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Mechanism of pyocyanin abolishment caused by mvaT mvaU double knockout in Pseudomonas aeruginosa PAO1.

ABSTRACT: MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.

SUBMITTER: Dong L

PROVIDER: S-EPMC6961724 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa is an important opportunistic pathogen that is lethal to cystic fibrosis (CF) patients. Glycerol generated during the degradation of phosphatidylcholine, the major lung surfactant in CF patients, could be utilized by P. aeruginosa. Previous studies have indicated that metabolism of glycerol by this bacterium contributes to its adaptation to and persistence in the CF lung environment. Here, we investigated the metabolic mechanisms of glycerol and its important metabolic intermediate glycerol 3-phosphate (G3P) in P. aeruginosa PAO1. We found that G3P homeostasis plays an important role in the growth and virulence factor production of P. aeruginosa PAO1. The G3P accumulation caused by the mutation of G3P dehydrogenase (GlpD) and exogenous glycerol led to impaired growth and reductions in pyocyanin synthesis, motilities, tolerance to oxidative stress, and resistance to kanamycin. Transcriptomic analysis indicates that the growth retardation caused by G3P stress is associated with reduced glycolysis and adenosine triphosphate (ATP) generation. Furthermore, two haloacid dehalogenase-like phosphatases (PA0562 and PA3172) that play roles in the dephosphorylation of G3P in strain PAO1 were identified, and their enzymatic properties were characterized. Our findings reveal the importance of G3P homeostasis and indicate that GlpD, the key enzyme for G3P catabolism, is a potential therapeutic target for the prevention and treatment of infections by this pathogen. IMPORTANCE In view of the intrinsic resistance of Pseudomonas aeruginosa to antibiotics and its potential to acquire resistance to current antibiotics, there is an urgent need to develop novel therapeutic options for the treatment of infections caused by this bacterium. Bacterial metabolic pathways have recently become a focus of interest as potential targets for the development of new antibiotics. In this study, we describe the mechanism of glycerol utilization in P. aeruginosa PAO1, which is an available carbon source in the lung environment. Our results reveal that the homeostasis of glycerol 3-phosphate (G3P), a pivotal intermediate in glycerol catabolism, is important for the growth and virulence factor production of P. aeruginosa PAO1. The mutation of G3P dehydrogenase (GlpD) and the addition of glycerol were found to reduce the tolerance of P. aeruginosa PAO1 to oxidative stress and to kanamycin. The findings highlight the importance of G3P homeostasis and suggest that GlpD is a potential drug target for the treatment of P. aeruginosa infections.

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Mechanism of pyocyanin abolishment caused by mvaT mvaU double knockout in Pseudomonas aeruginosa PAO1.

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