The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell‐to‐cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium‐host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine‐specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.

The prfA gene ofListeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatOdylfiititol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L.monocytogenes.

We have isolated, by molecular cloning and

The expression of listeriolysin, a major virulence factor of the gram-positive facultative intracellular pathogen Listeria monocytogenes, is positively regulated by a transcriptional activator, the prfA gene product. We had previously shown that mutations within the prfA gene lead to loss of listeriolysin production. In The gram-positive facultative intracellular bacterium Listeria monocytogenes is an opportunistic pathogen, particularly in immunocompromised persons, pregnant women, and newborns (32). The consumption of contaminated food has been shown to be a significant infection route for human listeriosis, and several recent epidemics have underscored the significance of L. monocytogenes in food-borne infections (8, 19, 31). Listerial infections can result in septicemia, meningitis, meningoencephalitis, and death. The virulence of L. monocytogenes is attributed to its ability to survive and replicate in cells of the infected host (20). Following oral uptake of infected food, L. monocytogenes is able to cross the intestinal barrier and spread to the internal organs, where it causes the formation of granulomatous foci. In severe infections, the endothelial barriers to the brain or the placenta may be bridged. Hence, the intruding bacterium must possess an array of factors that permit invasion and assist and promote evasion from immune defenses.The best-characterized listerial virulence factor necessary for intracellular survival is listeriolysin, a hemolytic cytolysin, that allows the invading bacterium to escape from the phagosome by lysing the phagolysosomal membrane (9, 11, 27). We have recently localized the listeriolysin gene (lisA) in a cluster of genes that is uniquely present in the pathogenic species L. monocytogenes. This region includes the mpl gene encoding a metalloprotease and a gene encoding a phosphatidylinositol-specific phospholipase C (pic) located downstream and upstream of the listeriolysin gene (lisA), respectively (5, 17). Mutations within any one of these genes lead to a loss of virulence of L. monocytogenes (4,9,11,13 Plasmids. Plasmids pMG14 and pGK20 were obtained from J. Kok

A cloned cytolytic determinant from the genome of Bacillus cereus Bacillus cereus, a common soil saprophyte, has been recognized as an opportunistic pathogen of increasing importance (reviewed in reference 41). Although food-borne gastroenteritis is the most common malady attributed to B. cereus (41), the most devastating is B. cereus endophthalmitis (1, 4, 17). B. cereus elaborates a variety of extracellular membrane-active enzymes and cytolytic toxins. These membrane-active proteins include a phospholipase C (34), sphingomyelinase (22), phosphatidylinositol phospholipase C (23), cereolysin (7; a cytolysin of the streptolysin 0, thiol-activated class), and a second, heat stabile cytolysin about which little is known (8, 10, 37). Phospholipase C, sphingomyelinase, and cereolysin have been highly purified and used in studies of membrane structure (6,29,43) and in studies on the evolution of cytolysins produced by diverse genera of gram-positive bacteria (13,38). Phospholipase C is a Zn metalloenzyme of 23,000 daltons (34) which shows a high degree of stability in chaotropic agents and surfactants (27,28). The sphingomyelinase produced by B. cereus is a protein of between 41,000 and 23,300 daltons, depending on the method of analysis used (40), and requires divalent cations for activity (21).As a first step in determining the contribution that extracellular membrane-active proteins make to the ecology and virulence of B. cereus, a gene bank was established. Identification of a cloned cytolytic determinant from this B. cereuls GP-4 library has been reported (25). To gain insight into the relationship of the cloned cytolysin determinant to membrane-active proteins of B. cereus, nucleotide sequence and enzyme activity analyses were undertaken. The results reported here show that the cytolytic determinant cloned from B. cereus is composed of tandemly arranged genes for two distinct protein products, the activities of both being required to effect target cell lysis (hemolysis as tested). Moreover, the individual cytolysin components possess * Corresponding author. phospholipase C and sphingomyelinase activity, respectively. These data suggest that although the sphingomyelinase and phospholipase C of B. cereus have been studied in detail individually, their function in nature appears to be as a cytolytic unit representing the heat-stabile hemolysin previously observed. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in these experiments are listed in Table 1. Escherichia coli strains were routinely cultured in 2XYT medium (31) with aeration. Bacillus subtilis cultures were grown in HGP broth as previously described (25). Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was incorporated into liquid and solid (1.2% agar) media at concentrations of 10 ,ug/ml for selection of resistant B. subtilis and E. coli strains. Ampicillin (Sigma) was used at 100 pRg/ml to select for recombinants cloned into the vectors pUC8,45). In addition, to screen for insertional ina...