Title: LPS as a virulence factor of Burkholderia cepacia during plant and human infection

Jack Klahr Biology

Elizabeth Danka Assistant Professor of Biology

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Burkholderia cepacia is a gram-negative bacterium that is the causative agent of sour skin rot in onion crops, and is an opportunistic pathogen in patients with cystic fibrosis. There are limited treatments available for this bacterium due to its innate resistance to antibiotics, and its virulence mechanisms are not well understood. This work aims to identify and describe virulence factors necessary for B. cepacia pathogenesis. Transposon mutagenesis in B. cepacia ATCC 25416, followed by a virulence screen in an onion infection model identified mutants that generated smaller wounds at 24 hours post-infection. We chose to follow up on mutant 169 and determined that the transposon inserted in the rfbB gene of chromosome one. Expression analysis suggested that the entire rfbBDCE operon is likely disrupted. rfbB is homologous to genes encoding glucose dehydratase, an enzyme in the lipopolysaccharide (LPS) biosynthesis pathway. Production of altered LPS was confirmed in the mutant, and current work is focused on determining whether this disruption affects the pathogen’s ability to interact with host cells. Initially, we focused on designing a bacterial adhesion and invasion assay in the onion host, but this model proved inconsistent. We are currently developing a new assay using a human epithelial cell line with the goal of determining whether the altered LPS impedes binding and/or invasion into host cells. This quantitative assay will be supplemented with direct visualization of bacterial binding and invasion using confocal microscopy. Together, these data establish the B. cepacia rfbBDCE operon as critical for virulence.

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