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Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

ABSTRACT: In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we performed a combination of in silico analysis and experimental validation to identify a core set of Tat substrates in the opportunistic pathogen Pseudomonas aeruginosa. In silico analysis predicted 44 putative Tat signal peptides in the P. aeruginosa PA14 proteome. We developed an improved amidase-based Tat reporter assay to show that 33 of these are real Tat signal peptides. In addition, in silico analysis of the full translated genome revealed a Tat candidate with a missassigned start codon. We showed that it is a new periplasmic protein in P. aeruginosa. Altogether we discovered and validated 34 Tat substrates. These show little overlap with Escherichia coli Tat substrates, and functional analysis points to a general role for the P. aeruginosa Tat system in the colonization of environmental niches and pathogenicity.

SUBMITTER: Gimenez MR

PROVIDER: S-EPMC6085387 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

Gimenez Maxime Rémi MR Chandra Govind G Van Overvelt Perrine P Voulhoux Romé R Bleves Sophie S Ize Bérengère B

Scientific reports 20180809 1

In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we pe ...[more]

PMID: 30093651

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Project description:Pseudomonas aeruginosa is an opportunistic pathogen prevalent in the environment and in health care settings. Transmission in the health care setting occurs through human-human interactions and/or contact with contaminated surfaces. Moist surfaces such as respirators, sink and tub drains, and even disinfectants can serve as reservoirs. Dry surfaces such as plastic and stainless steel could also serve as a reservoir but would necessitate some degree of tolerance to desiccation. Using an assay to measure P. aeruginosa tolerance to desiccation on plastic and stainless-steel surfaces, we found that only 0.05 to 0.1% of the desiccated cells could be recovered 24 h postdesiccation. We took advantage of the strong selection imposed by desiccation to identify genes important for tolerance using Tn-seq. A highly saturated Tn-seq library was desiccated on plastic and stainless-steel surfaces. NexGen sequencing of the recovered cells identified 97 genes important for survival. Comparing cells desiccated under low- and high-nutrient conditions allowed for differentiation of genes important for desiccation tolerance. The 53 genes identified in the latter analysis are involved in maintenance of cell envelope integrity, purine and pyrimidine biosynthesis, tricarboxylic acid (TCA) cycle, and the hydrolysis of misfolded proteins. The Tn-seq findings were validated by competition experiments with wild-type (WT) cells and select Tn insertion mutants. Mutants lacking carB and surA demonstrated the largest fitness defects, indicating that pyrimidine biosynthesis and outer membrane integrity are essential for desiccation tolerance. Increased understanding of desiccation tolerance could provide insight into approaches to control environmental reservoirs of P. aeruginosa. IMPORTANCE Health care-associated infections (HAIs) caused by Pseudomonas aeruginosa result in significant morbidity and mortality and are a significant economic burden. Moist environments that promote biofilm formation are an important reservoir for P. aeruginosa. Dry environments may also serve as a reservoir but would require some degree of desiccation tolerance. Here, we took a genome-wide approach to identify genes important for desiccation tolerance on plastic and stainless-steel surfaces. Genes involved in assembly of outer membrane proteins and pyrimidine biosynthesis were particularly important. Strains lacking these functions were unable to tolerate surface desiccation. These findings suggest that inhibitors of these pathways could be used to prevent P. aeruginosa survival on dry surfaces.

Project description:Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms.We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to < 1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity.Most PCSK enzymes display strong positive expression correlation with predicted target proteins in our genome-wide analysis. We also show that expression correlation screen combined with a cleavage site-prediction analysis can be used to identify novel bona fide target molecules for PCSKs. Exploring the positively correlating genes can thus offer additional insights into the biology of proprotein convertases.

Dataset Information

Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

Publications

Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

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