Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage.

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24(B) integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24(B) int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24(B) must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24(B) prophage is proposed.

Short communication: Characterization of Shiga toxin 2-carrying bacteriophages induced from Shiga-toxigenic Escherichia coli isolated from Italian dairy products.

Forty samples of raw milk cheese and 25 samples of raw milk itself were subjected to enrichment culture for Shiga-toxigenic Escherichia coli (STEC), and a single Shiga toxin 2- (Stx(2)) positive strain was obtained from one of the cheese samples. Thus, aged cheeses in which the curd is subsequently heat treated (48°C) cannot be presumed to be STEC free. Infective Stx(2) bacteriophages were induced from 3 STEC strains isolated elsewhere from raw milk and 1 STEC strain from aged cheese sourced in Italy. Data on E. coli host range, morphology, genome size, and genetic variation determined by restriction fragment length polymorphism and multi-locus genotyping are presented. Although all 4 bacteriophages were found to be short-tailed Podoviridae, they exhibited considerable variation in both genome size and content. This extended to the Stx(2) genes themselves, whose sequences contained several point mutations, but these did not translate to amino acid substitutions.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Effects of a nanoparticulate silica substrate on cell attachment of Candida albicans.

AIMS: To investigate the influence of silica nanoparticles on the attachment and growth of Candida albicans cells. METHODS AND RESULTS: Spherical silica nanoparticles with diameters of 4, 7, 14 or 21 nm were attached to tissue culture polystyrene by a polycationic binding layer using a simple deposition procedure. The modified surfaces were shown to reduce the attachment and growth of C. albicans cells by a range of different measurements including microscopy, staining cells and measuring the amount of dye taken up and total cell activity measured using a dye reduction assay. For those cells that did attach and grow, the nanoparticle-coated surface inhibited the yeast to hyphal transition that is induced in the presence of serum. The greatest effect was observed for 7 and 14 nm diameter silica particles and we propose that the mechanism for these effects are related to either the topography of the surface or the slow dissolution of the bound silica. CONCLUSIONS: The attachment and growth of C. albicans is reduced by surface modification with silica nanoparticles. SIGNIFICANCE AND IMPACT OF THE STUDY: The modification of surfaces by nanoparticulate coatings is a simple process that may have applications in reducing the prevalence of Candida sp. cells on medical devices thus, limiting the incidence of this pathogenic yeast in clinical environments.

Cloning and characterization of a Prevotella melaninogenica hemolysin.

Hemolysins have been proven to be important virulence factors in many medically relevant pathogenic organisms. Their production has also been implicated in the etiology of periodontal disease. Hemolytic strain 361B of Prevotella melaninogenica, a putative etiologic agent of periodontal disease, was used in this study. The cloning, sequencing, and characterization of phyA, the structural gene for a P. melaninogenica hemolysin, is described. No extensive sequence homology could be identified between phyA and any reported sequence at either the nucleotide or amino acid level. As predicted from sequence analysis, this gene produces a 39-kDa protein which has hemolytic activity as measured by zymogram analysis. Unlike many Ca2+-dependent bacterial hemolysins, both the cloned and native PhyA proteins were enhanced by the presence of EDTA in a dose-dependent fashion with 40 mM EDTA allowing maximum activity. Ca2+ and Mg2+ were found to be inhibitory. The hemolytic activity also was found to have a dose-dependent endpoint. Through recovery of hemolytic activity from a spent reaction, this endpoint was shown to be the result of end product inhibition. This is the first report describing the cloning and sequencing of a gene from P. melaninogenica.

Characterization of TPI gene expression in isogeneic wild-type and gcr1-deletion mutant strains of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the enzymes of glycolysis constitute 30-60 percent of the soluble protein. GCR1 gene function is required for high level glycolytic gene expression. We have undertaken a biochemical and genetic characterization of TPI, a gene affected by gcr1 lesions. Northern analysis showed that steady-state levels of TPI transcripts are severely reduced in gcr1 mutant strains. However, primer extension experiments revealed that TPI transcripts isolated from wild-type and gcr1 mutant strains have identical 5' ends. To map the 5' boundary of TPI controlling region, we employed a TPI::lacZ gene fusion carrying 3.5 kb 5' to the translational start of the TPI structural gene. Nuclease Bal31 deletion analysis demonstrated that sequences sufficient for high level expression of TPI reside within 392 nucleotides preceding the start of the structural gene. We have identified GRF1/RAPI/TUF-binding site positioned 339 to 349 bp 5' to the translation start of TPI. DNA band shift assays were carried out with wild-type and gcr1 deletion mutant strains, and similar patterns of band shifting were observed.

Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches.

OBJECTIVE: To determine whether autoantibodies directed against muscarinic M3 receptors are present in the serum of patients with primary Sjogren's syndrome (pSS), and if so whether these autoantibodies inhibit secretion by intact salivary acinar cells. METHODS: IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca(2+) activity ([Ca(2+)](i)) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3). RESULTS: We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca(2+) signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjogren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca(2+)](i). CONCLUSION: These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.