Dataset Information

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa.

ABSTRACT: The Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA from Salmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants of Salmonella enterica, Escherichia coli, and Saccharomyces cerevisiae that lack a functional RidA exhibit growth defects, suggesting that 2AA metabolic stress is similarly conserved. The PubSEED database shows Pseudomonas aeruginosa (PAO1) encodes eight members of the Rid superfamily. Mutants of P. aeruginosa PAO1 lacking each of five Rid proteins were screened, and the mutant phenotypes that arose in the absence of PA5339 were dissected. A PA5339::Tn mutant has growth, motility, and biofilm defects that can all be linked to the accumulation of 2AA. Further, the PA5339 protein was demonstrably a 2AA deaminase in vitro and restored metabolic balance to a S. enterica ridA mutant in vivo The data presented here show that the RidA paradigm in Pseudomonas aeruginosa had similarities to those described in other organisms but was distinct in that deleting only one of multiple homologs generated deficiencies. Based on the collective data presented here in, PA5339 was renamed RidA.IMPORTANCE RidA is a widely conserved protein that prevents endogenous metabolic stress caused by 2-aminoacrylate (2AA) damage to pyridoxal 5'-phosphate (PLP)-dependent enzymes in prokaryotes and eukaryotes. The framework for understanding the accumulation of 2AA and its consequences have largely been defined in Salmonella enterica We show here that in P. aeruginosa (PAO1), 2AA accumulation leads to reduced growth, compromised motility, and defective biofilm formation. This study expands our knowledge how the metabolic architecture of an organism contributes to the consequences of 2AA inactivation of PLP-dependent enzymes and identifies a key RidA protein in P. aeruginosa.

SUBMITTER: Irons J

PROVIDER: S-EPMC6199476 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa.

Irons Jessica J Hodge-Hanson Kelsey M KM Downs Diana M DM

Journal of bacteriology 20181023 22

The Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA from Salmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants of Salmonella enterica, Escherichia coli, and Saccharomyces cerevisiae that lack a functional RidA exhibit growth defects, suggesting that 2AA meta ...[more]

PMID: 30181125

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Project description:UNLABELLED:Human pathogens frequently use protein mimicry to manipulate host cells in order to promote their survival. Here we show that the opportunistic pathogen Pseudomonas aeruginosa synthesizes a structural homolog of the human ?2-macroglobulin, a large-spectrum protease inhibitor and important player of innate immunity. Small-angle X-ray scattering analysis demonstrated that the fold of P. aeruginosa MagD (PA4489) is similar to that of the human macroglobulin and undergoes a conformational modification upon binding of human neutrophil elastase. MagD synthesis is under the control of a general virulence regulatory pathway including the inner membrane sensor RetS and the RNA-binding protein RsmA, and MagD undergoes cleavage from a 165-kDa to a 100-kDa form in all clinical isolates tested. Fractionation and immunoprecipitation experiments showed that MagD is translocated to the bacterial periplasm and resides within the inner membrane in a complex with three other molecular partners, MagA, MagB, and MagF, all of them encoded by the same six-gene genetic element. Inactivation of the whole 10-kb operon on the PAO1 genome resulted in mislocalization of uncleaved, in trans-provided MagD as well as its rapid degradation. Thus, pathogenic bacteria have acquired a homolog of human macroglobulin that plays roles in host-pathogen interactions potentially through recognition of host proteases and/or antimicrobial peptides; it is thus essential for bacterial defense. IMPORTANCE:The pathogenesis of Pseudomonas aeruginosa is multifactorial and relies on surface-associated and secreted proteins with different toxic activities. Here we show that the bacterium synthesizes a 160-kDa structural homolog of the human large-spectrum protease inhibitor ?2-macroglobulin. The bacterial protein is localized in the periplasm and is associated with the inner membrane through the formation of a multimolecular complex. Its synthesis is coregulated at the posttranscriptional level with other virulence determinants, suggesting that it has a role in bacterial pathogenicity and/or in defense against the host immune system. Thus, this new P. aeruginosa macromolecular complex may represent a future target for antibacterial developments.

Project description:Pseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNA-sequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, β-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.

Dataset Information

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa.

Publications

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa.

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