Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic 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 (CNFY 709-1014 ).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 SummaryThe RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection 3 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.

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

“…The respective segment is significantly different in CNF Y (Fig EV1), which may explain why CNF Y does not interact with Lu/BCAM. Instead, a recent study has shown that a C‐terminal fragment of CNF Y (residues 709–1,014) employs glycosaminoglycans as receptors and is sufficient for endosomal uptake (preprint: Kowarschik et al , 2020), in line with the observations made here. Thus CNF Y , similar to CNF1 (Piteau et al , 2014; Reppin et al , 2017), contains two distinct host cell binding sites, one each at the N‐ and C‐terminus.…”

“…The receptor of the N‐terminal part of CNF Y is still unknown, but it has been shown that binding of CNF1 to host cells has no effect on CNF Y uptake (Blumenthal et al , 2007), suggesting that both toxins use different host cell factors for endocytosis. This has also been corroborated in a recent study that identified glycosaminoglycans as interaction partners of C‐terminal fragments of CNF Y (preprint: Kowarschik et al , 2020). The CNFs are taken up into endosomes and their release into the host cytoplasm requires two hydrophobic sequence motifs within the N‐terminal half of the toxin that have been predicted to form α‐helices.…”