Modular AB-type bacterial protein toxins target mammalian host cells with high specificity and deliver their toxic cargo into the cytosol. Hence, these toxins are being explored as agents for targeted cytosolic delivery in biomedical and research applications. The cytotoxic necrotizing factor (CNF) family is unique among these toxins in that their homologous sequences are found in a wide array of bacteria, and their activity domains are packaged in various delivery systems. Here, to study how CNF cargo and delivery modules can be assembled for efficient cytosolic delivery, we generated chimeric toxins by swapping functional domains among CNF1, CNF2, CNF3 and CNFy. Chimeras with a CNFy delivery vehicle were more stably expressed, but were less efficient at cargo delivery into HEK293-T cells. We also found that CNFy cargo is the most universally compatible and that CNF3 delivery vehicle is the most flexible and efficient at delivering cargo. These findings suggest that domains within proteins can be swapped and accommodate each other for efficient function, and that an individual domain could be engineered for compatibility with multiple partner domains. We anticipate that our insights could help inform chemical biology approaches to develop toxin-based cargo-delivery platforms for cytosolic cargo delivery of therapeutics or molecular probes into mammalian cells.

The zoonotic pathogen produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα-protein targets. The N-terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide of three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has as-of-yet undefined function, and C3 catalyzes deamidation of a specific active-site glutamine residue in Gα-protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers into the cytosol non-native GFP cargo, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105-1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

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Correction for Clemons et al., "Cytosolic delivery of multidomain cargos by the N terminus of Pasteurella multocida toxin." Infect Immun 86:e00596-18.

Bacterial toxin‐inspired drug delivery (BTIDD) exploits the cellular entry and cytosolic delivery mechanisms of bacterial protein toxins to facilitate intracellular delivery of therapeutic agents. The Gln‐deamidase activity domains of the cytotoxic necrotizing factor (CNF) family are found in a diverse array of protein toxins (6 known AB‐type toxins), effectors of specialized secretion systems (T3SS and T6SS), and a number of putative homologs. This suggests that CNF‐like domains might be universal toxic cargos that can be swapped readily among various delivery systems. The CNF family of highly conserved modular toxins presents a unique opportunity to gain insights on how a delivery platform evolved for efficient delivery of its cognate cargo. We have swapped functional domains among CNF1, CNF2, CNF3 and CNFy at various joining sites to determine the effect on efficiency of cargo delivery into the host cell cytosol, using a cell‐based SRE‐luciferase downstream reporter assay. We first refined the limits of modularity among the CNF domains by testing three amino acid joining sites and found the most effective joining site is not the canonical catalytic domain start site, but rather sites surrounding a C‐terminal secondary binding domain. Under our defined assay conditions, CNFy is less efficient than CNF1 or CNF2. CNFy1 or CNFy2 chimeras with a CNFy delivery domain are less efficient than CNF1 or CNF2, suggesting the delivery domain dictates delivery efficiency. However, the delivery domains of CNF1 and CNF2 were unable to enhance delivery efficiency of the CNFy cargo. Combined, these results show that CNFy is limiting as both the cargo and the delivery system. We hypothesized that rather than one domain being responsible for the efficiency of cargo delivery, that cooperativity between the delivery domain and its cognate cargo is rate limiting, whereby specific amino acid residues within these domains recognize and interact with each other to facilitate an efficient translocation event. To test this, we generated chimeric constructs with CNF3 cargo delivered by CNF1, CNF2, and CNFy. Since CNF3 is most closely related to CNFy, as expected CNFy is most efficient at delivering CNF3 cargo. Although the CNF toxin family has high amino acid identity, the seemingly small variations in each peptide may account for subtle compensatory changes that enhance the delivery of cognate cargo over related domains. Using our cellular assays coupled with examining the alignment of these high identity proteins, we can trace these evolutionary adaptations and gain insight into the protein determinants that define toxin‐based delivery of cargo. This knowledge offers basis for generating tunable BTIDD systems to enhance delivery of specific cargo for clinical and research applications.Support or Funding InformationDeBoer Fellowship UIUCResearch Assistantship UIUCNIH/NAID AI038396Chemistry Biology Interface Training Program Fellowship UIUCNational Institute Of General Medical Sciences of the National Institutes of Health T32GM070421