Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RII! and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtiis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikficus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.Listena monocytogenes is a gram-positive facultative intracellular bacterium which can cause severe food-borne infections in humans and animals (16). Virulent strains of L. monocytogenes are able to survive and multiply within host macrophages and can invade, replicate, and multiply in nonprofessional phagocytes such as mouse 3T6 fibroblasts (35) and the human colon carcinoma cell line Caco-2 (23). A pathway of entry into epithelial cells is mediated by internalin, an 80-kDa surface protein which has been identified recently (22). After uptake, listeriae escape from the phagosome via the action of a cytolytic toxin, listeriolysin, which is an essential virulence factor (23,35,43). In the host cell cytoplasm, the bacteria replicate, become coated with actin filaments, and spread intracellularly and from cell to cell by forming a tail of polymerized actin (40,52,53). Other L. monocytogenes virulence factors with less well-defined roles include a phosphatidylinositol-specific phospholipase C, a metalloprotease, a lecithinase, and ActA, a gene product involved in actin polymerization (12, 13, 31, 38, and 57; for review, see reference 42). The genes for these virulence factors are located in a gene cluster and are positively regulated at the transcriptional level by the regulatory protein PrfA (39).We have previously described spontaneously occurring rough mutants of L. monocytogenes which produce greatly reduced levels of a 60-kDa major extracellular protein termed p60 (34). These mutants show a rough colony morphology and form long chains of cells separated by double septa (34). Rough mutants have reduced virulence in the * Corresponding author.

Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

We have used RT-PCR and GFP-mediated uorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more eciently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s).

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an ''escape'' step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfAregulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells. M any pathogenic bacteria are able to trigger their uptake by mammalian cells, which is followed by efficient multiplication of the internalized bacteria inside of the host cells. Internalization of these bacteria involves normal phagocytosis when the host cells are professional phagocytes, e.g., macrophages, or triggered phagocytosis in the case of nonprofessional phagocytic host cells, such as epithelial cells, hepatocytes, fibroblasts, and endothelial cells (1, 2). After internalization, most intracellular bacteria reside and replicate inside membrane-bound vacuoles that are specifically modified by the different bacteria (3, 4). Salmonella enterica, Legionella pneumophila, members of the Mycobacterium tuberculosis complex, Mycobacterium leprae, Brucella spp., Chlamydia, Rhodococcus equi, and several others belong to this group of intracellular bacteria. A smaller group of intracellular bacteria, including Shigella spp., the closely related enteroinvasive Escherichia coli (EIEC), Listeria monocytogenes, Listeria ivanovii, and Ricksettia spp., can escape from the primary phagosome into the host cell cytosol where the bacteria proficiently replicate. These latter bacteria synthesize specific proteins that disrupt the phagosomal membrane, thus allowing bacterial entry into the cytosol. In L. monocytogenes, the required proteins are best characterized and comprise the pore-forming lysteriolysin (LLO) and two phospholipases C, PlcA and PlcB (5, 6).It has been reported that the introduction and expression of the listerial hly gene (encoding LLO) in Bacillus subtilis leads to the release of these avirulent bacteria into the cytosol of mammalian cells wher...