N-Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells.

Tc toxin is an exotoxin composed of three subunits named TcA, TcB and TcC. Structural analysis revealed that TcA can form homopentamer that mediates the cellular recognition and delivery processes, thus contributing to the host tropism of Tc toxin. N-glycans and heparan sulfates have been shown to act as receptors for several Tc toxins. Here, we performed two independent genome-wide CRISPR-Cas9 screens, and have validated glycans and sulfated glycosaminoglycans (sGAGs) as Tc toxin receptors also for previously uncharacterized Tc toxins. We found that TcdA1 form Photorhabdus luminescens W14 (TcdA1W14) can recognize N-glycans via the RBD-D domain, corroborating previous findings. Knockout of N-glycan processing enzymes specifically blocks the intoxication of TcdA1W14-assembled Tc toxin. On the other hand, our results showed that sGAG biosynthesis pathway is involved in the cell surface binding of TcdA2TT01 (TcdA2 from P. luminescens TT01). Competition assays and biolayer interferometry demonstrated that the sulfation group in sGAGs is required for the binding of TcdA2TT01. Finally, based on the conserved domains of representative TcA proteins, we have identified 1,189 putative TcAs from 1,039 bacterial genomes. These TcAs are categorized into five subfamilies. Each subfamily shows a good correlation with both genetic organization of the TcA protein(s) and taxonomic origin of the genomes, suggesting these subfamilies may utilize different mechanisms for cellular recognition. Taken together, our results support the previously described two different binding modalities of Tc toxins, leading to unique host targeting properties. We also present the bioinformatics data and receptor screening strategies for TcA proteins, provide new insights into understanding host specificity and biomedical applications of Tc toxins.