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

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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A GENETICALLY DISTINCT FORM OF CYCLIC AMP PHOSPHODIESTERASE ASSOCIATED WITH CHROMOMERE 3D4 IN DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/91.3.521 ◽

1979 ◽

Vol 91 (3) ◽

pp. 521-535

Author(s):

John A Kiger ◽

Eric Golanty

Keyword(s):

Drosophila Melanogaster ◽

Cyclic Amp ◽

Structural Gene ◽

Regulatory Gene ◽

Heat Stability ◽

Normal Female ◽

Dependent Manner ◽

Cyclic Amp Phosphodiesterase ◽

Heat Stable ◽

Camp Levels

ABSTRACT Two cyclic AMP phosphodiesterase enzymes (E.C.3.1.4.17) are present in homogenates of adult Drosophila melanogaster. The two enzymes differ from one another in heat stability, affinity for Mg++, Ca++ activation and molecular weight. They do not differ markedly in their affinities for cyclic AMP, and both exhibit anomalous Michaelis-Menten kinetics. The more heatlabile enzyme is controlled in a dosage-dependent manner by chromomere 3D4 of the X chromosome and is absent in flies that are deficient for chromomere 3D4. Chromomere 3D4 is also necessary for the maintenance of normal cAMP levels, for male fertility, and for normal female fertility and oogenesis. The structural gene(s) for the more heat-stable enzyme is located outside of chromomeres 3C12-3D4. Whether 3D4 contains a structural gene, or a regulatory gene necessary for the presence of the labile enzyme, remains to be determined.

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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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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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The SagS sensory protein modulates biofilm formation and c-di-GMP levels by Pseudomonas aeruginosa in response to glucose-6-phosphate.

10.5194/biofilms9-122 ◽

2020 ◽

Author(s):

Soyoung Park ◽

Jozef Dingemans ◽

Madison Gowett ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Genetic Analysis ◽

Signaling Pathway ◽

Biofilm Formation ◽

Phosphate Concentration ◽

Dependent Manner ◽

Diguanylate Cyclase ◽

Swarming Motility ◽

Two Component ◽

Insight Into

<p>In <em>Pseudomonas aeruginosa</em>, the orphan two-component sensor SagS contributes to both, the transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment as glucose-6-phosphate likewise enhanced biofilm formation. We show that exposure to glucose-6-phosphate results in decreased swarming motility but increased cellular c-di-GMP levels in biofilms. Genetic analysis indicated that the diguanylate cyclase NicD is an activator of biofilm formation and is not only required for enhanced biofilm formation in response to glucose-6-phosphate but also interacts with SagS. Our findings indicate glucose-6-phosphate to likely mimic a signal or conditions sensed by SagS to activate its motile-sessile switch function. Additionally, our findings provide new insight into the interfaces between the ligand-mediated TCS signaling pathway and c-di-GMP levels.</p>

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Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00188-17 ◽

2017 ◽

Vol 199 (16) ◽

Cited By ~ 13

Author(s):

Ryan N. C. Buensuceso ◽

Martin Daniel-Ivad ◽

Sara L. N. Kilmury ◽

Tiffany L. Leighton ◽

Hanjeong Harvey ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cyclic Amp ◽

Response Regulator ◽

Opportunistic Pathogen ◽

Regulatory Elements ◽

Twitching Motility ◽

Camp Phosphodiesterase ◽

Content Type ◽

Polar Localization ◽

Camp Levels

ABSTRACT FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds—lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB—pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility. IMPORTANCE Pseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements—including the PilSR two-component system—to cell poles for optimal function.

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Cyclic AMP and calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle

The Journal of Cell Biology ◽

10.1083/jcb.200505117 ◽

2005 ◽

Vol 170 (4) ◽

pp. 551-557 ◽

Cited By ~ 95

Author(s):

Flávio H. Beraldo ◽

Fabiana M. Almeida ◽

Aline M. da Silva ◽

Célia R.S. Garcia

Keyword(s):

Cell Cycle ◽

Plasmodium Falciparum ◽

Cyclic Amp ◽

Signaling Pathways ◽

Second Messengers ◽

Parasite Cell ◽

Camp Analogue ◽

Cell Permeable Peptide ◽

Dependent Protein ◽

Camp Levels

The host hormone melatonin increases cytoplasmic Ca2+ concentration and synchronizes Plasmodium cell cycle (Hotta, C.T., M.L. Gazarini, F.H. Beraldo, F.P. Varotti, C. Lopes, R.P. Markus, T. Pozzan, and C.R. Garcia. 2000. Nat. Cell Biol. 2:466–468). Here we show that in Plasmodium falciparum melatonin induces an increase in cyclic AMP (cAMP) levels and cAMP-dependent protein kinase (PKA) activity (40 and 50%, respectively). When red blood cells infected with P. falciparum are treated with cAMP analogue adenosine 3′,5′-cyclic monophosphate N6-benzoyl/PKA activator (6-Bz-cAMP) there is an alteration of the parasite cell cycle. This effect appears to depend on activation of PKA (abolished by the PKA inhibitors adenosine 3′,5′-cyclic monophosphorothioate/8 Bromo Rp isomer, PKI [cell permeable peptide], and H89). An unexpected cross talk was found to exist between the cAMP and the Ca2+-dependent signaling pathways. The increases in cAMP by melatonin are inhibited by blocker of phospholipase C U73122, and addition of 6-Bz-cAMP increases cytosolic Ca2+ concentration, through PKA activation. These findings suggest that in Plasmodium a highly complex interplay exists between the Ca2+ and cAMP signaling pathways, but also that the control of the parasite cell cycle by melatonin requires the activation of both second messenger controlled pathways.

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Garlic (Allium sativum L.) Ethanolic Extract Capability to Inhibit Pseudomonas aeruginosa Biofilm Formation

Journal Of Medicine & Health ◽

10.28932/jmh.v3i1.3143 ◽

2021 ◽

Vol 3 (1) ◽

pp. 1-8

Author(s):

Lisa Gosal ◽

Suryani Hutomo ◽

Christiane M Sooai

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Allium Sativum ◽

Brain Heart Infusion ◽

Ethanolic Extract ◽

Assay Method ◽

Minimum Concentration ◽

Significant Difference ◽

Microplate Reader ◽

One Way Anova

Garlic extract (Allium sativum L.) is known to contain substances that can inhibitbacterial adhesion. This study aims to explore the ability of garlic (Allium sativum L.) extract toinhibit the adhesion of P. aeruginosa. Ethanolic garlic (Allium sativum L.) extract was made bythe maceration method. A bacterial adherence assay was performed used the static microtiter biofilm assay method on 96-well plates. Pseudomonas aeruginosa was inoculated in added garlic(Allium sativum L.) extract to brain heart infusion (BHI) broth in various concentrations. Biofilmbacteria on wall plates were stained with 0.1% Crystal violet and were extracted using 96%,subsequently were measured using a microplate reader with absorbance at 595 nm. Decreasedoptical density value equal with increased extract concentration. Statistical analysis using One-way ANOVA revealed a significant difference in the optical density (OD) value between thegroups (p<0.05). The minimum concentration obtained in this study was 156.25 ? g/ml.Conclusion, ethanolic garlic (Allium sativum L.) extract can inhibit adherence to P. aeruginosa.Keywords: Garlic ethanolic extract; Pseudomonas aeruginosa; adherence.

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Pseudomonas aeruginosa synthesizes the autoinducers of its oxylipin-dependent quorum sensing system extracellularly

10.1101/2021.09.08.459554 ◽

2021 ◽

Author(s):

Eriel Martínez ◽

Carlos J. Orihuela ◽

Javier Campos-Gomez

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Outer Membrane ◽

Biofilm Formation ◽

Octadecenoic Acid ◽

Secretion System ◽

Genetic Screen ◽

Dependent Manner ◽

Sensing System ◽

Quorum Sensing System

ABSTRACTThe oxylipin-dependent quorum sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two oxylipin autoinducers, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, and contrary to the prevailing notion that bacterial autoinducers are synthesized intracellularly, we show that 10-HOME and 7,10-DiHOME biosynthesis occurs extracellularly, and this requires the secretion of the oxylipin synthases. We implemented a genetic screen of P. aeruginosa strain PAO1, which identified fourteen genes required for the synthesis of oxylipins. Among the identified genes, four encoded components of the ODS system and the other ten were part of the Xcp type II secretion system (T2SS). We created a deletion mutant of xcpQ, which encodes the outer membrane component of Xcp, and found it recapitulated the impaired functionality of the transposon mutants. Upon further examination, the lack of ODS function was demonstrated to be caused by the blocking of the DS enzymes secretion. Notably, the xcpQ mutant activated the ODS system when exposed to 10-HOME and 7,10-DiHOME, indicating that the sensing component of this quorum sensing system remains fully functional. In contrast with the detrimental effect previously described for T2SS in biofilm formation, here we observed that T2SS was required for robust in vitro and in vivo biofilm formation in an ODS dependent manner. To the best of our knowledge, this study is the first to find QS autoinducers that are synthetized in the extracellular space and provides new evidence for the role of the T2SS for biofilm formation in P. aeruginosa.IMPORTANCEWe previously showed that the ODS quorum sensing system of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Herein, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp Type 2 secretion system of P. aeruginosa and thus, the biosynthesis of oxylipins occur extracellularly. Biofilm formation, which was thought to be impaired as result of Xcp activity, was found to be enhanced as result of ODS activation. This is a unique QS system strategy and reveals a new way by which P. aeruginosa interacts with the host environment.

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Antagonistic control of fluid secretion by the Malpighian tubules ofTenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers

Journal of Experimental Biology ◽

10.1242/jeb.205.4.493 ◽

2002 ◽

Vol 205 (4) ◽

pp. 493-501 ◽

Cited By ~ 7

Author(s):

U. I. M. Wiehart ◽

S. W. Nicolson ◽

R. A. Eigenheer ◽

D. A. Schooley

Keyword(s):

Cyclic Amp ◽

Cyclic Gmp ◽

Second Messengers ◽

Fluid Secretion ◽

Tenebrio Molitor ◽

Inhibitory Effect ◽

Malpighian Tubules ◽

Dependent Manner ◽

Response Curves

SUMMARYFluid secretion by insect Malpighian tubules is controlled by haemolymph-borne factors. The mealworm Tenebrio molitor provides the first known example of antagonistic interactions between endogenous neuropeptides acting on Malpighian tubules. The two corticotropin-releasing-factor (CRF)-related diuretic peptides previously isolated from Tenebrio molitor, Tenmo-DH37 and Tenmo-DH47, were found to stimulate Tenebrio molitor tubules in vitro in a dose-dependent manner with EC50 values of 0.12 nmol l–1 and 26 nmol l–1 respectively. However, no synergistic or additive effect was observed when these two peptides were tested simultaneously. We then investigated antagonism between second messengers: dose–response curves were constructed for stimulation of Tenebrio molitor tubules by cyclic AMP and their inhibition by cyclic GMP. When both cyclic nucleotides were included in the bathing Ringer, the stimulatory effect of cyclic AMP was neutralised by cyclic GMP. Similarly, the stimulatory effect of Tenmo-DH37 was reversed on addition of an antidiuretic peptide (Tenmo-ADF), which was recently isolated from Tenebrio molitor and acts via cyclic GMP. The cardioacceleratory peptide CAP2b, originally isolated from Manduca sexta, also increases intracellular cyclic GMP levels and inhibited fluid secretion by Tenebrio molitor tubules, with an EC50 value of 85 nmol l–1. This inhibitory effect was reversed by Tenmo-DH37. Endogenous diuretic and antidiuretic peptides, effective at low concentrations and acting via antagonistic second messengers, have the potential for fine control of secretion rates in the Malpighian tubules of Tenebrio molitor.

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Antibacterial Effects of Bicarbonate in Media Modified to Mimic Cystic Fibrosis Sputum

International Journal of Molecular Sciences ◽

10.3390/ijms21228614 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8614

Author(s):

Pongsiri Jaikumpun ◽

Kasidid Ruksakiet ◽

Balázs Stercz ◽

Éva Pállinger ◽

Martin Steward ◽

...

Keyword(s):

Cystic Fibrosis ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Culture Media ◽

Anion Channel ◽

Ambient Air ◽

Hereditary Disease ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cell Counts

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene encoding an epithelial anion channel. In CF, Cl− and HCO3− hyposecretion, together with mucin hypersecretion, leads to airway dehydration and production of viscous mucus. This habitat is ideal for colonization by pathogenic bacteria. We have recently demonstrated that HCO3− inhibits the growth and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus when tested in laboratory culture media. Using the same bacteria our aim was to investigate the effects of HCO3− in artificial sputum medium (ASM), whose composition resembles CF mucus. Control ASM containing no NaHCO3 was incubated in ambient air (pH 7.4 or 8.0). ASM containing NaHCO3 (25 and 100 mM) was incubated in 5% CO2 (pH 7.4 and 8.0, respectively). Viable P. aeruginosa and S. aureus cells were counted by colony-forming unit assay and flow cytometry after 6 h and 17 h of incubation. Biofilm formation was assessed after 48 h. The data show that HCO3− significantly decreased viable cell counts and biofilm formation in a concentration-dependent manner. These effects were due neither to extracellular alkalinization nor to altered osmolarity. These results show that HCO3− exerts direct antibacterial and antibiofilm effects on prevalent CF bacteria.

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