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The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa.

ABSTRACT: The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP) levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC), which catalyzes the synthesis of the second messenger c-di-GMP - an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network.

SUBMITTER: Strempel N

PROVIDER: S-EPMC5702645 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa.

Strempel Nikola N Nusser Michael M Neidig Anke A Brenner-Weiss Gerald G Overhage Joerg J

Frontiers in microbiology 20171122

The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancemen ...[more]

PMID: 29213262

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Project description:With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

Project description:BackgroundPseudomonas aeruginosa (P. aeruginosa) is a common pathogenic bacterium which causes pleural empyema, and infection of P. aeruginosa is often associated with biofilm. The aim of this study was to establish a model of rabbit empyema infected by P. aeruginosa to determine whether it causes the formation of biofilm in the pleural cavity. Furthermore, we investigated the effect of cyclic diguanosine monophosphate (c-di-GMP) on biofilm formation in this P. aeruginosa empyema model.MethodsTwenty rabbits were used and randomly divided into five groups: PAO1, PAO1ΔwspF, and PAO1/p lac-yhjH infection groups, and Luria-Bertani (LB) broth and turpentine control groups. A drainage catheter was implanted into the pleural cavity through thoracentesis. The three infection groups were respectively infected with PAO1, PAO1ΔwspF, and PAO1/p lac-yhjH strains, which caused empyema. The two control groups were injected with LB or turpentine. After 4 days of infection, we sacrificed the rabbits. We evaluated the pathology of pleura through hematoxylin-eosin staining. Colony count and crystal violet assay were used to analyze the biofilm formation on the surface of catheters. Scanning electron was used to observe the biofilm on the surface of the pleura. Peptide nucleic acids-fluorescence in situ hybridization (PNA-FISH) was used to observe the biofilm in the fibrinous deposition.ResultsBy the PNA-FISH assay, biofilms were observed in the fibrinous deposition of the three infection groups. The red fluorescence area of the PAO1ΔwspF infection group was larger than that of the PAO1 and PAO1/p lac -yhjH infection groups. Through electron microscopy, we observed that PAO1 strains were embedded in an electron-dense extracellular matrix on the surface of pleural tissue, and appeared to be biofilm-like structures. For the crystal violet assay, the optical density values of different groups were significantly different: PAO1ΔwspF > PAO1 > PAO1/p lac-yhjH > control groups (P<0.05).ConclusionsTo the best knowledge of the authors, this is the first study to report P. aeruginosa forming biofilm in a novel animal model of pleural empyema. In addition, c-di-GMP signaling molecules played an important role in biofilm formation in the pleural cavity.

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Project description:Light is known to trigger regulatory responses in diverse organisms, including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial infections. We grew P. aeruginosa strain PA14 in a colony morphology assay and found that growth under prolonged exposure to low-intensity blue light inhibited biofilm matrix production and thereby the formation of vertical biofilm structures (i.e., "wrinkles"). Light-dependent inhibition of biofilm wrinkling was correlated with low levels of cyclic di-GMP (c-di-GMP), consistent with the role of this signal in stimulating matrix production. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified c-di-GMP phosphodiesterases that contribute to light-dependent inhibition of biofilm wrinkling. One of these, RmcA, was previously characterized by our group for its role in mediating the effect of redox-active P. aeruginosa metabolites called phenazines on biofilm wrinkle formation. Our results suggest that an RmcA sensory domain that is predicted to bind a flavin cofactor is involved in light-dependent inhibition of wrinkling. Together, these findings indicate that P. aeruginosa integrates information about light exposure and redox state in its regulation of biofilm development.IMPORTANCE Light exposure tunes circadian rhythms, which modulate the immune response and affect susceptibility to infection in plants and animals. Though molecular responses to light are defined for model plant and animal hosts, analogous pathways that function in bacterial pathogens are understudied. We examined the response to light exposure in biofilms (matrix-encased multicellular assemblages) of the nonphotosynthetic bacterium Pseudomonas aeruginosa We found that light at intensities that are not harmful to human cells inhibited biofilm maturation via effects on cellular signals. Because biofilm formation is a critical factor in many types of P. aeruginosa infections, including burn wound infections that may be exposed to light, these effects could be relevant for pathogenicity.

Dataset Information

The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa.

Publications

The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in <i>Pseudomonas aeruginosa</i>.

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