Investigating the role of the phenylacetic acid pathway in the quorum sensing-regulated virulence of cystic fibrosis pathogens

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Lightly, Tasia
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For successful bacterial infection, bacteria need to sense and respond to their environment. Quorum sensing (QS) allows bacteria to regulate their virulence in a cell-density dependent manner. Polymicrobial infections are prevalent in cystic fibrosis (CF) infections but the interactions between these microbes are not well understood. The phenylacetic acid (PAA) pathway is upregulated in CF-like conditions and PAA was linked to the attenuation of virulence in two major CF pathogens, Burkholderia cenocepacia and Pseudomonas aeruginosa. However, the mechanism of attenuation remains unknown. The goals of my thesis were to investigate the role of PAA release in CF pathogen interactions, identify the PAA pathway metabolite responsible for the attenuation of virulence, and determine if the mechanism of inhibition is due to an effect on the CepIR QS system (either directly or indirectly). I investigated the accumulation of PAA in CF pathogens. I determined that B. cenocepacia was the most likely candidate for the accumulation of PAA but that PAA did not affect the pathogen interactions. I then characterized knockout mutants of the first steps of the PAA pathway (PaaK or PaaABCDE) to determine which PAA metabolite(s) are responsible for the attenuation of virulence. While loss of the paaABCDE operon resulted in decreased virulence, a paaK deficient strain had increased virulence compared to wild type despite the fact that both mutations resulted in higher levels of external PAA. Although we found no evidence of direct cepI or cepR downregulation by PAA or PAA-CoA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. Whereas removal of the paaABCDE operon in the cepR mutant had no bearing on its attenuated phenotype. These results demonstrate that B. cenocepacia can elicit a pathogenic response if PAA-CoA is not produced. Conversely, the accumulation of PAA-CoA appears to attenuate pathogenicity. In summary, this thesis demonstrates that a metabolic signal can activate virulence in B. cenocepacia when the classical QS system is not functioning. This finding challenges the classical view of virulence activation by QS, providing new insight into the relationship between metabolism and virulence in opportunistic pathogens.
Phenylacetic acid, Quorum Sensing, Burkholderia cenocepacia, Cystic fibrosis, Phenylacetyl-CoA, Microbiology, Gram-negative, Pseudomonas aeruginosa, Aspergillus fumigatus, Metabolism, Virulence
Pribytkova, T., Lightly, T. J., Kumar, B., Bernier, S. P., Sorensen, J. L., Surette, M. G., & Cardona, S. T. (2014). The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid. Molecular Microbiology, 94(3), 522–536. 32.
Lightly, T. J., Phung, R. R., Sorensen, J. L., & Cardona, S. T. (2017). Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production. Canadian Journal of Microbiology, 63(5), 1–12. 27.
Lightly, T. J., Frejuk, K. L., Groleau, M.-C., Chiarelli, L. R., Ras, C., Buroni, S., Déziel, E., Sorensen, J. L., & Cardona, S. T. (2019). Phenylacetyl-CoA, not phenylacetic acid, attenuates CepIR-regulated virulence in Burkholderia cenocepacia. Applied and Environmental Microbiology, 85:e01594-19.