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 (TcdA1(W14)) can recognize N-glycans via the RBD-D domain, corroborating previous findings. Knockout of N-glycan processing enzymes specifically blocks the intoxication of TcdA1(W14)-assembled Tc toxin. On the other hand, our results showed that sGAG biosynthesis pathway is involved in the cell surface binding of TcdA2(TT01) (TcdA2 from P. luminescens TT01). Competition assays and biolayer interferometry demonstrated that the sulfation group in sGAGs is required for the binding of TcdA2(TT01). 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. Author summary The Toxin complexes, also referred to as Tc toxins, are a family of A(5)BC exotoxins widely distributed among Gram-negative and positive bacteria. First identified in Entomopathogenic bacteria as key virulence factors to combat insect hosts, putative Tc toxin loci are also encoded by a range of human pathogens such as Salmonella and Yersinia. Previous studies indicated that several Tc toxins can target invertebrate and vertebrate cells via binding with N-glycans and heparan sulfates. Here our genome-wide CRISPR-Cas9 screens validated that different Tc toxins utilized distinct receptors for the adhesion to their targets, which is determined by TcA homopentamer. For example, TcdA1 from Photorhabdus luminescens W14 (TcdA1(W14)) relies on N-glycan binding to exert its toxic effects, while sulfate groups of sulfated glycosaminoglycans are critical for the cell targeting of other TcAs such as TcdA2(TT01) (TcdA2 from P. luminescens TT01). Consistent with the previously described different binding modalities of Tc toxins, our results confirm that the receptor selectivity of TcAs contribute to the cellular tropism of Tc toxins. Furthermore we has also identified 1,189 TcA homologues and categorized them into five subfamilies. Each TcA subfamily shows a good correlation with the taxonomic origin of the genomes, suggesting these subfamilies are linked to diverse host tropisms via different binding modalities. Together, our findings provide mechanistic insights into understanding host specificity of distinct Tc toxins and the development of therapeutics for Tc toxin-related infections, as well as the adaptation of Tc-injectisomes as potential biotechnology tools and pest-control weapons.

Automated summary

Tc toxins are exotoxins composed of TcA, TcB, and TcC subunits. They recognize host cells through N-glycans and heparan sulfates. Different Tc toxins use distinct receptors to bind to target cells, determined by TcA homopentamer. The identification of TcA homologues and their categorization into subfamilies provides insights into host specificity and potential therapeutic applications.