The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.

The spoOJ gene of BaciUlus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spoOJ is suppressed by a null mutation in the gene located directly upstream from spoOJ, soj (suppressor of spoOJ). These results indicate that Soj inhibits the initiation of sporulation and that SpoOJ antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spoOA. In addition, the temperature-sensitive sporulation phenotpe caused by the fts4279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and SpoOJ are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2. spoOJ was found to be required for normal chromosome partitioning as well as for sporulation. spoOJ null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of SpoOJ could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.The generation of new cells during growth or development requires proper replication, repair, and segregation of chromosomes. Conditions that perturb chromosome replication or damage DNA often inhibit cell cycle progression or development through checkpoint mechanisms. Such regulatory mechanisms are present in organisms as diverse as bacteria, yeasts, and mammals and act to prevent the unproductive formation of cells lacking intact chromosomes.Spore formation by the gram-positive bacterium Bacillus subtilis is a developmental process requiring two different cell types. Each cell type has an intact chromosome and a characteristic and distinct pattern of gene expression. The two cell types are created by formation of an asymmetric division septum early during development. The smaller cell, known as the forespore, develops into the mature spore while enclosed in the larger mother cell (30).One of the most important early events necessary for the initiation of sporulation is the activation of the developmental transcription factor encoded by spoOA. SpoOA is activated by phosphorylation, and SpoOA-P induces expression of several genes, including spoIL4, spoIIE, and spoIIG (4,6,46,47,55,58) that are essential for sporulation and the establishment of cell type-specific gene expression (30). Phosphorylation of SpoOA requires histidine protein kinases, i.e., KinA, -B, and -C (2, 27, 38, 56), and two phospho-transfer proteins, SpoOF and SpoOB (7). The histidine protein kinases autophosphorylate on a histidine residue, and phosphate is transferred to SpoOF, and then from

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

Introductory Paragraph Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin motility process to spread between mammalian cells1. Actin ‘comet tails’ propel Listeria through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which Listeria overcomes cortical tension to generate protrusions is unknown. Here, we identify bacterial and host proteins that directly regulate protrusions. We show that efficient spreading between polarized epithelial cells requires the secreted Listeria virulence protein InlC. We next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP2. InlC promotes protrusion formation by inhibiting Tuba and N-WASP, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells3. We demonstrate that, by inhibiting Tuba and N-WASP, InlC makes taut apical junctions become slack. Experiments with myosin II inhibitors indicate that InlC-mediated perturbation of junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, our results suggest that InlC promotes bacterial dissemination by relieving cortical tension, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions.

The Gram-positive pathogen Listeria monocytogenes induces its own internalization into some non-phagocytic mammalian cells by stimulating host tyrosine phosphorylation, phosphoinositide (PI) 3-kinase activity, and rearrangements in the actin cytoskeleton. Entry into many cultured cell lines is mediated by the bacterial protein InlB. Here we investigate the role of InlB in regulating mammalian signal transduction and cytoskeletal structure. Treatment of Vero cells with purified InlB caused rapid and transient increases in the lipid products of the PI 3-kinase p85-p110, tyrosine phosphorylation of the mammalian adaptor proteins Gab1, Cbl, and Shc, and association of these proteins with p85. InlB also stimulated large scale changes in the actin cytoskeleton (membrane ruffling), which were PI 3-kinase-dependent. These results identify InlB as the first reported non-mammalian agonist of PI 3-kinase and demonstrate similarities in the signal transduction events elicited by this bacterial protein and known agonists such as epidermal growth factor.Infection of viral, bacterial, or protozoan intracellular pathogens often involves subversion of mammalian signaling pathways. The first step in the life cycle of an intracellular pathogen is internalization by the host cell. While some microbial pathogens are taken up only by professional phagocytes (macrophages or neutrophils), others induce their own uptake ("enter") into cells that are not normally phagocytic, such as epithelial cells underlying mucosal surfaces or endothelial cells inside blood vessels (reviewed in Ref. 1). Entry into non-phagocytic cells can permit traversal of tissue-specific barriers and promote survival of the pathogen by providing access to a nutrient-rich environment that is protected from host antibody or complement.Listeria monocytogenes is a food-borne bacterial pathogen that causes severe illnesses leading to meningitis or abortions in immunocompromised individuals or pregnant women (2).This faculative intracellular pathogen enters into cells that are non-phagocytic, including epithelial cells and hepatocytes. Entry into such cells is likely to play an important role in traversing the intestinal, blood-brain, and placental barriers, and in colonization of the liver (reviewed in Ref. 3).Entry of L. monocytogenes into non-phagocytic cells involves specific bacterial surface proteins that exhibit cell tropism. The bacterial protein InlA (internalin) promotes entry into the intestinal epithelial cell line Caco-2, whereas the protein InlB mediates internalization into several other cultured cell lines, including Vero, HEp-2, HeLa, and some hepatocytes (4 -6). InlB has a role in virulence in the mouse model, as a bacterial mutant (⌬inlB) deleted for the inlB gene is defective in colonization of the liver (4, 5, 7). InlA promotes bacterial entry by interacting with its mammalian receptor, the cell-cell adhesion molecule E-cadherin (8). The mammalian receptor for InlB is not known.Entry of L. monocytogenes requires tyrosine phosphorylation and other sig...