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Enzymic hydrolysis of sphingomyelin in the presence of bile salts.

ABSTRACT: Sphingomyelin in mixed dispersion with bile salts was hydrolysed by the solubilized sphingomyelinase of rat brain lysosomes. In parallel studies, physical properties of these dispersions were determined. The kinetic curves that described the rate of hydrolysis as a function of increasing concentrations of bile salt were multiphasic. A region of very low activity was followed by an ascending portion, a peak, a descending portion, a trough and a second ascending portion. The positions of the initiation points, peaks and troughs were found to be a function of the respective ratios of the bile salt to sphingomyelin for the detergent sodium taurodeoxycholate, but of the absolute concentration of the detergent for sodium taurocholate. Turbidity studies suggested that hydrolysis of sphingomyelin begins at a bile salt concentration that solubilizes the lipid and incorporates it into a mixed micelle with the detergent. Ultracentrifugation studies suggested that the sizes of the mixed aggregates of detergent and lipid were a function of the ratio of taurodeoxycholate to sphingomyelin, but of the absolute concentration of the bile salt, for sodium taurocholate.

SUBMITTER: Yedgar S

PROVIDER: S-EPMC1161453 | biostudies-other | 1980 Mar

REPOSITORIES: biostudies-other

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Project description:Enterohemorrhagic Escherichia coli (EHEC), including serotype O157:H7, cause severe food-borne illness. On route to the human colon, they encounter and resist, numerous anti-microbial ingestion stresses. We hypothesize that these stresses cue EHEC to alter virulence properties. This study investigated the impact of bile salts on virulence properties and examined the genetic basis of the phenotypes. Established assays were used to examine adhesion to human epithelial cells, motility, verotoxin (VT) production and antimicrobial resistance with/without bile salt stress. Bacteria treated for 90 minute in DMEM plus 0.15% (w/v) bile salt mix demonstrated significantly enhanced adhesion to epithelial cells and resistance to several antibiotics but did not increase motility or VT production. To determine the genetic basis of these phenotypes a microarray experiment was conducted. EHEC strain 86-24, in mid-log phase of growth, were grown in DMEM pH 7.4 (control), or DMEM plus bile salt mix (0.15% w/v), for 90 minutes, statically at 37˚C, 5% CO2 prior to harvesting RNA for the microarray study. Four biological replicates were produced for each treatment. Microarray and gene expression analysis (semi-quantitative RT-PCR and beta-galactosidase reporter assays) of bile salt-treated EHEC revealed significant up-regulation of genes for lipid A modification, fimbriae, an efflux pump, and a two-component regulatory system relative to the bacteria grown in DMEM alone. This work points to several mechanisms that EHEC employs to resist the stresses of the human small intestine, notably efflux, antimicrobial resistance, and outer membrane alterations. Bile salts enhanced the virulence-related properties of increased adhesion and resistance to antimicrobials but not VT production or motility. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms this pathogen uses to successfully colonize and infect the human host.

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Enzymic hydrolysis of sphingomyelin in the presence of bile salts.

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