AdvisorCarbonetti, Nicholas H.
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AbstractPertussis toxin (PT), an AB5 exotoxin and an important virulence factor of Bordetella pertussis, has been hypothesized to traffic along a retrograde transport pathway in mammalian cells. This model includes endosomal uptake, transport to the Golgi apparatus and endoplasmic reticulum (ER), ATP-stimulated dissociation of the holotoxin in the ER, and translocation of the A subunit (S1) to the cytosol. PT has been detected in the Golgi apparatus by immunofluorescence microscopy and cell fractionation, but transport beyond the Golgi has not been detected by these methods. To gain biochemical evidence for the retrograde pathway, target sites for tyrosine sulfation (a trans-Golgi network (TGN)-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained enzymatic activity as assessed by in vitro and cellular ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. To investigate the importance of the interaction of PT with ATP, a form of PT was engineered with changes to two amino acid residues in S5 thought to be involved in the interaction. This form of PT was reduced in its ability to be dissociated by interaction with ATP in vitro; it was also substantially reduced in activity in an in vitro enzymatic assay and in a cellular ADP-ribosylation assay. When cells were treated with PT possessing the ATP-binding mutations and the tyrosine sulfation and N-glycosylation target sequences, the N-glycosylation patterns of the S1 and S4 subunits were altered, consistent with an inability of the holotoxin to dissociate in the ER. This form of PT also colocalized substantially with ER markers when observed under indirect immunofluorescence microscopy. Overall, these results comprise evidence in support of the hypothesized retrograde pathway of PT intracellular transport.
DescriptionUniversity of Maryland, Baltimore. Microbiology. Ph.D. 2007
retrograde transport pathway
Virulence Factors, Bordetella