Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains — which proteolyse cytoskeleton proteins — and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.
SummaryAutoclaving (15 min, 121 °C, 1 atm overpressure) and high‐intensity ultrasound (3 pulses of 1 min, 750 W, 20 kHz, 80% amplitude) were evaluated individually or combined in cassava pulp to reduce the digestible starch and increase the fibre content. Ultrasound alone produced minimal modifications, and the subsequent autoclaving increased the oil‐binding capacity and swelling. Autoclaving alone significantly increased total dietary fibre (TDF) from 14.7% to 21.4%, and the ratio of hemicellulose to lignocellulose. It also reduced the extractable starch from 42.7% to 7.5% and the resistant starch from 7.4% to 3.1%. Despite the higher digestibility, the starch digestion rate with pancreatic α‐amylase was slower. Starch solubility increased at 25, 68, and 90 °C. Ultrasonication after autoclaving reduced the hemicellulose percentage, but the sorption properties remained unchanged. Resistant starch substantially increased to 39.9%, mainly composed of RS1 (inaccessible starch), and starch solubility decreased at the three temperatures assayed.
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