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Francisella tularensis is a highly infectious pathogen that infects animals and humans to cause the disease tularemia. The primary targets of this bacterium are macrophages, in which it replicates in the cytoplasm after escaping the initial phagosomal compartment. The ability to replicate within macrophages relies on the tightly regulated expression of a series of genes. One of the most commonly used means of coordinating the regulation of multiple genes in bacteria consists of the association of dedicated alternative sigma factors with the core of the RNA polymerase (RNAP). In silico analysis of the F. tularensis LVS genome led us to identify, in addition to the genes encoding the RNAP core (comprising the [alpha]1, [alpha]2, [beta], [beta]' and [omega] subunits), one gene (designated rpoD) encoding the major sigma factor [[sigma].sup.70], and a unique gene (FTL_0851) encoding a putative alternative sigma factor homologue of the [[sigma].sup.32] heat-shock family (designated rpoH). Hence, F. tularensis represents one of the minority of bacterial species that possess only one or no alternative sigma factor in addition to the main factor [[sigma].sup.70]. In the present work, we show that FTL_0851 encodes a genuine [[sigma].sup.32] factor. Transcriptomic analyses of the F. tularensis LVS heat-stress response allowed the identification of a series of orthologues of known heat-shock genes (including those for Hsp40, GroEL, GroES, DnaK, DnaJ, GrpE, CIpB and CIpP) and a number of genes implicated in Francisella virulence. A bioinformatic analysis was used to identify genes preceded by a putative [[sigma].sup.32]-binding site, revealing both similarities to and differences from RpoH-mediated gene expression in Escherichia coil Our results suggest that RpoH is an essential protein of F. tularensis, and positively regulates a subset of genes involved in the heat-shock response.

Listeria monocytogenes is a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome of L. monocytogenes EGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (Imo1272 and Imo1273), encoding proteins sharing significant similarities with the YIqF and RnhB proteins, respectively, of Bacillus subtilis. Imo1273 could complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation of Imo1273 provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of the Imo 1273 mutant in target organs was significantly impaired. However, the mutation did not impair L. monocytogenes intracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with an Imo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-N-acetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent.

Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91--99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K1 03A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope ([DELTA]91-99), as well as the substitution of two, three or four of the four lysine residues (K1 03 to K1 06) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91-99 region in the production of a stable and functional LLO. L[D.sub.50] determinations in the mouse model suggest a possible link between LLO stability and virulence.

The role of type I signal peptidases (SPases I) is to remove the signal peptides of preproteins exported by the general secretory pathway. The genome of Listeria monocytogenes contains a locus encoding three contiguous SPases I (denoted SipX, SipY and SipZ). The authors recently showed that SipX and SipZ perform distinct functions in protein secretion and bacterial pathogenicity. Here, the regulation of sip gene expression in broth and in infected eukaryotic cells was studied. The results show that expression of the three sip genes is (i) controlled by two distinct promoter regions that respond differently to growth phase and temperature variations, and (ii) influenced by PrfA (the transcriptional activator regulating most of the virulence genes of L. monocytegenes) and the stress proteins ClpC and ClpP. It was found that sip gene expression was strongly upregulated upon infection of eukaryotic cells when bacteria were still entrapped in the phagosomal compartment. This upregulation is compatible with the need of L. monocytogenes to optimize its production of virulence factors in the early stage of the intracellular cycle.

Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The [DELTA]srtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.

Research has been conducted on the listeriolysin O, the virulence factor secreted by the pathogenic Listeria monocytogenes acting as a pore-forming cytolysin. The truncated listeriolysin O proteins have been secreted into the L. monocytogenes culture supernatant to bind to cell membranes and the results of the experiment are reported.

Research has been conducted on the bacteriophage lambda that interacts with cell-surface receptor LamB. The fusion of the C-terminal portion of the tail fiber protein of lambda to maltose-binding protein is described.

The levanase operon of Bacillus subtilis was found to be also inducible by mannose and to enhance mannose uptake in B. subtilis as well as in Escherichia coli. Also, B. subtilis cytoplasmic components IIC(super Fru) and IID(super Fru) could substitute for their E. coli mannose homologs to permit phage lambda DNA injection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not need the mannose transporter for infection.

Topological analysis of an integral membrane protein LamB from Escherichia coli porin has detected nine external loops, five of which were identified by random and directed epitope tagging. Data confirmed four periplasmic loops that have been predicted in previous studies. A membrane protein topological model has been developed to represent the topology of the E. coli outer membrane protein.

A gene cluster in the Escherichia coli chromosome's 67.4-76.0 min region was revealed via systematic genome sequencing. Five genes encode homologues of the phosphotransferase system's mannose permease. Other genes encode a putative N-acetylgalactosamine 6-phosphate metabolic pathway that includes a deacetylase, an aldolase, a putative carbohydrate kinase and an isomerizing deaminase. A transcriptional regulatory protein is homologous to DeoR family members. Results suggest that the aldolase-encoding gene found in the cluster is the previously designated kba gene that encodes tagatose-1,6-biphosphate aldolase.

LamB, the integral outer membrane protein and cell receptor site for bacteriophage lambda in E. coli K12, is a 42-amino acid polypeptide. Residue 151 of LamB forms the accessibility gate closest to the phage binding site. The residue at site 151 affects changes in the entry of phage to the binding site according to the size of the substitute side chain. The phage block by the accessibility gate is relieved by a series of amino acid changes in the distal part of J genes of extended host-range mutants of lambda.

J protein mutated residues maybe involved in overcoming the LamB mutation-induced phage adsorption blockage. The J protein distal part contains seven residues involved in lambda mutants host range properties, indicating the participation of the final C-terminal region in the phage adsorption on LamB. The two-step extended host range mutants exhibit double or triple point mutations at the J protein C-terminal.

Fifteen enterobacterial species were studied in terms of the immunological relatedness of LamB proteins by using antibodies against E. coli K12 and K. pneumoniae LamBproteins. A maltose-inducible protein related to E. coli maltoporin was detected by anti-LamBE.c. serum in all Mal+ enterobacteria. Only one protein was not recognized by anti LamBK.p. serum. Peptide regions E 2, E 5, E 3 and E 4 were highly variable and also antigenetically polymorphic. Except for poor recognition in species least related to E. coli, no correlations were observed between the pattern of antibody recognition and phylogenetic relationships betweens strains.

Abstract Background Regulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far. Results We have used a combination of experimental assays and in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen. Conclusion Our findings suggest that F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend.

Abstract Background Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested. Results We show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin. Discussion Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic F. tularensis ssp tularensis, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating F. tularensis infection.