Listeria monocytogenes B Contributes to Invasion of Human Intestinal Epithelial Cells

The gram-positive facultative intracellular food-borne pathogen Listeria monocytogenes is associated with serious invasive infections in humans and animals (10). To establish a foodborne bacterial infection, a pathogen must survive rapidly changing environmental conditions encountered in the gastrointestinal tract, such as exposure to bile salts, organic acids, and changing osmolarity. Survival of extreme and rapidly changing conditions requires timely and appropriate alterations in bacterial gene expression and protein activity. At the transcriptional level, these alterations are often controlled by alternative sigma factors and the catalytic core of RNA polymerase (24). Alternative sigma factors differentially associate with the core polymerase and essentially reprogram promoter recognition specificities of the enzyme, thus allowing expression of new sets of target genes. For L. monocytogenes, the alternative sigma factor B (encoded by sigB) contributes to bacterial resistance to environmental stress conditions, such as reduced temperature, oxidative stress, carbon starvation, and low pH (2, 3, 12, 13, 39), as well as to virulence in the mouse (29, 39). L. monocytogenes is capable of survival and growth under widely varying environmental conditions (11). Further, this organism is able to invade and multiply in a wide range of professional and nonprofessional phagocytic mammalian cells (38). Two cell wall-anchored proteins, internalin A (InlA) and internalin B (InlB), are necessary for efficient invasion of human nonprofessional phagocytic cells (7, 16). The intracellular infectious cycle is characterized by escape from the primary vacuole, intracytosolic replication, actin-based motility, and direct spread to neighboring cells, where a new cycle initiates (17, 36). Several virulence factors responsible for these cellular events have been identified. A pore-forming hemolysin (listeriolysin O) (17), along with two phospholipases (PI-PLC and PC-PLC) (6, 37), is involved in bacterial escape from vacuoles, and the ActA surface protein is responsible for F-actin polymerization and intracellular bacterial motility (21). A DNA binding protein, PrfA, controls expression of these virulence genes (5, 8, 22, 23, 25, 33), but the precise mechanism of regulation is not well understood. prfA is transcribed from three independent promoters, PplcA, P1prfA, and P2prfA (15). PrfA has been suggested to exist in functionally active or inactive forms, depending on environmental conditions (5, 19, 27, 31, 32), and to interact differently at various target promoters (15, 40). The presence of -dependent promoters upstream of prfA (P2prfA [29]) and inlA (P4inlA [20]) suggests that B contributes to L. monocytogenes virulence. Further, Milohanic et al. (28) have reported that a number of PrfA-regulated genes have putative -dependent promoters. In the present study, the role of B in infection of human intestinal epithelial cells was investigated. B contributes to early stages of infection in human intestinal epithelial cells. Intracellular growth of the wild-type L. monocytogenes strain 10403S (4) as well as sigB (FSL A1-254) (39) and P2prfA (DP-L1957) (14) isogenic mutant strains was examined in two intestinal epithelial cell lines, Henle-407 and Caco-2. The assay was performed as previously described (30). Briefly, bacteria were grown at 30°C overnight in brain heart infusion (BHI) broth (with shaking at 250 rpm) and washed in phosphate-buffered saline (PBS) before infection. Host cell infections were performed at 37°C in a 5% CO2 atmosphere. Monolayers of host cells on glass coverslips were infected with a multiplicity of infection (MOI) of approximately 30 and 3 for Henle-407 and Caco-2 cells, respectively. Cells were washed at 1 h postinfection, and gentamicin was added at 1.5 h postinfection. Gentamicin concentrations were experimentally optimized to 150 g/ml (Henle-407) or 50 g/ml (Caco-2) to achieve effective killing of extracellular bacteria within 30 min. The number of intracellular bacteria per coverslip was determined at various time points during infection by plating appropriate serial dilutions of infected host cell lysates onto Luria-Bertani (LB) agar plates. Intracellular doubling times were evaluated by one-way analysis of variance (ANOVA), and individual comparisons were made with Bonferroni’s multiple comparison test to determine whether a mean doubling time for one strain was significantly different from those for other strains. Intracellular growth rates were not significantly different among the various strains tested (Fig. 1; Table 1). However, at 2.5 h postinfection intracellular sigB numbers were consistently 2.0to 2.5-fold lower than those of the wild-type strain in both cell lines. Intracellular P2prfA * Corresponding author. Mailing address: Department of Microbiology and Immunology, VMC 5-169, Cornell University, Ithaca, NY 14853. Phone: (607) 253-3273. Fax: (607) 253-3384. E-mail: hm72 @cornell.edu.