Abstract
Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SignificanceOur findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.
Published Version
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