Abstract
BackgroundThe genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles.ResultsPhenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically.ConclusionsOur results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.
Highlights
The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/ Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence
The mutant strain demonstrated a delayed production of several virulence factors, on the test plates containing glycerol where the growth of the mutant was significantly slower when compared to the wild-type strain PAO1
The mutant strain secreted a reduced amount of pyoverdine (57% of the amount of the parent strain) when both strains were grown in liquid Casamino Acids medium (CAA) medium (Table 2)
Summary
The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/ Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Protein phosphorylation is considered the universal language for inter- and intra-cellular communication in all living organisms This process, catalysed by protein kinases, enables the translation of extracellular signals into cellular responses and allows for adaptation to a constantly changing environment. A number of bacterial eukaryotic-type Ser/Thr protein kinases (STPKs) and phosphoprotein phosphatases have been identified. These enzymes operate in many bacteria, in which they constitute a signalling network that is independent of the canonical two-component system circuits. Prokaryotic STPKs regulate various cellular functions, such as developmental processes [5,6], primary and secondary metabolism [7,8], stress responses [9], and biofilm formation [10]
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