A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes.

Shiga toxin-producing Escherichia coli (STEC) colonizes the human intestine, causing haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Treatment options are limited to intravenous fluids in part because sublethal doses of some antibiotics have been shown to stimulate increased toxin release and enhance the risk of progression to HUS. Preventative antimicrobial agents, especially those that build on the natural antimicrobial action of the host defence, may provide a better option. In order to survive the acid stress of gastric passage, STEC is equipped with numerous acid resistance and DNA repair mechanisms. Inhibition of acid-induced DNA repair may offer a strategy to target survival of ingested STEC. We report here that brief pretreatment with a novel antimicrobial peptide, which was previously shown to inhibit bacterial DNA repair, significantly and profoundly reduces survival of acid-stressed O157 : H7 and non-O157 : H7 STEC seropathotypes that are highly associated with HUS. Reduction in survival rates of STEC range from 3 to 5 log. We also show that peptide/acid treatment results in little or no increase in toxin production, thereby reducing the risk of progression to HUS. This study identifies the peptide wrwycr as a potential new candidate for a preventative antimicrobial for STEC infection.

Hormonal control of plasminogen activator inhibitor-1 gene expression and production in human adipose tissue: stimulation by glucocorticoids and inhibition by catecholamines.

Plasma levels of type 1 plasminogen activator inhibitor (PAI-1), a risk factor for cardiovascular disease, are elevated in obese subjects, especially those with omental fat accumulation. We investigated the hormonal control of PAI-1 gene expression and secretion in cultured human adipose tissue. We more particularly focused on the effects of glucocorticoids, insulin, cAMP, and catecholamines in explants from the omental region. The addition of dexamethasone to the culture medium increased PAI-1 secretion in a time-dependent manner for up to 24 h. The stimulation by the glucocorticoid was preceded by a 2-fold rise in PAI-1 messenger ribonucleic acid levels between 4-8 h of culture. The effectiveness of the glucocorticoid was concentration dependent, with a half-maximal effect within a physiological range. This stimulation was also observed in sc fat, but dexamethasone-stimulated as well as basal PAI-1 secretion rates were always higher in omental fat. Unlike dexamethasone, 24-h insulin did not modify PAI-1 secretion while accelerating glucose consumption. In contrast, 24-h cAMP inhibited PAI-1 gene expression and protein production under basal conditions and in the presence of dexamethasone. This inhibition was already detectable after 1 h and was maximal after 4 h at the level of gene expression. It occurred in both omental and sc adipose tissues. Importantly, epinephrine dose dependently inhibited PAI-1 parameters, an effect that was reproduced by isoproterenol. Dexamethasone- and cAMP-induced changes in PAI-1 messenger ribonucleic acid abundance were similar in explants and isolated fat cells. In isolated stromal-vascular cells, only dexamethasone was effective. In conclusion, we provide evidence for a reciprocal regulation of PAI-1 by dexamethasone (positive effector) and cAMP/catecholamines (negative effectors) in cultured human adipose tissue. The stimulation by glucocorticoids could contribute to enhanced production of PAI-1 by adipose tissue and high plasma levels of PAI-1 associated with central obesity and thereby be a link between this disorder and cardiovascular disease. Impaired inhibition by catecholamines could also contribute, as in vivo adipose tissue responses to these hormones are usually blunted in obese individuals.

Transposon Tn917PF1 mutagenesis in Bacillus licheniformis.

The plasmid pTnPFl containing the transposon Tn917PF1 was introduced into the protoplasts of two Bacillus licheniformis strains in the presence of polyethylene glycol. Transpositions were produced at high temperature which inhibited plasmid replication and kanamycin was used for selection.Transposon Tn917PF1 was inserted randomly into the bacterial chromosome,producing different auxotrophic, prophage BLF and bacitracin-non-producing mutants. The auxotrophic mutant phenotypes were characterized by the Holliday-test and some mutations by hybridization with a transposon DNA probe. Insertions for the entire chromosome or for the prophage genophore were found at random, but preferred target sites were detected within limited regions, like the bacitracin synthetase or sulphate reductase genes. The partial physical map of the chromosomal region of bacitracin synthetase was constructed based on the hybridization patterns of insertion mutants.