Bis-(3'-5')-cyclic dimeric GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa.
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ABSTRACT:
SUBMITTER: Chua SL
PROVIDER: S-EPMC3632963 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The second messenger cyclic dimeric GMP (c-di-GMP) regulates surface attachment and biofilm formation by many bacteria. For Pseudomonas fluorescens Pf0-1, c-di-GMP impacts the secretion and localization of the adhesin LapA, which is absolutely required for stable surface attachment and biofilm formation by this bacterium. In this study we characterize LapD, a unique c-di-GMP effector protein that controls biofilm formation by communicating intracellular c-di-GMP levels to the membrane-localized attachment machinery via its periplasmic domain. LapD contains degenerate and enzymatically inactive diguanylate cyclase and c-di-GMP phosphodiesterase (EAL) domains and binds to c-di-GMP through a degenerate EAL domain. We present evidence that LapD utilizes an inside-out signaling mechanism: binding c-di-GMP in the cytoplasm and communicating this signal to the periplasm via its periplasmic domain. Furthermore, we show that LapD serves as the c-di-GMP receptor connecting environmental modulation of intracellular c-di-GMP levels by inorganic phosphate to regulation of LapA localization and thus surface commitment by P. fluorescens.
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Project description:Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.
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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.
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Project description:Movement over and colonization of surfaces are important survival strategies for bacteria, and many find it advantageous to perform these activities as a group, using quorum sensing to sample population size and synchronize behavior. It is puzzling however, that swarming-proficient and virulent strains of Vibrio parahaemolyticus are silenced for the vibrio archetypal pathway of quorum sensing. Here we describe the S-signal, a pheromone that can be communicated between cells in coculture to regulate surface colonization. This signal was harvested in cell-free supernatants and demonstrated to stimulate swarming gene expression at low cell density. The S-signal was generated by the pyridoxal phosphate-dependent aminotransferase ScrA; signal reception required the periplasmic binding protein ScrB and the membrane-bound GGDEF-EAL domain-containing protein ScrC. ScrC is a bifunctional enzyme that has the ability to form and degrade the second messenger bis-(3'-5') cyclic dimeric GMP (c-di-GMP). ScrA in neighboring cells was able to alter the activity of ScrC in a ScrB-dependent manner, transforming ScrC's repressing ability to inducing activity with respect to swarming. Conversely, cell-cell signaling repressed capsule gene expression. In summary, we report that quorum sensing can stimulate swarming in V. parahaemolyticus; it does so via an alternative pathway capable of generating an autoinducing signal that influences c-di-GMP, thereby expanding the lexicon and language of cell-cell communication.
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Project description:The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action.
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Project description:Biofilms are highly structured, surface-associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that ΔsagS biofilm cells harbour the secondary messenger c-di-GMP at reduced levels similar to those observed in wild-type cells grown planktonically rather than as biofilms. Restoring c-di-GMP levels to wild-type biofilm-like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of ΔsagS biofilm cells. We likewise found that increasing c-di-GMP levels present in planktonic cells to biofilm-like levels (≥ 55 pmol mg(-1) ) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c-di-GMP levels of biofilm cells to ≤ 40 pmol mg(-1) correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signalling pathway involving a specific c-di-GMP pool regulated by SagS contributes to the resistance of P. aeruginosa biofilms.
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Project description:UnlabelledThe opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels.ImportanceOur work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.
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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.
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Project description:Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.
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