Pseudomonas aeruginosa secreted factors impair biofilm development in Candida albicans

Signal-mediated interactions between the human opportunistic pathogens Pseudomonas aeruginosa and Candida albicans affect virulence traits in both organisms. Phenotypic studies revealed that bacterial supernatant from four P. aeruginosa strains strongly reduced the ability of C. albicans to form biofilms on silicone. This was largely a consequence of inhibition of biofilm maturation, a phenomenon also observed with supernatant prepared from non-clinical bacterial species. The effects of supernatant on biofilm formation were not mediated via interference with the yeast–hyphal morphological switch and occurred regardless of the level of homoserine lactone (HSL) produced, indicating that the effect is HSL-independent. A transcriptome analysis to dissect the effects of the P. aeruginosa supernatants on gene expression in the early stages of C. albicans biofilm formation identified 238 genes that exhibited reproducible changes in expression in response to all four supernatants. In particular, there was a strong increase in the expression of genes related to drug or toxin efflux and a decrease in expression of genes associated with adhesion and biofilm formation. Furthermore, expression of YWP1, which encodes a protein known to inhibit biofilm formation, was significantly increased. Biofilm formation is a key aspect of C. albicans infections, therefore the capacity of P. aeruginosa to antagonize this has clear biomedical implications.

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Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1865-1873.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1865-1873 ◽

Cited By ~ 341

Author(s):

Teresa R. De Kievit ◽

Richard Gillis ◽

Steve Marx ◽

Chris Brown ◽

Barbara H. Iglewski

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Expression Patterns ◽

Homoserine Lactone ◽

Lower Percentage ◽

Spatial Studies ◽

Green Fluorescent ◽

Biofilm Development

ABSTRACT Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). InPseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI andrhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development,lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI andrhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.

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Control of Candida albicans Metabolism and Biofilm Formation by Pseudomonas aeruginosa Phenazines

mBio ◽

10.1128/mbio.00526-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 128

Author(s):

Diana K. Morales ◽

Nora Grahl ◽

Chinweike Okegbe ◽

Lars E. P. Dietrich ◽

Nicholas J. Jacobs ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Candida Albicans ◽

Biofilm Formation ◽

Carbon Sources ◽

Metabolic Effects ◽

Respiratory Metabolism ◽

Fermentation Products ◽

Content Type ◽

Opportunistic Fungal Pathogen ◽

Biofilm Development

ABSTRACTCandida albicanshas developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions betweenCandida albicansandPseudomonas aeruginosathrough the action ofP. aeruginosa-produced phenazines. While phenazines are toxic toC. albicansat millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development ofC. albicanswrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impairedC. albicansgrowth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease.IMPORTANCEMany of the infections caused byCandida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study ofC. albicansinteractions with the bacteriumPseudomonas aeruginosa, which often coinfects withC. albicans, we have found thatP. aeruginosa-produced phenazines modulateC. albicansmetabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure ofC. albicansbiofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treatC. albicansinfections.

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Cyclic di-GMP Is Integrated Into a Hierarchal Quorum Sensing Network Regulating Antimicrobial Production and Biofilm Formation in Roseobacter Clade Member Rhodobacterales Strain Y4I

Frontiers in Marine Science ◽

10.3389/fmars.2021.681551 ◽

2021 ◽

Vol 8 ◽

Author(s):

April C. Armes ◽

Alison Buchan

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Roseobacter Clade ◽

Partial Restoration ◽

Microbial Biofilms ◽

Expression Of Genes ◽

Genes Encoding ◽

Biofilm Development ◽

And Function

Microbial biofilms associated with marine particulate organic matter carry out transformations that influence local and regional biogeochemical cycles. Early microbial colonizers are often hypothesized to “set the stage” for biofilm structure, dynamics, and function via N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS). Production of AHLs, as well as antimicrobials, contributes to the colonization success of members of the Roseobacter clade. One member of this group of abundant marine bacteria, Rhodobacterales sp. Y4I, possesses two QS systems, phaRI (QS1) and pgaRI (QS2). Here, we characterize mutants in both QS systems to provide genetic evidence that the two systems work in hierarchical fashion to coordinate production of the antimicrobial indigoidine as well as biofilm formation. A mutation in pgaR (QS2) results in decreased expression of genes encoding both QS systems as well as those governing the biosynthesis of indigoidine. In contrast, mutations in QS1 did not significantly influence gene expression of QS2. Addition of exogenous AHLs to QS1 and QS2 mutants led to partial restoration of indigoidine production (45–60% of WT) for QS1 but not QS2. Mutational disruptions of QS1 had a more pronounced effect on biofilm development than those in QS2. Finally, we demonstrate that c-di-GMP levels are altered in QS and indigoidine biosynthesis Y4I mutants. Together, these results indicate that pgaRI (QS2) is at the top of a regulatory hierarchy governing indigoidine biosynthesis and that the global regulatory metabolite, c-di-GMP, is likely integrated into the QS circuitry of this strain. These findings provide mechanistic understanding of physiological processes that are important in elucidating factors driving competitiveness of Roseobacters in nature.

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Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00137-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5383-5386 ◽

Cited By ~ 187

Author(s):

Yumiko Sakuragi ◽

Roberto Kolter

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Wild Type ◽

Wild Type Level ◽

Dna Release ◽

Biofilm Development ◽

Development Regulation ◽

Biofilm Matrix

ABSTRACT Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.

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Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

Applied and Environmental Microbiology ◽

10.1128/aem.03551-14 ◽

2015 ◽

Vol 81 (7) ◽

pp. 2328-2338 ◽

Cited By ~ 23

Author(s):

Soo-Kyoung Kim ◽

Ha-Young Park ◽

Joon-Hee Lee

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Shear Force ◽

Degradation Products ◽

Early Stage ◽

Bacterial Species ◽

Extracellular Polysaccharide ◽

Content Type ◽

Tryptophan Degradation ◽

Biofilm Development

ABSTRACTAnthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation ofPseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product ofP. aeruginosa, had an opposite effect onP. aeruginosabiofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment ofP. aeruginosaat the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole onP. aeruginosabiofilm formation.

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Conditional antagonism in co-cultures of Pseudomonas aeruginosa and Candida albicans: an intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

10.1101/2020.04.20.050765 ◽

2020 ◽

Cited By ~ 1

Author(s):

Georgia Doing ◽

Katja Koeppen ◽

Patricia Occipinti ◽

Deborah A. Hogan

Keyword(s):

Pseudomonas Aeruginosa ◽

Candida Albicans ◽

Carboxylic Acid ◽

Microbial Interactions ◽

Phosphate Limitation ◽

Nutrient Concentrations ◽

Opportunistic Pathogens ◽

Kegg Pathways ◽

Expression Of Genes ◽

Anti Fungal

AbstractPseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here we describe the focal role of ethanol in mixed-species co-cultures by dual RNA-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analyses using KEGG-pathways and the previously published eADAGE method revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low phosphate response mediated by PhoB. C. albicans wild-type, but not C. albicans adh1Δ/Δ, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells. We first demonstrate that PhoB is required for this interaction and that PhoB hyperactivity, via deletion of pstB, leads to increased production of 5-MPCA even when phosphate concentrations are high, but only in the presence of ethanol. Second, we show that ethanol is only sufficient to promote 5-MPCA production at permissive phosphate concentrations. The intersection of ethanol and phosphate in co-culture is mirrored in C. albicans; the adh1Δ/Δ mutant had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate which we confirmed by showing the adh1Δ/Δ strain had elevated Pho4-dependent phosphatase activity. The dual-dependence on ethanol and phosphate concentrations for anti-fungal production highlights how environmental factors modulate microbial interactions and dictate antagonisms such as those between P. aeruginosa and C. albicans.Author SummaryPseudomonas aeruginosa and Candida albicans are opportunistic pathogens that are frequently isolated from co-infections. Using a Dual-Seq approach in combination with genetics approaches, we found that ethanol produced by C. albicans stimulates the PhoB regulon in P. aeruginosa asynchronously with activation of the Pho4 regulon in C. albicans. In doing so, we demonstrate that eADAGE-based analysis can improve the understanding of the P. aeruginosa response to ethanol-producing C. albicans as measured by transcriptomics: we identify a subset of PhoB-regulated genes as differentially expressed in response to ethanol. We validate our result by showing that PhoB is necessary for multiple roles in co-culture including the competition for phosphate and the production of 5-methyl-phenazine-1-carboxylic acid, and that the P. aeruginosa response to C. albicans-produced ethanol depends on phosphate availability. The conditional stimulation of virulence production in response to sub-inhibitory concentrations of ethanol only under phosphate limitation highlights the importance of considering nutrient concentrations in the analysis of co-culture interactions.

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Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

International Journal of Bioassays ◽

10.21746/ijbio.2017.01.007 ◽

2016 ◽

Vol 6 (01) ◽

pp. 5218

Author(s):

Laxmi Mohandas ◽

Anju T. R. ◽

Sarita G. Bhat*

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity ◽

Opportunistic Pathogens ◽

Redox Active ◽

Sem Imaging ◽

The Impact

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

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Sodium Salicylate Influences the Pseudomonas aeruginosa Biofilm Structure and Susceptibility Towards Silver

International Journal of Molecular Sciences ◽

10.3390/ijms22031060 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1060

Author(s):

Erik Gerner ◽

Sofia Almqvist ◽

Peter Thomsen ◽

Maria Werthén ◽

Margarita Trobos

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Sodium Salicylate ◽

Treatment Strategies ◽

Cell Aggregation ◽

Wound Fluid ◽

Global Challenge ◽

3D Collagen ◽

Biofilm Development ◽

Biofilm Structure

Hard-to-heal wounds are typically infected with biofilm-producing microorganisms, such as Pseudomonas aeruginosa, which strongly contribute to delayed healing. Due to the global challenge of antimicrobial resistance, alternative treatment strategies are needed. Here, we investigated whether inhibition of quorum sensing (QS) by sodium salicylate in different P. aeruginosa strains (QS-competent, QS-mutant, and chronic wound strains) influences biofilm formation and tolerance to silver. Biofilm formation was evaluated in simulated serum-containing wound fluid in the presence or absence of sodium salicylate (NaSa). Biofilms were established using a 3D collagen-based biofilm model, collagen coated glass, and the Calgary biofilm device. Furthermore, the susceptibility of 48-h-old biofilms formed by laboratory and clinical strains in the presence or absence of NaSa towards silver was evaluated by assessing cell viability. Biofilms formed in the presence of NaSa were more susceptible to silver and contained reduced levels of virulence factors associated with biofilm development than those formed in the absence of NaSa. Biofilm aggregates formed by the wild-type but not the QS mutant strain, were smaller and less heterogenous in size when grown in cultures with NaSa compared to control. These data suggest that NaSa, via a reduction of cell aggregation in biofilms, allows the antiseptic to become more readily available to cells.

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Green Tea Polyphenols and Padma Hepaten Inhibit Candida albicans Biofilm Formation

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/1690747 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Yosi Farkash ◽

Mark Feldman ◽

Isaac Ginsburg ◽

Doron Steinberg ◽

Miriam Shalish

Keyword(s):

Candida Albicans ◽

Green Tea ◽

Crystal Violet ◽

Biofilm Formation ◽

Host Cells ◽

Green Tea Polyphenols ◽

Crystal Violet Staining ◽

Biofilm Inhibition ◽

Therapeutic Concept ◽

Biofilm Development

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

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