Interaction of invasive bacteria with host signaling pathways.

Many bacterial pathogens exploit mammalian cell functions in order to promote their adherence to or uptake by host cells. Recent work has led to the identification of some of the bacterial and mammalian proteins involved in these processes. Although specific mechanisms differ among pathogens, a common aspect appears to be regulation of signaling pathways that control the actin cytoskeleton.

A role for phosphoinositide 3-kinase in bacterial invasion.

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.

Host-pathogen interactions during entry and actin-based movement of Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium that induces its own uptake into mammalian cells, and spreads from one cell to another by an actin-based motility process. Entry into host cells involves the bacterial surface proteins InlA (internalin) and InlB. The receptor for InlA is the cell adhesion molecule E-cadherin. InlB-mediated entry requires activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. Actin-based movement of L. monocytogenes is mediated by the bacterial surface protein ActA. The N-terminal region of this protein is necessary and sufficient for polymerization of host cell actin. Other host proteins involved in bacterial motility include profilin, Vasodilator-Stimulated Phosphoprotein (VASP), the Arp2/Arp3 complex, and cofilin. Studies of entry and intracellular movement of L. monocytogenes could lead to a better understanding of receptor-ligand signaling and dynamics of actin polymerization in mammalian cells.

DNA-related conditions controlling the initiation of sporulation in Bacillus subtilis.

In the Gram-positive bacterium Bacillus subtilis, the developmental process of spore formation occurs in response to nutrient deprivation and requires the generation of two different cell types with distinct programs of gene expression. Entry into sporulation is regulated primarily by activation (phosphorylation) of the transcription factor encoded by spo0A. The phosphorylation state of Spo0A is controlled by a multi-component phospho-transfer pathway and by at least one phosphatase. Recent experiments indicate that several intracellular conditions that decrease the fidelity of chromosome transmission inhibit production of Spo0A approximately P. These conditions include inhibition of DNA replication, DNA damage, and some alterations in the chromosome partitioning and cell division machinery. Coupling accumulation of a critical level of Spo0A approximately P to these conditions seems to serve as a developmental checkpoint to ensure that cells do not attempt to sporulate unless they are able to provide an intact, undamaged chromosome for each of the two cell types needed for sporulation.

Coupling between gene expression and DNA synthesis early during development in Bacillus subtilis.

Endospore formation in the bacterium Bacillus subtilis involves generation of two cell types, each with different developmental fates. Each cell type contains an active chromosome, and treatments that inhibit DNA synthesis at the beginning of development inhibit spore formation. We describe experiments demonstrating that gene expression early during sporulation is coupled to DNA synthesis. Expression of several genes that are induced early during sporulation, before the formation of two cell types, is inhibited when DNA synthesis is inhibited. Genes that are affected require the transcription factor encoded by spo0A for normal induction. Spo0A protein is normally activated early in development by a multicomponent phosphorylation pathway, or phospho-relay. Altered function mutations in spo0A that bypass the need for the phospho-relay allow early sporulation gene expression, even when DNA synthesis is inhibited. These results indicate that inhibition of DNA synthesis prevents activation of the Spo0A transcription factor by inhibiting a step in the phospho-relay. It seems likely that coupling early developmental gene expression to DNA synthesis is a general mechanism to prevent inappropriate or unnecessary gene expression.

Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis.

The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.

A developmental checkpoint couples the initiation of sporulation to DNA replication in Bacillus subtilis.

Spore formation in Bacillus subtilis requires the generation of two distinct cell types, each with an active chromosome that becomes committed to a defined program of gene expression. Here we show that a developmental checkpoint couples the initiation of sporulation, and the subsequent formation of these two cell types, to DNA replication early during development. Inhibiting the initiation of chromosomal replication prevents the onset of sporulation and inhibits expression of several genes that are normally induced early during development. This defect in gene expression is due to inhibition of the multi-component phosphorylation pathway needed to activate the developmental transcription factor encoded by spo0A. The target affected by inhibiting the initiation of replication is neither Spo0A nor the major kinase, KinA, needed for production of Spo0A approximately P. Rather, the target appears to be one of the proteins that transfers phosphate from the kinase to the Spo0A transcription factor. The signal that couples activity of the phosphorelay to the initiation of DNA replication is different from the previously described DNA damage signal that inhibits the phosphorelay during SOS induction in a recA-dependent response. Thus, DNA replication as well as DNA damage signals control production of Spo0A approximately P and initiation of sporulation.

The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase.

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.

Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis.

The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn917 mutation was also found to be an allele of ald. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.

spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis.

The spo0J gene of Bacillus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J). These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A. In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and Spo0J 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, spo0J was found to be required for normal chromosome partitioning as well as for sporulation. spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase.

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.

Possible role for the essential GTP-binding protein Obg in regulating the initiation of sporulation in Bacillus subtilis.

We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.

The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence.

Spore formation in Bacillus subtilis is a dramatic response to environmental signals that is controlled in part by a two-component regulatory system composed of a histidine protein kinase (SpoIIJ) and a transcriptional regulator (Spo0A). The spo0K locus plays an important but undefined role in the initiation of sporulation and in the development of genetic competence. spoIIJ spo0K double mutants had a more severe defect in sporulation than either single mutant. Overproduction of the spoIIJ gene product resulted in the suppression of the sporulation defect, but not the competence defect, caused by mutations in the spo0K locus. On the basis of the phenotype of the spoIIJ spo0K double mutant and the effect of overproduction of the spoIIJ gene product, a transposon insertion in the spo0K locus was isolated. The spo0K locus was cloned and sequenced. spo0K proved to be an operon of five genes that is homologous to the oligopeptide permease (opp) operon of Salmonella typhimurium and related to a large family of membrane transport systems. The requirement for the transport system encoded by spo0K in the development of competence was somewhat different than its requirement in the system encoded by spo0K in the development of competence was somewhat different than its requirement in the initiation of sporulation. Disruption of the last open reading frame in the spo0K operon caused a defect in competence but had little or no effect on sporulation. We hypothesize that the transport system encoded by spo0K may have a role in sensing extracellular peptide factors that we have shown are required for efficient sporulation and perhaps in sensing similar factors that may be necessary for genetic competence.

Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation.

Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Krebs cycle function is required for activation of the Spo0A transcription factor in Bacillus subtilis.

Expression of genes early during sporulation in Bacillus subtilis requires the activity of the transcription factor encoded by spo0A. The active, phosphorylated form of Spo0A is produced through the action of a multicomponent pathway, the phosphorelay. A mutant defective in the first three enzymes of the Krebs citric acid cycle was unable to express early sporulation genes, apparently because of a failure to activate the phosphorelay. Cells that produce an altered Spo0A protein that can be phosphorylated by an alternative pathway were not dependent on Krebs cycle function for early sporulation gene expression. These findings suggest that Krebs cycle enzymes transmit a signal to activate the phosphorelay and that B. subtilis monitors its metabolic potential before committing itself to spore formation.

Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor.

Multiple physiological and environmental signals are needed to initiate endospore formation in Bacillus subtilis. One key event controlling sporulation is activation of the Spo0A transcription factor. Spo0A is a member of a large family of conserved regulatory proteins whose activity is controlled by phosphorylation. We have isolated deletion mutations that remove part of the conserved amino terminus of Spo0A and make the transcription factor constitutively active, indicating that the amino terminus normally functions to keep the protein in an inactive state. Expression of an activated gene product is sufficient to activate expression of several sporulation genes in the absence of signals normally needed for initiation of sporulation. Our results indicate that nutritional, cell density, and cell-cycle signals are integrated through the phosphorylation pathway that controls activation of Spo0A.