Dissertation Defense: Noah Souza

Title: AB5 Toxins: From Salmonella Pathogenesis to Staphylococcus aureus Vaccine Development

Program: Biomolecular Sciences PhD

Committee Chair: Juliette Tinker

Committee: Juliette Tinker, Ken Cornell, Javier Ochoa-Repáraz

Abstract: AB5 toxins are a structurally conserved family of bacterial proteins that contribute to host-pathogen interactions through an enzymatically active A-subunit and a pentameric B-subunit responsible for host-cell binding. Several well-characterized AB5 toxins, including cholera toxin (CT) from Vibrio cholerae, pertussis toxin (PT) from Bordetella pertussis, and heat-labile toxin (LT) from enterotoxigenic Escherichia coli (ETEC), function as major virulence factors by altering host-cell signaling and physiology. However, their structural and immunomodulatory properties have also made AB5 toxins attractive components for vaccine development. We hypothesized that AB5 toxins exert distinct effects on host-cell physiology during bacterial pathogenesis and that their structural and immunomodulatory properties can be adapted for vaccine development. This dissertation examines these contrasting roles by investigating the cellular signaling and functional activity of the Salmonella AB5 toxin ArtAB and expanding novel CT-derived vaccine platforms to prevent Staphylococcus aureus infection. Previous work from our laboratory purified ArtAB and its B-subunit, ArtB, from multidrug-resistant Salmonella Typhimurium definitive type 104 (DT104) and demonstrated distinct morphological effects in mammalian cells. To further characterize these effects, multi-omics and functional analyses were used to examine host-cell responses to ArtAB and ArtB. ArtAB produced minimal cytotoxicity but induced substantial host-cell remodeling and disrupted epithelial barrier integrity, supporting a role in modulating host physiology without overt cell death. In contrast, the mechanism of action of CT is well known, and it has been extensively studied for its adjuvant properties. Our laboratory uses a CT-derived CTA2/B chimeric vaccine platform in which the toxic CTA1 domain is replaced with an antigen of interest while retaining the CTA2 linking domain and non-toxic B-subunit (CTB). This platform was expanded and optimized for S. aureus vaccine development through recombinant chimera production, evaluation in a bovine mastitis vaccination and challenge model, and investigation of microneedle (MN)-based vaccine delivery for human applications. Platform optimization improved the production of selected recombinant vaccine constructs, while bovine vaccination elicited strong systemic antigen-specific serum IgG responses that were maintained through challenge. MN vaccination in mice produced limited antibody responses under the conditions tested but established feasibility for optimization of the delivery platform. Together, these studies demonstrate how AB5 toxins can function as pathogenic effectors while also providing adaptable platforms for vaccine development.