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Dostert, Melanie

University of British Columbia

Antibiotic therapy frequently fails when treating biofilm-associated infections, which are aggregated bacterial communities embedded in a protective, extracellular matrix. Bacterial biofilms cause 65% of bacterial infections in humans and are inherently resistant to antimicrobials. Despite their clinical and economical significance, there are no clinically approved drugs selectively targeting biofilms. To understand the biofilm regulatory gene network, I set out to identify genes phenotypically affecting biofilm growth in the global priority pathogen Pseudomonas aeruginosa that causes multiple different biofilm infections in humans. A transposon insertion sequencing (TnSeq) approach comparing biofilm and planktonic growth of P. aeruginosa PA14 was employed to identify genes in different models from in vitro hydroxyapatite (HA) and in vivo-like human skin organoids to in vivo murine abscesses. Biofilm genes detected in the HA model indicated the requirement of several one-component transcriptional regulators (OCRs), which are the most abundant and versatile group of bacterial regulators but a poorly studied one in the context of biofilms. This led to the identification of six regulators required for biofilm growth without affecting motility phenotypes, namely yeaG, bosR, arsR, merD, PA14_36180 and PA14_56430. The putative oxidative stress regulator bosR was required for biofilm growth in another divergent phylogenetic P. aeruginosa lineage and further confirmed by deletion and complementation. Biofilm growth on in vivo-like human skin organoids involved several known biofilm regulators of well-established biofilm pathways, such as the Gac-Rsm and the alginate regulatory pathway, that showed discrepancies in mutant phenotypes compared with in vitro HA biofilms, suggesting environment-dependent remodeling of the biofilm gene network. In the murine abscess model, TnSeq was employed to support the presence of P. aeruginosa biofilms. Accordingly, many genes previously implicated in biofilm growth were shown to affect growth in murine abscesses in a similar manner, including two dozen characterized biofilm genes. Furthermore, gene cbfA was identified as a novel biofilm gene required in all three biofilm models used in this study. These findings provided new insights into the mechanisms governing biofilm growth in P. aeruginosa and a broad range of genes representing novel environment-specific targets for the design of antibiofilm therapies.

Text

2023-07-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85285

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/