Author ORCID Identifier

https://orcid.org/0000-0003-4103-3151

Semester

Spring

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Slawomir Lukomksi

Committee Member

Mariette Barbier

Committee Member

Jennifer Franko

Committee Member

Paul LaSala

Committee Member

Karen Martin

Abstract

Burkholderia spp. are Gram‐negative intrinsically resistant bacteria that are responsible for environmental and nosocomial infections of clinical and biodefense concern. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the BSL-3 pathogens Burkholderia pseudomallei and Burkholderia mallei (Figure 1). The protein sequence of Bucl8 contains tandem outer-membrane efflux protein domains (OEP) typical of efflux systems and a unique collagen-like domain (CL) consisting of a repeating sequence of Glycine-Alanine-Serine (GAS). Because efflux pumps are often a mechanism of pathogenicity and antibiotic resistance, we investigated Bucl8 further. Here, we determined that the locus of bucl8 contains genes encoding a novel tripartite efflux pump that confers resistance to fusaric acid and pHBA, but not clinically-relevant antibiotics. Furthermore, deletion of the pump resulted delayed growth, suggesting a physiological role of the pump in human infectious strains. In addition to the prototypical barrel of outer membrane efflux proteins, Bucl8 has an extracellular GAS(n)-CL domain that forms a triple helix and binds fibrinogen. Moreover, we developed Burkholderia vaccine formulations based on the predicted immunogenic eipitopes of Bucl8, including the conserved, surface-exposed loops, L1 and L2, and portions of the collagen-like and C-terminal domains. Several combinations with different antigens, adjuvants, peptide conjugates, and routes of administration were tested for immunogenicity and/or protection in an intranasal challenge model utilizing outbred CD-1 mice and BSL-2 surrogate, B. thailandensis. The combination that reduced inflammation and disease the most was an intranasally administrated formulation of synthetic peptide of L1 conjugated to cross-reacting material 197 (CRM197) and adjuvanted with fluorinated cyclic diguanylate monophosphate (FCDG). Correlating disease outcomes with IgG antibody subtypes, T helper cell cytokines, and different myeloid cells, we determined that IL-10 correlated with morbidity, IgG2a with improved outcomes, and vaccination with reduced TH1/TH17 tissue-damaging cytokines. These humoral and cell-mediated correlates of protection could be used as biomarkers for diseases or applied to other therapeutics.

Embargo Reason

Publication Pending

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