Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Microbiology, Immunology, and Molecular Genetics

First Advisor

Dr. Erin C. Garcia

Abstract

Contact-dependent growth inhibition (CDI) systems mediate interbacterial competition. The genes encoding these systems are widespread among Gram-negative bacteria, including Burkholderia pathogens. CDI systems of Burkholderia species are composed of two-partner secretion pathway proteins and function to deliver the toxic C-terminus of a polymorphic surface-exposed exoprotein BcpA (Burkholderia CDI protein A) to the cytoplasm of neighboring recipient bacteria upon cell-cell contact. Specific outer and inner membrane proteins facilitate BcpA translocation both out of the donor bacterium and into the recipient cell cytoplasm. Most Burkholderia species-specific CDI translocation factors in recipient cells are unknown. BcpA intoxication functions as a mechanism by which ‘non-kin’ and ‘kin’ bacteria are differentiated. Survival of ‘kin bacteria’ is, in part, dependent on the presence of a small cognate immunity protein, BcpI, in the recipient bacterium. BcpI acts in an allele-specific manner to protect against BcpA from a particular CDI encoding locus.

Through this body of work, key components of both the donor and recipient bacteria are identified, adding further insight into the function of Burkholderia species-specific CDI mechanisms. Findings are broken into three overarching results. Utilizing Burkholderia dolosa as the primary model, comparisons of distinct CDI systems observed minimal differences in intoxication efficiency and secondary functions associated with CDI system proteins such as biofilm formation and motility. The pre-toxin region within the BcpA protein is demonstrated to be required for CDI of recipient cells. The final experiments identified proteins required in Burkholderia dolosa recipient cells for maximum intoxication by donor bacteria. Using transposon mutagenesis and whole genome sequencing approaches, CDI sensitivity candidate genes in quorum sensing regulation, stress response, metabolism, and lipopolysaccharide (LPS) structure and production genes were identified. Mutations in both the quorum sensing regulator and the LPS biosynthesis associated genes conferred resistance against both Burkholderia dolosa CDI system-1 and system-2. The other identified CDI candidates conferred partial protection against specific BcpA intoxication. All together this dissertation adds key information to the field, building a more robust understanding of the uniqueness of the Burkholderia CDI components and donor-recipient interactions. The spectrum of sensitivity each of the identified factors contributes to recipient susceptibility adds further insight into the complexity of the CDI mediated competition.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.451

Funding Information

This body of work was supported by grants from the National Institutes of Health (K22AI118949 and R01AI150767 awarded to Dr. E.C. Garcia) and start-up funding from the University of Kentucky.

Available for download on Saturday, December 16, 2023

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