scholarly journals Ethanol decreases Pseudomonas aeruginosa flagellar motility through a cyclic-di-GMP- and stator-dependent pathway

2018 ◽  
Author(s):  
Kimberley A. Lewis ◽  
Amy E. Baker ◽  
Annie I. Chen ◽  
Colleen E. Harty ◽  
Sherry L. Kuchma ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Microscopic Analysis ◽  
Fold Increase ◽  
Bioactive Metabolites ◽  
Flagellar Motility ◽  
Planktonic Cells ◽  
Motile Cells ◽  
Diguanylate Cyclases ◽  
Surface Sensing ◽  
In The Wild

AbstractPseudomonas aeruginosa frequently encounters microbes that produce bioactive metabolites including ethanol. At concentrations that do not affect growth, we found that ethanol reduces P. aeruginosa motility by 30% in a swim agar assay and this decrease is accompanied by a 2.5-fold increase in levels of cyclic diguanylate (c-di-GMP), a second messenger that represses motility, in planktonic cells. A screen of mutants lacking genes involved in c-di-GMP metabolism identified SadC and GcbA as diguanylate cyclases involved in swim zone reduction by ethanol and ethanol-induced c-di-GMP production. The reduction of swimming in response to ethanol also required the stator set, MotAB, two PilZ-domain proteins (FlgZ and PilZ), PilY1-a proposed surface-sensing protein, and PilMNOP, which comprises the pilus alignment complex and these proteins have been previously implicated in the control of motility on agar surfaces. Microscopic analysis of the fraction of quiescent cells in swim medium showed that ethanol decreased the portion of motile cells in the wild type, but had opposite effects in the ∆pilY1, ∆pilMNOP, ∆motAB, and ∆pilZ∆flgZ mutants. Together, these data indicate ethanol induces a regulated change in motility in planktonic cells at concentrations similar to those produced by other microbes. We propose that this ethanol-responsiveness may contribute to the co-localization of P. aeruginosa with ethanol-producing microbes.

scholarly journals Ethanol Decreases Pseudomonas aeruginosa Flagellar Motility through the Regulation of Flagellar Stators

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Kimberley A. Lewis ◽  
Amy E. Baker ◽  
Annie I. Chen ◽  
Colleen E. Harty ◽  
Sherry L. Kuchma ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Single Cells ◽  
Microscopic Analysis ◽  
Soft Agar ◽  
Biologically Active ◽  
Time Interval ◽  
Flagellar Motility ◽  
Content Type ◽  
Swimming Motility ◽  
Potential Marker

ABSTRACT Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol. IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions

2020 ◽  
Vol 21 (5) ◽  
pp. 438-450
Author(s):  
Ramya Ramchandran ◽  
Swetha Ramesh ◽  
Anviksha A ◽  
RamLal Thakur ◽  
Arunaloke Chakrabarti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pathogenic Fungi ◽  
Fold Increase ◽  
Production Yield ◽  
Yield Enhancement ◽  
Bioactive Metabolites ◽  
Candida Auris ◽  
Media Formulation ◽  
Static Conditions

Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

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

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
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.

scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms

2021 ◽  
Author(s):  
Rossana de Aguiar Cordeiro ◽  
Bruno Nascimento da Silva ◽  
Ana Luiza Ribeiro de Aguiar ◽  
Livia Maria Galdino Pereira ◽  
Fernando Victor Monteiro Portela ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Fungal Infections ◽  
Tolerance Induction ◽  
Microscopic Analysis ◽  
Fungal Species ◽  
Planktonic Cells ◽  
Persister Cells ◽  
Candida Spp ◽  
Patients At Risk

Abstract Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980’s, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. Lay abstract This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

scholarly journals Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence

2006 ◽  
Vol 103 (8) ◽  
pp. 2839-2844 ◽  
Author(s):  
H. Kulasakara ◽  
V. Lee ◽  
A. Brencic ◽  
N. Liberati ◽  
J. Urbach ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cyclic Gmp ◽  
Diguanylate Cyclases

scholarly journals c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Bridget R Kulasekara ◽  
Cassandra Kamischke ◽  
Hemantha D Kulasekara ◽  
Matthias Christen ◽  
Paul A Wiggins ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Histidine Kinase ◽  
Molecular Basis ◽  
Individual Cell ◽  
Cell Heterogeneity ◽  
Flagellar Motility ◽  
Bacterial Populations ◽  
Phosphorylation State ◽  
Asymmetric Partitioning

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors.

scholarly journals Effects of Selection to Diflubenzuron and Bacillus thuringiensis Var. Israelensis on the Overwintering Successes of Aedes albopictus (Diptera: Culicidae)

Insects ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 822
Author(s):  
Charalampos S. Ioannou ◽  
Christos Hadjichristodoulou ◽  
Varvara A. Mouchtouri ◽  
Nikos T. Papadopoulos
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Albopictus ◽  
Selection Process ◽  
Mosquito Species ◽  
Fold Increase ◽  
Development Of Resistance ◽  
Control Programmes ◽  
Invasive Mosquito ◽  
In The Wild ◽  
Successive Generations

Aedes albopictus is an invasive mosquito species responsible for local transmission of chikungunya and dengue viruses in Europe. In the absence of available treatments, insecticides-based control remains one of the most important viable strategies to prevent emerging problems. Diflubenzuron (DFB) and Bacillus thuringiensis var. israelensis (Bti) are among the most commonly used larvicides for Ae. albopictus control with consequent concerns for the potential development of resistance. Studies on the resistance emergence in Ae. albopictus and its persistence in the wild to both DFB and Bti are essential for the efficient and sustainable planning of the control programmes. In this context, larvae from a recently laboratory established population were subjected to increasing selective pressure for nine successive generations using both DFB and Bti. The resistance levels and the overwintering success of the selected populations relative to control (colonies that received no selection) were determined. Results revealed an 8.5- and 1.6-fold increase on the resistance levels following selection with DFB and Bti, respectively. The selection process to both larvicides had no apparent impacts on the overwintering capability relative to control, suggesting the successful persistence of the selected individuals in the wild on an annual base.

scholarly journals Author response: Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations

2019 ◽  
Author(s):  
Catherine R Armbruster ◽  
Calvin K Lee ◽  
Jessica Parker-Gilham ◽  
Jaime de Anda ◽  
Aiguo Xia ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Division Of Labor ◽  
Author Response ◽  
Surface Sensing
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