Glycosaminoglycans are specific endosomal receptors for Yersinia pseudotuberculosis Cytotoxic Necrotizing Factor

Abstract

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author Summary The RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.View Full Text