Distinguishing delivery and catalytic activity differences among G-protein deamidating protein toxins of the cytotoxic necrotizing factor (CNF) and Pasteurella multocida toxin (PMT) family through comparative analyses

AB toxins are a class of bacterial protein exotoxins characterized by a modular structure consisting of an 'A' active domain and a 'B' binding domain. These toxins are adept at delivering potent payloads into the cytosol of target cells, making them attractive candidates for Bacterial Toxin-Inspired Drug Delivery (BTIDD) platforms. This thesis focuses on a family of G-protein deamidating toxins, including Pasteurella multocida toxin and the cytotoxic necrotizing factor (CNF) family members, as models for investigating the structural and functional determinants for cytosolic cargo delivery. The diversity of this family, including the thirteen full-length homologs of the CNF toxin family, various CNF-like catalytic domains attached to different toxin systems, and the prevalence of the PMT N-terminal delivery motif throughout bacterial genomes offers a rich source for exploring the modularity and adaptability of AB toxins. A critical aspect of AB toxin-mediated delivery is the ability to escape endosomal degradation, a common challenge in cytosolic delivery of biologics. The CNF family is notable among AB toxins for the high homology yet distinct behaviors in binding, delivery, and activity among the studied homologs. The best studied CNF toxins, CNF1, CNF2, CNF3, and CNFy, all also have different responses to endosomal acidification. These studies found that modifying specific acidic amino acid residues in the CNFy translocation domain with those from CNF3 improved endosomal escape at higher pH levels, leading to more efficient cytosolic delivery. Furthermore, using small molecule inhibitors of the endosomal trafficking pathway, we showed that CNFy leaves the endosome at a later stage of the acidification process than CNF3, potentially leading to decreased efficiency in cargo delivery. These insights into the amino acid determinants of bacterial toxins can be leveraged to optimize the cytosolic delivery of biologic cargos using these toxins as BTIDD platforms. Once inside the cytosol, the CNF activity domains activate Rho GTPases through deamidation of a glutamine residue at the active site, with each CNF variant showing distinct substrate preferences. The data here shows that CNFx, a novel and phylogenetically distant member of the CNF family identified in cetain E. coli strains, modifies RhoA through transglutamination rather than deamidation, akin to the activity of dermonecrotic toxin (DNT). This unique activity profile of CNFx provided a valuable opportunity to study the structural determinants that differentiate these two reaction mechanisms catalyzed by these toxins. Our research identified critical active-site residues in CNFx that govern its enzymatic preference. We also identified a C-terminal cysteine residue that impeded cargo delivery, resulting in a higher EC50 value compared to other CNFs. The CNF family shares an N-terminal delivery vehicle with PMT, PMT-N. Previous studies in the laboratory by Nathan Clemons have shown that PMT-N is able to facilitate the entry of exogenous proteins, such as GFP, into the cytosol. However, the native PMT cargo domains, C1-C2-C3, require both C1 and C2 for the effective delivery of the catalytic C3 domain, indicating the presence of an accessory module that assists in cytosolic delivery. PMT-N homology is found in a wide range of bacterial genomes, linked to various C-terminal enzymatic motifs. This suggests a role for the accessory module in adapting cargo to the PMT-N delivery vehicle. Through luciferase reporter assays, we show that the CNFs also require an accessory module for delivery of their cargo domains, though a significantly smaller region than for PMT is required to restore delivery. The presence of accessory modules in PMT-like delivery systems suggests a promising strategy for improving the delivery of diverse cargos. These results offer an understanding of the amino acid sequence characteristics of bacterial toxins that can be leveraged to improve the cytosolic delivery of biological cargos using BTIDD platforms.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Handy, Nicholas Bayne

Contributors dc:contributor

• Wilson, Brenda A
• Blanke, Steven R
• Olsen, Gary J
• Slauch, James M

• MICROBIOLOGY
• TOXIN
• AB TOXIN
• CYTOTOXIC NECROTIZING FACTOR
• CNF
• PASTEURELLA MULTOCIDA TOXIN
• PMT

Statement dc:rights

• Copyright 2025 Nicholas Handy

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129619

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129619