Host cell-based screening assays for identification of molecules targeting Pseudomonas aeruginosa cyclic di-GMP signaling and biofilm formation
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PROVIDER: S-EPMC10684931 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the DeltabifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the DeltabifA mutant but does not contribute to the observed swarming defect. The DeltabifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.
| S-EPMC2168662 | biostudies-literature
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.
| S-EPMC7576012 | biostudies-literature
Project description:Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling.
| S-EPMC11197132 | biostudies-literature
Project description:Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.
| S-EPMC2847200 | biostudies-literature
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.
| S-EPMC7317040 | biostudies-literature
Project description:During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.
| S-EPMC2837407 | biostudies-literature
Project description:The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.
| S-EPMC4933438 | biostudies-literature
Project description: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.
| S-EPMC5702645 | biostudies-literature
Project description:Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.
| S-EPMC3008239 | biostudies-literature
Project description:Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.
| S-EPMC3719571 | biostudies-literature