Date of Award
Winter 12-8-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology and Immunology
First Advisor
Jonathan Allen
Abstract
Bacteria compete for limited nutrients and space in polymicrobial environmental and host niches. One mechanism of interbacterial competition involves the production of toxins that antagonize neighboring cells in contact-dependent or independent manners. These holotoxins can be composed of different protein domains that function in secretion, delivery, and effector roles. As the toxic effector domains often vary between individual strains within a species, toxins with this architecture are referred to as polymorphic. Polymorphic toxin-producing cells and their siblings are protected from intoxication due to an encoded cognate immunity factor that neutralizes the effector. Our work characterized a gene cluster in Pseudomonas aeruginosa that encodes a new class of polymorphic toxins. The first two genes in this cluster, which we termed polymorphic toxins with rhs elements (pxr)B and pxrA, encode rhs elements, a series of tyrosine-aspartate-rich repeating motifs that together form a hollow, cage-like structure. Examples of characterized bacterial Rhs proteins include tripartite toxin complexes, which intoxicate insect epithelial cells, and specific type VI secretion system effectors, which intoxicate other bacteria. In each example, the Rhs shell acts similarly to a chaperone, encapsulating an unfolded C-terminal toxic effector domain. We demonstrated that the novel PxrBA protein complex appears to function as a diffusible holotoxin to antagonize neighboring bacteria of the same and distinct species. Our data indicate that the C-terminus of PxrA contains variable effector domains, which are positively selected for at numerous distinct genomic loci and seem to move by horizontal gene transfer and recombination. We identified one of our novel effectors as a probable toxic DNA nuclease. These effector domains are neutralized by cognate immunity factors encoded by pxrI, the third gene in this cluster, likely by the direct binding we observed between the two proteins. The N-terminus of PxrB shares homology with type IV pilus-binding domains of filamentous phage minor coat proteins, suggesting that binding to these pili is important for intoxication. In support of this, we found that type IV pili-deficient strains are no longer susceptible to PxrBA growth inhibition. Additionally, pxr mutant strains have increased susceptibility to phage infection that is also dependent on type IV pili. This may indicate that binding of the PxrBA holotoxin to pili both mediates toxin delivery and antagonizes phage propagation. Finally, we showed that mutant pxr strains are attenuated for virulence in a mouse bacteremia model, and that the decrease in host survival from wildtype Pa infection was independent of bacterial burden. Thus, we have identified a new class of polymorphic Rhs toxins in Pseudomonas aeruginosa with pleiotropic effects on bacterial fitness. PxrBA mediates interbacterial competition, antagonizes bacteriophage infection, and impacts mammalian virulence. By studying the ways bacteria antagonize each other, we could engineer these toxin delivery systems to target and deliver antimicrobials as an alternative to antibiotic therapies. Furthermore, if we understand what bacteria target to antagonize each other, we may also be able to design molecules that disrupt those same targets. This is especially important in a time of growing bacterial antibiotic resistance and a lack of new antibiotic development.
Recommended Citation
Banas, Abigail Nicole, "Discovery of a New Class of Polymorphic Rhs Toxins in Pseudomonas aeruginosa with Pleotropic Effects on Interbacterial Competition, Bacteriophage Antagonism, and Host Virulence" (2025). Dissertations. 4295.
https://ecommons.luc.edu/luc_diss/4295
