterologous antigens in live vaccines, an area that has been previously problematic. Finally, we expect that our methodology will be used to uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.

Monoclonal immunoglobulin a prevents adherence and invasion of polarized epithelial cell monolayers by Salmonella typhimurium

BACKGROUND/AIMS Invasion of the intestinal epithelium is considered a critical step in Salmonella pathogenesis. Infection by Salmonella of cultured monolayers of polarized Madin-Darby canine kidney (MDCK) cells has been established as a simple in vitro system that mimics the invasion of intestinal enterocytes in vivo. This study analyzes the protective role of secretory immunoglobulin (Ig) A antibodies against epithelial invasion. METHODS Salmonella typhimurium was applied to MDCK cell monolayers in the presence or absence of a monoclonal, polymeric IgA antibody (Sal4) directed against an antigenic determinant exposed on the surface of wild-type S. typhimurium. RESULTS In the presence of Sal4 IgA, confluent monolayers of MDCK cells were protected against apical invasion by wild-type S. typhimurium but not against a mutant strain that lacks the Sal4 epitope. Protection was Sal4-specific, dependent on the concentration of Sal4 in the apical medium, and occurred at IgA concentrations at which agglutination of IgA-bacterial complexes was observed. When MDCK cell monolayers were formaldehyde-fixed before incubation with Salmonella to prevent bacterial invasion, adhesion of Salmonella occurred in the absence of IgA and in the presence of control IgA but not in the presence of Sal4 IgA. CONCLUSIONS IgA alone can prevent bacterial adherence and invasion of epithelial cells in the absence of other immune or nonimmune protective mechanisms.

Mucosal Immunity: The Role of Secretory Immunoglobulin a in Protection Against the Invasive Pathogen Salmonella typhimurium

Many pathogens gain entry into a host organism by crossing the epithelia of the digestive, respiratory or genital tracts. The main defense against this entry is the mucosal immune system. In the gut mucosa, antigen sampling sites contain organized mucosal lymphoid tissue including lymphoid follicles. These cellular assemblies, which form large aggregates in Peyer’s patches, sample lumenal antigens, resulting in the stimulation of both T cells and B lymphoblasts, committed to IgA synthesis. This leads to the production of secretory antibodies of the IgA isotype. Secretory IgA (slgA) antibodies are thought to act by immune exclusion. That is, slgA prevents the pathogen from contacting the mucosal surface by agglutination, entrapment of immune complexes in the mucus, and clearance by peristalsis (Brandtzaeg, 1989; Childers, et al., 1989; Mestecky, 1988).