Inoculum composition and Salmonella pathogenicity island 1 regulate M-cell invasion and epithelial destruction by Salmonella typhimurium

Abstract

In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S. typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S. typhimurium. Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S. typhimurium. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems.

FIG. 1

SEM images of mouse Peyer’s patch tissues infected for 60 min with wild-type S. typhimurium strain IR715 resuspended in PBS (a and b) or LB (c to f). (a and b) Bacteria in PBS fail to exert a cytotoxic effect. Shown are whole dome (a) and localized area of a dome surface (b), both with an intact FAE. M cells in panel b either display a normal microvillous morphology (M) or possess membrane ruffles (arrow). (c to f) Destruction of FAE by bacteria resuspended in LB. (c and d) Whole domes, with loss of extensive areas of FAE in panel c (arrows) and loss of localized, peripheral regions of FAE in panel d (arrows). (e) Localized region of FAE showing relatively unaffected areas interspersed by damaged regions (arrows). (f) High-power view of FAE, depicting an area of FAE loss (lower left), an intermediate zone of cells denuded of microvilli (arrows), and a relatively unaffected region (upper mid-right). Bacteria are visible in the damaged areas. Bars: a, c, and d, 100 μm; b and f, 10 μm; e, 20 μm.

FIG. 2

CLSM images of mouse Peyer’s patch FAE infected for 15 min with wild-type S. typhimurium strain IR715 suspended in LB and dually stained for M cells (a and c) and bacteria (b and d). (a and b) Surface views. Adhesion of three bacteria to the surface of an M-cell is accompanied by redistribution of M-cell surface UEA1 staining (a, arrow). (c and d) Images at 6-μm depth. A bacterium (d) has invaded an M cell (c, arrow); localization of bacteria within the M cell is facilitated by weak UEA1 staining of the M-cell cytoplasm. Bar = 5 μm.

FIG. 3

SEM images of mouse Peyer’s patch tissues infected for 60 min with wild-type S. typhimurium strain SR11 (a and b) or its isogenic invA mutant SB111 (c and d), both suspended in LB. (a and b) Destruction of FAE by SR11. (a) Whole dome, showing peripheral regions of destruction (arrows); (b) localized region of FAE, depicting an area of FAE destruction (arrows) characterized by cell loss, microvillous denudation and the presence of associated bacteria, and a relatively unaffected area of FAE (upper region). (c and d) The invA mutant SB111 fails to exert a cytotoxic effect. Shown are whole dome (c) and localized area of a dome surface, both with an intact FAE. In panel d, the FAE is composed of enterocytes, a goblet cell (G), and M cells displaying a normal microvillous morphology (M) or surface membrane ruffles (arrows). Bars: a and c, 100 μm; b and d, 10 μm.