Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa

AbstractBacteria typically occur either as free-swimming planktonic cells or within a sessile, biofilm mode of growth. In Pseudomonas aeruginosa, the transition between these lifestyles is known to be modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP). We are interested in the control of distinct biofilm-relevant phenotypes in P. aeruginosa through the modulation of intracellular c-di-GMP. Here, we characterise motility and associated biofilm formation and dispersal in two pairs of related multi-domain proteins with putative c-di-GMP turnover domains, selected to contain additional PAS (Per-Arnt-Sim) homology domains known for their ability to process environmental stimuli. The enzymes PA0861 (RbdA) and PA2072 have distinct functions despite their similar domain structures. The ΔrbdA deletion mutant showed significantly increased biofilm formation while biofilm formation was impaired in ΔPA2072. Using a GFP transcriptional reporter fused to the cyclic di-GMP-responsive cdrA promoter, we show correlation between biofilm phenotype and c-di-GMP levels. Both proteins are shown to play a role in nitric oxide (NO) induced biofilm dispersal. We further studied pseudo-enzymes of similar architecture. PA5017 (DipA) is an inactive cyclase, and PA4959 (FimX) is described here as an inactive cyclase/phosphodiesterase. Loss of swimming and twitching motilities, respectively, is observed in deletion variants, which correlated with NO-induced biofilm dispersal phenotypes, as ΔdipA dispersed less well while ΔfimX dispersed better than wild type. The study highlights how Pseudomonas differentiates c-di-GMP output – in this case motility – using structurally very similar proteins and underlines a significant role for pseudo-enzymes in motility regulation and associated biofilm dispersal.ImportanceBacterial biofilms exert pervasive economic and societal impact across a range of environmental, engineered and clinical contexts. The secondary messenger cyclic guanosine di-phosphate, c-di-GMP, is known to control the ability of many bacteria to form biofilms. The opportunistic human pathogen Pseudomonas aeruginosa PAO1 has 38 putative enzymes that can regulate c-di-GMP turnover, and these proteins modulate various cellular functions and influence bacterial lifestyle. The specific protein sensory domains and mechanisms of motility that lead to biofilm dispersal remain to be fully understood. Here we studied multi-domain proteins with the PAS (Per-Arnt-Sim) homology domains, these being classic sensors to environmental stimuli. Our study demonstrates the significant roles for the pseudo-enzymes PA4959 (FimX) and PA5017 (DipA) in regulation of biofilm phenotype and motility. Further, enzymes with highly homologous structures, such as PA0861 (RbdA) and PA2072, have almost orthogonal function in biofilm and motility control.

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Attenuation of Quorum Sensing Controlled Virulence Factors and Biofilm Formation by Edible Fruit Extract of Coccinia indica against Pseudomonas aeruginosa

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i47b33180 ◽

2021 ◽

pp. 756-764

Author(s):

R. Shruthi Devi ◽

P. Sankar Ganesh ◽

A. S. Smiline Girija ◽

J. Vijayashree Priyadharshini

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Crystal Violet Staining ◽

Chronic Infections ◽

Fruit Extract ◽

Fluorescent Microscope ◽

Edible Fruit ◽

Coccinia Indica ◽

Opportunistic Human Pathogen

Background: Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that mainly infects immunocompromised individuals and patients with urinary tract infection and chronic infections of the respiratory pathways, including cystic fibrosis. Many quorum sensing (QS) controlled components such as bio surfactants and swarming motilities play an important role in the establishment of biofilms. Targeting these factors through anti-QS strategies prevent biofilm formation and treating infections. Coccinia indica commonly called little gourd is used to treat diabetes, wound, burn infections and has antioxidant, antibacterial and antitussive properties. Methods: The methanolic fruit extract of C. indica was prepared and screened for anti-QS and anti-biofilm formation activity. Pyocyanin inhibition, rhamnolipid, crystal violet staining assay tests was performed and the extract was observed under fluorescent microscope. Results: The results obtained are as follows - the fruit extract inhibits the pyocyanin at 58.13% and 42.27% at 0.5 mg/ml and 1.0 mg/ml, biofilm at 69.86% and 49.06% at 0.5 mg/ml and 1.0 mg/ml, inhibits rhamnolipid assay and under fluorescent microscope it is seen scattered whereas control produce biofilm matrix like appearance. Conclusion: Since less study has been made on the quorum sensing and biofilm activity of C.indica our study aimed to fulfil it and it was found that it exhibits good biofilm formation and thus can be used for treating infections.

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Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02431-20 ◽

2021 ◽

Author(s):

Jens Bo Andersen ◽

Kasper Nørskov Kragh ◽

Louise Dahl Hultqvist ◽

Morten Rybtke ◽

Martin Nilsson ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Single Cell ◽

Biofilm Formation ◽

Ectopic Expression ◽

Second Messenger ◽

Flow Cell ◽

Diguanylate Cyclases ◽

Biofilm Dispersal ◽

High Level

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. C-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We have previously presented evidence that ectopic expression in Pseudomonas aeruginosa of the Escherichia coli phosphodiesterase YhjH results in biofilm dispersal. More recently, however, evidence has been presented that induction of native c-di-GMP phosphodiesterases does not lead to dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for development of anti-biofilm drugs. However, here we demonstrate that induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed does result in dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and in a flow-cell biofilm system.

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Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain

Applied and Environmental Microbiology ◽

10.1128/aem.01233-10 ◽

2010 ◽

Vol 76 (24) ◽

pp. 8160-8173 ◽

Cited By ~ 103

Author(s):

Shuwen An ◽

Ji'en Wu ◽

Lian-Hui Zhang

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Regulatory Protein ◽

Phosphodiesterase Activity ◽

Low Oxygen ◽

Ggdef Domain ◽

Biofilm Dispersal ◽

Biofilm Development

ABSTRACT 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.

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Genome-Wide Mapping Reveals Complex Regulatory Activities of BfmR in Pseudomonas aeruginosa

Microorganisms ◽

10.3390/microorganisms9030485 ◽

2021 ◽

Vol 9 (3) ◽

pp. 485

Author(s):

Ke Fan ◽

Qiao Cao ◽

Lefu Lan

Keyword(s):

Pseudomonas Aeruginosa ◽

Chromatin Immunoprecipitation ◽

Biofilm Formation ◽

High Throughput Sequencing ◽

Response Regulator ◽

Cellular Functions ◽

Genome Wide ◽

Component Systems ◽

Genes Encoding ◽

Two Component Systems

BfmR is a response regulator that modulates diverse pathogenic phenotypes and induces an acute-to-chronic virulence switch in Pseudomonas aeruginosa, an important human pathogen causing serious nosocomial infections. However, the mechanisms of action of BfmR remain largely unknown. Here, using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we showed that 174 chromosomal regions of P. aeruginosa MPAO1 genome were highly enriched by coimmunoprecipitation with a C-terminal Flag-tagged BfmR. Integration of these data with global transcriptome analyses revealed that 172 genes in 106 predicted transcription units are potential targets for BfmR. We determined that BfmR binds to and modulates the promoter activity of genes encoding transcriptional regulators CzcR, ExsA, and PhoB. Intriguingly, BfmR bound to the promoters of a number of genes belong to either CzcR or PhoB regulon, or both, indicating that CzcRS and PhoBR two-component systems (TCSs) deeply feed into the BfmR-mediated regulatory network. In addition, we demonstrated that phoB is required for BfmR to promote the biofilm formation by P. aeruginosa. These results delineate the direct BfmR regulon and exemplify the complexity of BfmR-mediated regulation of cellular functions in P. aeruginosa.

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Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.01601-09 ◽

2010 ◽

Vol 192 (12) ◽

pp. 2973-2980 ◽

Cited By ~ 91

Author(s):

Rivka Glick ◽

Christie Gilmour ◽

Julien Tremblay ◽

Shirley Satanower ◽

Ofir Avidan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Essential Element ◽

Biosurfactant Production ◽

Twitching Motility ◽

Wild Type ◽

Bacterial Surface ◽

Surface Motility ◽

Opportunistic Human Pathogen ◽

Biofilm Development

ABSTRACT Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.

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Role of quorum sensing in UVA-induced biofilm formation in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.000932 ◽

2020 ◽

Vol 166 (8) ◽

pp. 735-750 ◽

Cited By ~ 1

Author(s):

Magdalena Pezzoni ◽

Ramón A. Pizarro ◽

Cristina S. Costa

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Type Species ◽

Transcriptional Level ◽

Content Type ◽

Link Type ◽

Opportunistic Human Pathogen ◽

Biofilm Development

Pseudomonas aeruginosa , a versatile bacterium present in terrestrial and aquatic environments and a relevant opportunistic human pathogen, is largely known for the production of robust biofilms. The unique properties of these structures complicate biofilm eradication, because they make the biofilms very resistant to diverse antibacterial agents. Biofilm development and establishment is a complex process regulated by multiple regulatory genetic systems, among them is quorum sensing (QS), a mechanism employed by bacteria to regulate gene transcription in response to population density. In addition, environmental factors such as UVA radiation (400–315 nm) have been linked to biofilm formation. In this work, we further investigate the mechanism underlying the induction of biofilm formation by UVA, analysing the role of QS in this phenomenon. We demonstrate that UVA induces key genes of the Las and Rhl QS systems at the transcriptional level. We also report that pelA and pslA genes, which are essential for biofilm formation and whose transcription depends in part on QS, are significantly induced under UVA exposure. Finally, the results demonstrate that in a relA strain (impaired for ppGpp production), the UVA treatment does not induce biofilm formation or QS genes, suggesting that the increase of biofilm formation due to exposure to UVA in P. aeruginosa could rely on a ppGpp-dependent QS induction.

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Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal

Folia Microbiologica ◽

10.1007/s12223-018-0585-4 ◽

2018 ◽

Vol 63 (4) ◽

pp. 413-432 ◽

Cited By ~ 31

Author(s):

Sinosh Skariyachan ◽

Vaishnavi Sneha Sridhar ◽

Swathi Packirisamy ◽

Supreetha Toplar Kumargowda ◽

Sneha Basavaraj Challapilli

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Molecular Basis ◽

Biofilm Dispersal

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The Nitrite Transporter Facilitates Biofilm Formation via Suppression of Nitrite Reductase and Is a New Antibiofilm Target in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00878-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Ji-Su Park ◽

Ha-Young Choi ◽

Won-Gon Kim

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Biofilm Formation ◽

No Production ◽

Content Type ◽

A Genome ◽

Biofilm Dispersal ◽

Gmp Production

ABSTRACT Biofilm-forming bacteria, including the Gram-negative Pseudomonas aeruginosa, cause multiple types of chronic infections and are responsible for serious health burdens in humans, animals, and plants. Nitric oxide (NO) has been shown to induce biofilm dispersal via triggering a reduction in cyclic-di-GMP levels in a variety of bacteria. However, how NO, at homeostatic levels, also facilitates biofilm formation is unknown. Here, we found that complestatin, a structural analog of vancomycin isolated from Streptomyces, inhibits P. aeruginosa biofilm formation by upregulating NO production via nitrite reductase (NIR) induction and c-di-GMP degradation via phosphodiesterase (PDE) stimulation. The complestatin protein target was identified as a nitrite transporter from a genome-wide screen using the Keio Escherichia coli knockout library and confirmed using nitrite transporter knockout and overexpression strains. We demonstrated that the nitrite transporter stimulated biofilm formation by controlled NO production via appropriate NIR suppression and subsequent diguanylate cyclase (DGC) activation, not PDE activity, and c-di-GMP production in E. coli and P. aeruginosa. Thus, this study provides a mechanism for NO-mediated biofilm formation, which was previously not understood. IMPORTANCE Bacterial biofilms play roles in infections and avoidance of host defense mechanisms of medically important pathogens and increase the antibiotic resistance of the bacteria. Nitric oxide (NO) is reported to be involved in both biofilm formation and dispersal, which are conflicting processes. The mechanism by which NO regulates biofilm dispersal is relatively understood, but there are no reports about how NO is involved in biofilm formation. Here, by investigating the mechanism by which complestatin inhibits biofilm formation, we describe a novel mechanism for governing biofilm formation in Escherichia coli and Pseudomonas aeruginosa. Nitrite transporter is required for biofilm formation via regulation of NO levels and subsequent c-di-GMP production. Additionally, the nitrite transporter contributes more to P. aeruginosa virulence than quorum sensing. Thus, this study identifies nitrite transporters as new antibiofilm targets for future practical and therapeutic agent development.

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Growth inhibition of adherent Pseudomonas aeruginosa by an N-butanoyl-l-homoserine lactone analog

Canadian Journal of Microbiology ◽

10.1139/w10-013 ◽

2010 ◽

Vol 56 (4) ◽

pp. 317-325 ◽

Cited By ~ 14

Author(s):

Pouneh Khalilzadeh ◽

Barbora Lajoie ◽

Salomé El Hage ◽

Aurélie Furiga ◽

Geneviève Baziard ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Communication Systems ◽

Homoserine Lactone ◽

Adherent Cells ◽

Dependent Manner ◽

Structure Activity ◽

Opportunistic Human Pathogen ◽

Dose Dependent ◽

Related Compounds

The discovery of quorum sensing (QS) communication systems regulating bacterial virulence has afforded a novel opportunity for controlling infectious bacteria by interfering with QS. Pseudomonas aeruginosa is an example of an opportunistic human pathogen for which N-acyl homoserine lactone (AHL)-related compounds have been described as potent inhibitors of biofilm formation and virulence factors, given their similarity to the natural QS autoinducers (AHLs). Our purpose was to design potent analogs of N-butanoyl-l-homoserine lactone (C4-HSL) and to screen them for biological activity. Eleven original compounds characterized by the modification of the lactone moiety were screened for their ability to impair biofilm formation. Among them, compound 11 was able to modify the growth kinetics and to restrict the number of adherent cells when added from the early stages of biofilm formation (i.e., adhesion and microcolony formation) in a dose-dependent manner. To demonstrate antagonism with C4-HSL, we showed that the inhibition of biofilm formation by compound 11 was impaired when C4-HSL was added. Structure–activity relationships are discussed with respect to the results obtained.

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Effects of Glycyrrhizin on Multi-Drug Resistant Pseudomonas aeruginosa

Pathogens ◽

10.3390/pathogens9090766 ◽

2020 ◽

Vol 9 (9) ◽

pp. 766

Author(s):

Nicholas J. Carruthers ◽

Sharon A. McClellan ◽

Mallika Somayajulu ◽

Ahalya Pitchaikannu ◽

Denise Bessert ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Type Iii Secretion ◽

Biofilm Formation ◽

Inhibitory Concentration ◽

Drug Resistant ◽

Liquid Chromatography Mass Spectrometry ◽

Corneal Epithelial ◽

Type Iii ◽

Biofilm Dispersal

The effects of glycyrrhizin (GLY) on multi-drug resistant (MDR) systemic (MDR9) vs. ocular (B1045) Pseudomonas aeruginosa clinical isolates were determined. Proteomes of each isolate with/without GLY treatment were profiled using liquid chromatography mass spectrometry (LC-MS/MS). The effect of GLY on adherence of MDR isolates to immortalized human (HCET) and mouse (MCEC) corneal epithelial cells, and biofilm and dispersal was tested. Both isolates were treated with GLY (0.25 minimum inhibitory concentration (MIC), 10 mg/mL for MDR9 and 3.75 mg/mL for B1045) and subjected to proteomic analysis. MDR9 had a greater response to GLY (51% of identified proteins affected vs. <1% in B1045). In MDR9 vs. controls, GLY decreased the abundance of proteins for: antibiotic resistance, biofilm formation, and type III secretion. Further, antibiotic resistance and type III secretion proteins had higher control abundances in MDR9 vs. B1045. GLY (5 and 10 mg/mL) significantly reduced binding of both isolates to MCEC, and B1045 to HCET. MDR9 binding to HCET was only reduced at 10 mg/mL GLY. GLY (5 and 10 mg/mL) enhanced dispersal for both isolates, at early (6.5 h) but not later times (24–72 h). This study provides evidence that GLY has a greater effect on the proteome of MDR9 vs. B1045, yet it was equally effective at disrupting adherence and early biofilm dispersal.

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