Year

2025

Season

Spring

Paper Type

Master's Thesis

College

College of Arts and Sciences

Degree Name

Master of Science in Biology (MS)

Department

Biology

NACO controlled Corporate Body

University of North Florida. Department of Biology

Committee Chairperson

Dr. Terri Ellis

Second Advisor

Dr. Judith Ochrietor

Third Advisor

Dr. David Waddell

Abstract

Klebsiella pneumoniae is the third most common cause of nosocomial infections in the United States. The capsular polysaccharides (CPS) are virulence factors allowing K. pneumoniae to evade host immune systems. The current model of capsule structure indicates that CPS fibrils interact with the external O-antigen of lipopolysaccharide molecules to enhance retention to the bacterial outer membrane. These interactions may influence pathogenicity in clinically relevant strains. This study characterized alterations to LPS and CPS structure, the impact of these changes on capsule retention, membrane permeability, biofilm formation, and macrophage interactions. LPS and CPS were quantified and visualized in four strains of K. pneumoniae: lab strain ATCC 43816, O-antigen knockout ΔwbbO, capsule knockout ΔwcaJ, and lab-evolved cephalothin resistant strain 14T which harbors multiple mutations, including in the capsule synthesis gene wcaJ. ΔwbbO exhibited significantly less CPS compared to 43816 despite exhibiting similar quantities of LPS core structures to 43816. Visualization of LPS from each strain via SDS-PAGE confirmed the absence of O-antigen associated LPS in the ΔwbbO strain. These results corroborate the suggested model that capsule retention is, in part, associated with the LPS O-antigen in K. pneumoniae. Strain 14T showed significantly increased LPS content compared to the three other strains, but similar CPS content to ΔwcaJ. Membrane permeability decreased only in ΔwbbO. Biofilm formation was significantly increased in both ΔwcaJ and 14T. This study also explored alterations to macrophage viability, bactericidal activity, and phagocytosis of bacteria, with variations in CPS and LPS. Infection with ΔwcaJ and 14T resulted in higher phagocytosis and increased bactericidal activity but similar drop in macrophage viability to 43816. Infection with ΔwbbO resulted in similar phagocytosis to 43816 but reduced bactericidal activity and increased macrophage viability. These results suggest that capsule retention is complex, associated with more than LPS, and may influence virulence and outcomes of infection.

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