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Impacts of Ser/Thr protein kinase Stk1 on proteome, twitching motility and competitive advantage in Pseudomonas aeruginosaImpacts of Ser/Thr protein kinase Stk1 on proteome, twitching motility and competitive advantage in Pseudomonas aeruginosa

ABSTRACT: Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the World Health Organization as one of the human pathogens that urgently need to develop new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has been proved to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed the stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a TMT labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in the ∆stk1 compared with the P. aeruginosa PAO1. Detailed bioinformatics analysis of these differentially expressed proteins were performed, including GO annotation, protein domain profile, the KEGG pathway, subcellular localization as well as enrichment analysis. Notably, downregulation of type Ⅳ pilus-related proteins and upregulation of T6SS-H1-related proteins were found in ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens the bacterial twitching motility and promotes a growth competition advantage, which is mediated by type Ⅳ pilus related proteins and T6SS-H1 related proteins, respectively. These findings contribute to a better understand the physiological role of Stk1, and the proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, though the detailed regulation and mechanism of Stk1 need to be further revealed.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Pseudomonas Aeruginosa Pak

TISSUE(S): Whole Body

SUBMITTER: Xianliang Ma

LAB HEAD: Jianyi Pan

PROVIDER: PXD027148 | Pride | 2021-07-07

REPOSITORIES: Pride

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Project description:Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the WHO as a human pathogen that urgently needs the development of new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has proven to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed a stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a Tandem Mass Tag (TMT) labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to the stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in ∆stk1 compared with P. aeruginosa PAO1. A detailed bioinformatics analysis of these differentially expressed proteins was performed, including GO annotation, protein domain profile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localization and enrichment analysis. Notably, the downregulation of type IV pilus-related proteins and upregulation of T6SS-H1-related proteins were found in the ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens bacterial twitching motility and promotes a growth competition advantage, which are, respectively, mediated by type IV pilus-related proteins and T6SS-H1-related proteins. These findings contribute to a better understanding of the physiological role of Stk1, and proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, although the detailed regulation and mechanism of Stk1 still need to be revealed.

Project description:Purpose: Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date post-transcriptional regulation has not been extensively investigated. Small non-coding RNAs (sRNA) are hypothesized to play post-transcriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. Overexpressing these revealed one sRNA, PA0805.1, that when cloned, transformed into PAO1 WT and overexpressed led to broad phenotypic changes. Methods: The sRNA PA0805.1 was cloned and overexpressed in PAO1 WT. Phenotypic screens including motility, cytotoxicity and adherence assays were performed. RNA-Seq and proteomics were performed under swarming conditions compared to the empty vector strain to discover which genes were influenced by the overexpression of PA0805.1 Results: Phenotypic screens revealed that PA0805.1 resulted in reduced swarming, swimming and twitching motility, as well as increased adherence, cytotoxicity, and tobramycin resistance. A deletion mutant ∆PA0805.1 was supersusceptible to tobramycin under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty vector strain by RNA-Seq and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles were revealed, encompassing 1121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD. Conclusions: The sRNA PA0805.1 appears to regulate diverse bacterial lifestyles, most likely through a regulatory cascade.

Project description:Oberhardt2008 - Genome-scale metabolic network of Pseudomonas aeruginosa (iMO1056) This model is described in the article: Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1. Oberhardt MA, Puchałka J, Fryer KE, Martins dos Santos VA, Papin JA. J. Bacteriol. 2008 Apr; 190(8): 2790-2803 Abstract: Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen. This model is hosted on BioModels Database and identified by: MODEL1507180020. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Impacts of Ser/Thr protein kinase Stk1 on proteome, twitching motility and competitive advantage in Pseudomonas aeruginosaImpacts of Ser/Thr protein kinase Stk1 on proteome, twitching motility and competitive advantage in Pseudomonas aeruginosa

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