Conaway, A. | bioRxiv Science

bioRxiv2024

Pseudomonas aeruginosa LasR-deficient mutants have increased methylglyoxal and hydrogen peroxide sensitivity due to low intracellular glutathione

The electrophile methylglyoxal (MG) is produced by microorganisms and host cells through central metabolic pathways. MG is a highly reactive electrophile, so it must be rapidly detoxified to prevent damaging modifications to macromolecules. Pseudomonas aeruginosa, a pathogen of concern due to its ability develop multidrug resistance, causes many types of infections that have been associated with elevated MG levels, including cystic fibrosis (CF). P. aeruginosa isolates commonly have mutations that lead to LasR loss-of-function (LasR-) and we found that lasR mutations confer sensitivity to MG in multiple strain backgrounds. LasR-strains have increased activity of the CbrAB two-component system which represses Crc regulation of metabolism. Here, we show that higher CbrAB activity and low Crc activity renders cells sensitive to MG. We found that P. aeruginosa LasR-strains are more sensitive to MG and have lower intracellular reduced glutathione (GSH) compared to their LasR+ comparators. Consistent with published reports, mutants lacking gloA3, which encodes a MG-glyoxalase, and mutants lacking GSH biosynthesis enzymes (gshA or gshB) were sensitive to MG. Exogenous GSH rescued MG sensitivity in LasR-strains and gshA or gshB mutants, but not in a gloA3 mutant strain. We propose that low GSH levels in LasR-strains contribute to increased sensitivity to MG and H2O2. SignificanceMethylglyoxal is a highly reactive metabolite that is detected in various disease states, including those where Pseudomonas aeruginosa is present and MG resistance requires the glutathione-dependent glyoxalase enzyme GloA3 enzyme. This study reveals that P.aeruginosa strains with LasR mutations, which are commonly found in clinical isolates, are more sensitive to methylglyoxal (MG) and hydrogen peroxide due to lower intracellular glutathione levels and high activity of the CbrAB-Crc regulatory pathway. This could be significant for understanding the selective pressures that drive P. aeruginosa evolution in infection sites, as well as a better understanding of LasR-strain metabolism in infections such as those associated with cystic fibrosis.

microbiology↗

bioRxiv2024

Loss of LasR function leads to decreased repression of Pseudomonas aeruginosa PhoB activity at physiological phosphate concentrations

While the Pseudomonas aeruginosa LasR transcription factor plays a role in quorum sensing (QS) across phylogenetically-distinct lineages, isolates with loss-of-function mutations in lasR (LasR- strains) are commonly found in diverse settings including infections where they are associated with worse clinical outcomes. In LasR- strains, the transcription factor RhlR, which is controlled by LasR, can be alternately activated in low inorganic phosphate (Pi) concentrations via the two-component system PhoR-PhoB. Here, we demonstrate a new link between LasR and PhoB in which the absence of LasR increases PhoB activity at physiological Pi concentrations and raises the Pi concentration necessary for PhoB inhibition. PhoB activity was also less repressed by Pi in mutants lacking different QS regulators (RhlR and PqsR) and in mutants lacking genes required for the production of QS-regulated phenazines suggesting that decreased phenazine production was one reason for decreased PhoB repression by Pi in LasR- strains. In addition, the CbrA-CbrB two-component system, which is elevated in LasR- strains, was necessary for reduced PhoB repression by Pi and a {Delta}crc mutant, which lacks the CbrA-CbrB-controlled translational repressor, activated PhoB at higher Pi concentrations than the wild type. The {Delta}lasR mutant had a PhoB-dependent growth advantage in a medium with no added Pi and increased virulence-determinant gene expression in a medium with physiological Pi, in part through reactivation of QS. This work suggests PhoB activity may contribute to the virulence of LasR- P. aeruginosa and subsequent clinical outcomes. ImportanceLoss-of-function mutations in the gene encoding the Pseudomonas aeruginosa quorum sensing (QS) regulator LasR occur frequently and are associated with worse clinical outcomes. We have found that LasR- P. aeruginosa have elevated PhoB activity at physiological concentrations of inorganic phosphate (Pi). PhoB activity promotes Pi acquisition as well as the expression of QS and virulence-associated genes. Previous work has shown that PhoB induce RhlR, another QS regulator, in a LasR-mutant in low Pi conditions. Here, we demonstrate a novel relationship wherein LasR represses PhoB activity, in part through the production of phenazines and Crc-mediated translational repression. This work suggests PhoB activity may contribute to the increased virulence of LasR- P. aeruginosa.

microbiology↗