Molecular Mechanisms Underlying the Regulation of Biofilm Formation and Swimming Motility by FleS/FleR in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a major cause of nosocomial infection, can survive under diverse environmental conditions. Its great adaptive ability is dependent on its multiple signaling systems such as the two-component system (TCS). A TCS FleS/FleR has been previously identified to positively regulate a variety of virulence-related traits in P. aeruginosa PAO1 including motility and biofilm formation which are involved in the acute and chronic infections, respectively. However, the molecular mechanisms underlying these regulations are still unclear. In this study, we first analyzed the regulatory roles of each domains in FleS/FleR and characterized key residues in the FleS-HisKA, FleR-REC and FleR-AAA domains that are essential for the signaling. Next, we revealed that FleS/FleR regulates biofilm formation in a c-di-GMP and FleQ dependent manner. Lastly, we demonstrated that FleR can regulate flagellum biosynthesis independently without FleS, which explains the discrepant regulation of swimming motility by FleS and FleR.

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Antibiofilm activity of aminopropanol derivatives against Pseudomonas aeruginosa

Farmatsevtychnyi zhurnal ◽

10.32352/0367-3057.1.17.12 ◽

2018 ◽

pp. 93-100

Author(s):

D. M. Dudikova ◽

Z. S. Suvorova ◽

V. V. Nedashkivska ◽

A. O. Sharova ◽

M. L. Dronova ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Biofilm ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Inhibition Activity ◽

Chronic Infections ◽

High Concentration ◽

Biofilm Inhibition

Bacterial biofilm, particularly formed by Pseudomonas aeruginosa, are a cause of severe chronic infectious diseases. Bacteria within a biofilm are phenotypically more resistant to antibiotics and the macroorganism immune system, making it an important virulence factor for many microbes. The aminopropanol derivatives with adamantyl (KVM-97) and N-alkylaryl radicals (KVM-194, KVM-204, KVM-261, and KVM-262) were used as study object. The aim of this study was to investigate the antibiofilm activity of compounds on biofilm formation and on mature biofilm of P. aeruginosa. The effects of the aminopropanol derivatives on the biofilm mass were evaluated by using crystal violet assay. Ciprofloxacin, meropenem, ceftazidime, gentamicin were used as reference substances. Reported results demonstrate that all compounds displayed antibiofilm activity at the tested concentrations. Remarkable reduction in biofilm formation of P. aeruginosa was found after treatment with KVM-97, KVM-261 and KVM-262 in high concentration (5× MIC), biofilm inhibition activity were 84.3%, 90.5% and 83.3% respectively. After a treatment with KVM-204 at 250 μg/ml (5× MIC) 76.6% of the preformed 24-hr biofilms were destroyed. Furthermore, compounds KVM-97, KVM-194, and KVM-261 in both concentrations showed potent antibiofilm activity against the P. aeruginosa, inhibition activity values being between 56.7 and 65.7%. All tested compounds in dose-dependent manner exhibited pronounced inhibition activity against mature 5-days P. аeruginosa biofilm. It was also observed that tested compounds show high antibiofilm activity in comparison to reference antimicrobials. The aminopropanol derivatives may provide templates for a new group of antimicrobial agents and potential future therapeutics for treating chronic infections.

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Quantitative mapping of mRNA 3’ ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3’ end formation in response to azithromycin

PLoS Genetics ◽

10.1371/journal.pgen.1009634 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009634

Author(s):

Salini Konikkat ◽

Michelle R. Scribner ◽

Rory Eutsey ◽

N. Luisa Hiller ◽

Vaughn S. Cooper ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Transcriptional Response ◽

Cost Effective ◽

Chronic Infections ◽

Biofilm Growth ◽

Persistent Infections ◽

Transcriptional Pausing

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3’ ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3’ end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3’ ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3’ end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3’ end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

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Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507245112 ◽

2015 ◽

Vol 112 (36) ◽

pp. E5048-E5057 ◽

Cited By ~ 75

Author(s):

Mona W. Orr ◽

Gregory P. Donaldson ◽

Geoffrey B. Severin ◽

Jingxin Wang ◽

Herman O. Sintim ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Active Site ◽

Half Life ◽

Biofilm Formation ◽

Degradation Pathway ◽

Dependent Manner ◽

Capillary Action ◽

Diguanylate Cyclases ◽

Active Site Mutants ◽

Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

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Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa

Biology ◽

10.3390/biology10060519 ◽

2021 ◽

Vol 10 (6) ◽

pp. 519

Author(s):

Kasidid Ruksakiet ◽

Balázs Stercz ◽

Gergő Tóth ◽

Pongsiri Jaikumpun ◽

Ilona Gróf ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cyclic Amp ◽

Biofilm Formation ◽

Second Messengers ◽

Brain Heart Infusion ◽

Ambient Air ◽

Dependent Manner ◽

Environmental Signals ◽

Common Causes ◽

Camp Levels

The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain–heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO3 in 5% CO2 (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.

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Putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00297-21 ◽

2021 ◽

Author(s):

Zhexian Liu ◽

Sarzana S. Hossain ◽

Zayda Morales Moreira ◽

Cara H. Haney

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune Responses ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Pathogen ◽

Guanosine Monophosphate ◽

Genetic Approach ◽

Chronic Infections ◽

Host Immune Responses ◽

Primary Mechanism

Pseudomonas aeruginosa , an opportunistic bacterial pathogen can synthesize and catabolize a number of small cationic molecules known as polyamines. In several clades of bacteria polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, L-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or through a metabolic derivative. Here we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa . Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that L-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation, but via a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and L-arginine induced a significant increase in the intracellular level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in P. aeruginosa . Importance: Biofilm formation allows bacteria to physically attach to a surface, confers tolerance to antimicrobial agents, and promotes resistance to host immune responses. As a result, regulation of biofilm is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switching that favors chronic infection.

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Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.00414-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5060-5069 ◽

Cited By ~ 124

Author(s):

Morten T. Rybtke ◽

Bradley R. Borlee ◽

Keiji Murakami ◽

Yasuhiko Irie ◽

Morten Hentzer ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Subsequent Development ◽

Cellular Level ◽

Host Immune System ◽

Chronic Infections ◽

Content Type ◽

Genes Encoding ◽

Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

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Peptide Mix from Olivancillaria hiatula Interferes with Cell-to-Cell Communication in Pseudomonas aeruginosa

BioMed Research International ◽

10.1155/2019/5313918 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Edward Ntim Gasu ◽

Hubert Senanu Ahor ◽

Lawrence Sheringham Borquaye

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Cell Communication ◽

Microtiter Plate ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Swarming Motility

Bacteria in biofilms are encased in an extracellular polymeric matrix that limits exposure of microbial cells to lethal doses of antimicrobial agents, leading to resistance. In Pseudomonas aeruginosa, biofilm formation is regulated by cell-to-cell communication, called quorum sensing. Quorum sensing facilitates a variety of bacterial physiological functions such as swarming motility and protease, pyoverdine, and pyocyanin productions. Peptide mix from the marine mollusc, Olivancillaria hiatula, has been studied for its antibiofilm activity against Pseudomonas aeruginosa. Microscopy and microtiter plate-based assays were used to evaluate biofilm inhibitory activities. Effect of the peptide mix on quorum sensing-mediated processes was also evaluated. Peptide mix proved to be a good antibiofilm agent, requiring less than 39 μg/mL to inhibit 50% biofilm formation. Micrographs obtained confirmed biofilm inhibition at 1/2 MIC whereas 2.5 mg/mL was required to degrade preformed biofilm. There was a marked attenuation in quorum sensing-mediated phenotypes as well. At 1/2 MIC of peptide, the expression of pyocyanin, pyoverdine, and protease was inhibited by 60%, 72%, and 54%, respectively. Additionally, swarming motility was repressed by peptide in a dose-dependent manner. These results suggest that the peptide mix from Olivancillaria hiatula probably inhibits biofilm formation by interfering with cell-to-cell communication in Pseudomonas aeruginosa.

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YbeY controls the type III and type VI secretion systems and biofilm formation through RetS in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.02171-20 ◽

2020 ◽

Author(s):

Yushan Xia ◽

Congjuan Xu ◽

Dan Wang ◽

Yuding Weng ◽

Yongxin Jin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Biofilm Formation ◽

Small Rnas ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Vi Secretion System ◽

Chronic Infections ◽

Type Iii ◽

Type Vi Secretion

YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa, we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS. Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa.

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Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.01222-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 832-843 ◽

Cited By ~ 170

Author(s):

Nekane Merino ◽

Alejandro Toledo-Arana ◽

Marta Vergara-Irigaray ◽

Jaione Valle ◽

Cristina Solano ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Protein A ◽

Surface Proteins ◽

Growth Media ◽

Dependent Manner ◽

Chronic Infections ◽

The Matrix ◽

Implanted Medical Devices ◽

Biofilm Development

ABSTRACT The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

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PYED-1 Inhibits Biofilm Formation and Disrupts the Preformed Biofilm of Staphylococcus aureus

Antibiotics ◽

10.3390/antibiotics9050240 ◽

2020 ◽

Vol 9 (5) ◽

pp. 240 ◽

Cited By ~ 1

Author(s):

Adriana Vollaro ◽

Anna Esposito ◽

Eliana Pia Esposito ◽

Raffaele Zarrilli ◽

Annalisa Guaragna ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Biofilm Formation ◽

Surface Proteins ◽

Transcriptional Analysis ◽

Capsular Polysaccharides ◽

Dependent Manner ◽

Chronic Infections ◽

Concentration Dependent Manner ◽

Expression Of Genes

Pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1 (PYED-1), a heterocyclic corticosteroid derivative of deflazacort, exhibits broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. Here, we investigated the effect of PYED-1 on the biofilms of Staphylococcus aureus, an etiological agent of biofilm-based chronic infections such as osteomyelitis, indwelling medical device infections, periodontitis, chronic wound infections, and endocarditis. PYED-1 caused a strong reduction in biofilm formation in a concentration dependent manner. Furthermore, it was also able to completely remove the preformed biofilm. Transcriptional analysis performed on the established biofilm revealed that PYED-1 downregulates the expression of genes related to quorum sensing (agrA, RNAIII, hld, psm, and sarA), surface proteins (clfB and fnbB), secreted toxins (hla, hlb, and lukD), and capsular polysaccharides (capC). The expression of genes that encode two main global regulators, sigB and saeR, was also significantly inhibited after treatment with PYED-1. In conclusion, PYED-1 not only effectively inhibited biofilm formation, but also eradicated preformed biofilms of S. aureus, modulating the expression of genes related to quorum sensing, surface and secreted proteins, and capsular polysaccharides. These results indicated that PYED-1 may have great potential as an effective antibiofilm agent to prevent S. aureus biofilm-associated infections.

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