Nutrient limitaton is a critical signal in Salmonela virulence gene regulation. The katF (rpoS) gene mediates the expression of the Salkionella spy p i virulence genes during bacterial starvation. A katF Salmonella mutant has Increased su bilit to nutrient deprivation, oxidative stress, acid stress, and DNA damage, conditions which are relevant to the intraphagnal environment of host macrophages. Moreover, the katF mutant has g ntly reduced virulence in mice. katF encodes an alternative a factor of RNA polymerase which coordinately regulates Sabnonela viruence.

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.Helicobacter pylori, the primary etiological agent of gastritis, colonizes both the surface of gastric epithelial cells and the mucus gel layer. However, several recent studies have also identified H. pylori in water distribution systems (18,29,34,38), surface wells or groundwater (16), rivers (11), and wastewater systems (17,25,29,30) in several countries. In addition, H. pylori has been found existing on the inner surface of water storage earthenware in Gambia (3). While the mechanism of transmission of H. pylori is thought to be primarily by the fecal-oral route, the possibility that it may exist as a biofilm (most likely mixed species) on surfaces exposed to water may provide another route of infection.Biofilms have been intensively studied as communities of bacteria able to survive in environments unfavorable to planktonic (free-living) bacteria (7,24). In response to quorumsensing signals, bacteria migrate and adhere to a surface, divide to form microcolonies, and expand laterally and vertically (8,40,48). Channels are formed in the biofilm to allow nutrients to reach bottom layers of bacteria. The mature biofilm is more resistant to killing by biocides and antibiotics, presumably due to the differentiation and survival of persister cells or slow growth rate (42). To further expand, sections of the biofilm shear off for colonization elsewhere (6).Two studies have alluded to the ability of H. pylori to form a biofilm. A polysaccharide-containing biofilm has been observed at the air-liquid interface when H. pylori is grown in a glass fermenter (43). H. pylori is als...

A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, SalmoneUa minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. The outer membrane proteins (OMPs) of gram-negative bacteria provide specialized functions enabling the organisms to interact with the environment, promoting survival. For pathogenic bacteria, a number of specific proteins required for virulence are located in the outer membrane. Gram-negative organisms causing invasive disease must resist the bactericidal action of complement present in normal human serum (22,46). The outer membrane is the initial site of interaction between complement and the bacterial cell, and both the structure of the lipopolysaccharide (LPS) and the presence of specific OMPs have been shown to be critical determinants of bacterial resistance to complement (22). In Salmonella spp., the number and length of the LPS side chains (O antigen) play a major role in complement resistance, since long-chain LPS leads to complement activation at a distance from the cell surface and precludes insertion of the membrane attack complex in the outer membrane (23, 24).Several genes encoded by Salmonella virulence plasmids have been implicated in serum resistance. These plasmids, 50 to 100 kb in size, are present in several invasive Salmonella serotypes, and all appear to contain a common 8.4-kb region required for progressive infection of the reticuloendothelial system, leading to septicemia and death in experimental animals (14). Genetic loci specifying serum resistance map outside this core virulence region and include a traT allele (42), a determinant affecting LPS structure (26), and the rsk region, which enhances the ability of smooth S. typhimurium to grow in serum when the virulence plasmid is integrated in the chromosome (50, 51).A distinct complement resistance locus was cloned from the S. typhimurium virulence plasmid by J. Hackett and colleagues (15). These workers showed that pADE016, containing a 2.4-kb ClaI-PstI fragment in a multicopy cosmid vector, confers high-level complement resistance to a serumsensitive Escherichia coli K-12 strain and to the rough, plasmid-cured S. typhimurium derivative of the wild-type parent strain (15). In addition, they found that expression of the serum resistance phenotype by pADE016 was associated with the production of an OMP. We have recently investigated the mechanisms by which this locus (designated rck, for resistance to complement killing) mediates serum resistance in gram-negative bacteria (17). These studies suggest that the rck gene product prevents complement-mediated bacteriolysis by inhibiting effective insertion of the terminal C5b-9 membrane attack complex into the gram-negative outer membrane, a mechanism of complement resistance similar to that noted in serum-resistant strains of Neisseria gonorrhoeae (2...

To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we have cloned the gene that encodes the two major surface-labeled trophozoite protein species. Sequence analysis of this gene reveals a single open reading frame specifying a hydrophilic, cysteine-rich (11.8%) protein of 72.5-kDa molecular mass with an amino-terminal signal peptide and a postulated hydrophobic membrane-spanning anchor region near the carboxyl terminus. Most of the cysteine residues (58 of 84) are in the motif Cys-Xaa-Xaa-Cys, which is dispersed 29 times throughout the sequence. Antibodies against the recombinant protein react with the entire surface of live trophozoites, including flagella and adhesive disc. These antibodies inhibit trophozoite attachment, prevent growth, and immunoprecipitate the major =66-and 85-kDa proteins from surface-labeled live trophozoites. The recombinant Escherichia coli also expresses polypeptides of -66-and 85-kDa molecular mass, which are not fusion proteins. This suggests that the processing and/or conformational changes that lead to production of these two peptide species in E. coli reflect those that occur in Giardia. The abundance of cysteine residues suggests that the native proteins on the parasite surface may contain numerous disulfide bonds, which would promote resistance to intestinal fluid proteases and to the detergent activity of bile salts and would help to explain the survival of Giardia in the human small intestine.

The virulence properties of various non-typhoid Salmonella serotypes depend on the presence of large plasmids 60-100 kb in size. We have shown previously that the virulence region on the 80 kb plasmid pSDL2 of Salmonella dublin Lane maps within a 14kb SalI fragment. In this report we show that an 8.2 kb region within this fragment is sufficient to express lethal disease in BALB/c mice. Sequence analysis of this segment revealed six sequential open reading frames designated vsdA-F, which encode putative proteins of 13-65kDa. Deletion analysis and location of Tn5-oriT inserts which abolish virulence suggest that vsdA, vsdC, vsdD and vsdE are essential for virulence expression. Downstream of vsdF we discovered a locus involved in stable plasmid maintenance. Deletion of that region resulted in plasmid multimerization and instability.