Adherence Of Yeast Cells Research Articles

The yeast Wickerhamomyces anomalus acts as a predator of the olive anthracnose-causing fungi, Colletotrichum nymphaeae, C. godetiae, and C. gloeosporioides.

Olive tree anthracnose is caused by infection with Colletotrichum fungi, which in Portugal are mostly C. nymphaeae, C. godetiae, and C. gloeosporioides s.s. Severe economic losses are caused by this disease that would benefit from a greener and more efficient alternative to the present agrochemical methods. Yeasts are serious candidates for pre-harvest/in field biocontrol of fungal infections. This work identified the yeast Wickerhamomyces anomalus as a strong antagonizer of the three fungi and studied in vitro this ability and its associated mechanisms. Antagonism was shown to not depend on the secretion of volatile compounds (VOCs), or siderophores or any other agar-diffusible compound, including hydrolytic enzymes. Rather, it occurred mostly in a cell-to-cell contact dependent manner. This was devised through detailed microscopic assessment of yeast-fungus cocultures. This showed that W. anomalus antagonism of the three Colletotrichum proceeded through (i) the adhesion of yeast cells to the phytopathogen hyphae, (ii) the secretion of a viscous extracellular matrix, and (iii) the emptying of the hyphae. Yeasts ultimately putatively feed on hyphal contents, which is supported by light microscopy observation of MB and PI co-culture-stained samples. Accordingly, numerous W. anomalus cells were observed packing inside C. godetiae emptied hyphae. This behaviour can be considered microbial predation and classified as necrotrophic mycoparasitism, more explicitly in the case of C. godetiae. The results support the prospect of future application of W. anomalus as a living biofungicide/BCA in the preharvest control of olive anthracnose.

P082 Synergistic activity, anti-adherence and anti-fungal abilities of fluconazole and voriconazole combined with thymol and carvacrol against C andida species

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThe current study aimed to assess the anti-adherence and antifungal activities of thymol and carvacrol against Candida albicans, C. glabrata, and C. krusei isolates obtained from patients with oral candidiasis concerning growth inhibition and fungal death as compared to the synthetic antifungals such as fluconazole and voriconazole.MethodsThe susceptibility assay for the test compounds was performed using the disk diffusion method against all Candida isolates. Also, anti-adherence activity was examined using a rapid and highly reproducible 96 well microtiter-based method.ResultsBoth natural phenols and antifungal drugs revealed various efficacies against studied Candida species. The susceptibility to fluconazole and voriconazole were 100% for C. albicans, 50% and 90% for C. glabrata, and 0% and 100% for C. krusei isolates, respectively. The mean diameter of the inhibition zone was greater for thymol than carvacrol in C. albicans (19.89-0.80 mm vs 17.05-0.61 mm), C. glabrata (18.87-0.71 mm vs 15.77-0.57 mm), and C. krusei (15.11-0.91 mm vs 13.91-1.04 mm) isolates tested.Thymol showed more effective inhibition on adherence of all Candida species than other treatments. The mean relative adherence ratios for C. albicans, C. glabrata, and C. krusei were 0.50, 0.60, and 0.64, respectively.ConclusionsThis study demonstrated significant inhibitory properties of thymol and carvacrol on the adherence and growth of azole susceptible- and -resistant Candida isolates. Also, thymol was more effective for preventing the adherence of yeast cells to polystyrene in comparison to carvacrol.

In Vitro and In Vivo Antibiofilm Potential of Eicosane Against Candida albicans.

Candida albicans is the most prevalent fungus in humans, producing infections ranging from mucosal to systemic. C. albicans colonizes mucosal surfaces asymptomatically as commensal, but, if the host environment is disrupted, or if the host immune system is compromised, C. albicans can multiply and infect almost all places in the host. The present study was aimed to identify a promising antibiofilm agent against Candida albicans biofilm. Through the molecular docking approach, it was identified that Eicosane was the top hit among the alkanes screened. Furthermore, in vitro analysis revealed that Eicosane at 100µg/mL was able to inhibit 60% of C. albicans biofilm without inhibiting the growth. Moreover, light microscopic investigation unveiled the significant reduction in the adhesion and colonization of yeast cells to the matrix on Eicosane-treated samples. The CLSM images showing a reduction in biomass and thickness of C. albicans biofilm in the presence of Eicosane were validated using COMSTAT. The results were well corroborated with SEM micrograph in which a pellucid gap between the cells was observed and colonization was considerably reduced. Further from qPCR analysis, the genes responsible for biofilm formation and hyphal growth were found to be downregulated in the presence of Eicosane. Similarly, Eicosane at BIC was able to significantly inhibit the adhesion and colonization of yeast cells on the chorion of the zebrafish embryos. Moreover, the binding ability of Eicosane to ALS3 was revealed through docking and molecular dynamics (MD) simulation studies.

Treatment with Candida albicans biotherapic influences in vitro fungal adhesion to Ma-104 cells

Background: Oral candidiasis is an opportunist fungal infection in humans, mainly caused by Candida albicans. It occurs when the host presents an imbalance in the immune system and Candida spp., normally found in human flora, become able to develop the infection [1]. This disease is very common in HIV patients, and in all individuals that present immunossupression, such as patients treated with chemotherapy. Considering this scenario, the development of new medicines to treat oral candidiasis is mandatory.
 
 Aims: The aim of this study was to evaluate citotoxicity, morphology and quantify the adhesion rates of C. albicans to biotherapic-treated Ma104 cells.
 
 Methodology: The biotherapic was prepared following the Roberto Costa technique and Brazilian Homeopathic Pharmacopeia protocol [2]. Briefly, biotherapic 1X was prepared with 1 mL of aqueous solution containing 108 yeasts of living Candida albicans plus 9 ml of sterile distilled water. This solution was submmited to 100 mechanical succussions. Biotherapic 2X was obtained after addition of 1 ml of 1X solution in 9 ml of sterile distilled water and it was also submitted to 100 mechanical succussions. This procedure was repeated until biotherapic 30X was obtained. As a control, sterile dynamized water (30X) was used. The inhibition of fungal growth induced by biotherapic was evaluated by MTT method after 24 hours of treatment. The morphological aspects of Ma104-biotherapic-treated cells were analyzed by Giemsa staining after 5, 10 and 60 days, and compared with control groups (water 30X and untreated cells). Additionally, Ma104 cells were treated during 5 and 30 days with biotherapic in parallel with respective controls, and the index adhesion of yeast cells was quantified. 
 
 Results: The biotherapic was not able to reduce the viability of treated C. albicans when compared with controls. On the other hand, Ma104 treated cells presented important morphological alterations after 60 days, such as: cytoplasmic vacuoles, halos around the nucleolus and elongation of the plasmatic membrane. These changes were not observed in ,untreated cells nor in ones treated with water 30X. The adhesion index to Ma104 cells was reduced around 27% after 5 and 30 days of treatment when compared to controls. 
 
 Conclusion: These results showed that the biotherapic did not present any citotoxicity, but was able to modify the morphological aspects of Ma-104 cells. Additionally, the interaction between host cells and ethilogic agent is directly influenced by biotherapic treatment, suggesting a promising antifungal potential of this medicine.

Quantification of the Adhesion Strength of Candida albicans to Tooth Enamel.

Caries is one of the most prevalent diseases worldwide, which is caused by the degradation of the tooth enamel surface. In earlier research the opportunistic pathogen Candida albicans has been associated with the formation of caries in children. Colonization of teeth by C. albicans starts with the initial adhesion of individual yeast cells to the tooth enamel surface. In this study, we visualized the initial colonization of C. albicans yeast cells on pellicle-covered enamel by scanning electron microscopy. To quantitatively unravel the initial adhesion strength, we applied fluidic force microscopy-based single-cell force spectroscopy to examine the key adhesion parameters adhesion force, rupture length and de-adhesion work. We analyzed single saliva-treated or untreated yeast cells on tooth enamel specimens with or without salivary pellicle. Under all tested conditions, adhesion forces in the lower nanonewton range were determined. Furthermore, we have found that all adhesion parameters were enhanced on the pellicle-covered compared to the uncovered enamel. Our data suggest that initial adhesion occurs through a strong interaction between yeast cell wall-associated adhesins and the salivary pellicle. Future SCFS studies may show whether specific management of the salivary pellicle reduces the adhesion of C. albicans on teeth and thus contributes to caries prophylaxis.

Anti-adherence and Anti-fungal Abilities of Thymol and Carvacrol Against Candida Species Isolated From Patients with Oral Candidiasis in Comparison with Fluconazole and Voriconazole

Background: Natural isopropyl cresols, such as thymol and carvacrol, have been known to have antifungal activities. Objectives: The current study aimed to investigate the anti-adherence and antifungal activities of thymol, carvacrol, fluconazole, and voriconazole against oral isolates of Candida albicans (C. albicans), C. glabrata, and C. krusei. Methods: The susceptibility assay for the test compounds was performed using the disk diffusion method against all Candida isolates. Also, anti-adherence activity was examined using a rapid and highly reproducible 96 well microtiter-based method. Results: Both natural phenols and antifungal drugs revealed various efficacies against studied Candida species. The susceptibility to fluconazole and voriconazole were 100% for C. albicans, 50% and 90% for C. glabrata, and 0% and 100% for C. krusei isolates, respectively. The mean diameter of the inhibition zone was greater for thymol than carvacrol in C. albicans (19.89 ± 0.80 mm versus 17.05 ± 0.61 mm), C. glabrata (18.87 ± 0.71 mm versus 15.77 ± 0.57 mm), and C. krusei (15.11 ± 0.91 mm versus 13.91 ± 1.04 mm) isolates tested. Thymol showed more effective inhibition on adherence of all Candida species than other treatments. The mean relative adherence ratios for C. albicans, C. glabrata, and C. krusei were 0.50, 0.60, and 0.64, respectively. Conclusions: This study demonstrated significant inhibitory properties of thymol and carvacrol on the adherence and growth of azole susceptible- and -resistant Candida isolates. Also, thymol was more effective for preventing the adherence of yeast cells to polystyrene in comparison to carvacrol.

Highly efficient antibiofilm and antifungal activity of green propolis against Candida species in dentistry materials.

This study evaluated the effect of green propolis extract on the adhesion and biofilm formation of Candida species in dentistry materials. Phytochemical analysis of green propolis extract was performed by high-performance liquid chromatography. Adhesion was quantified by counting the number of yeast cells adherent to dental material fragments in a Neubauer chamber. Biofilm formation was determined by counting colony-forming units recovered from dental material fragments. The intensity of biofilm adhesion was classified as negative, weak, moderate, strong, or very strong. Fifteen compounds, mainly flavonoids, were identified in green propolis extract. All strains adhered to and formed biofilms on the surfaces of the orthodontic materials studied. On steel and resin, yeast cell adhesion intensities were weak at all incubation times, except for those of Candida parapsilosis and C. tropicalis, which were moderate at 12 h. At 24 and 48 h, C. albicans formed biofilms on steel with moderate adhesion affinities; at 24 and 48 h, C. parapsilosis formed biofilms with very strong affinities. C. tropicalis formed biofilms with strong and very strong affinities at 24 and 48 h, respectively. On resin, all species displayed strong affinity for biofilm formation at 24 and 48 h, except for C. tropicalis, which displayed very strong affinity at only 48 h. Green propolis extract displayed antifungal activity and inhibited both adhesion and biofilm formation at 2.5 μg/mL. This study reinforces the idea that green propolis has antifungal activity and interferes with the virulence of Candida species.

Anti-inflammatory and anti-Candida Effects of Brazilian Organic Propolis, a Promising Source of Bioactive Molecules and Functional Food.

Brazilian organic propolis (BOP) is an unexplored Brazilian propolis that is produced organically and certified according to international legislation. Our results showed that BOP has strong anti-inflammatory effects and acts by reducing nuclear factor κB activation, tumor necrosis factor α release, and neutrophil migration. In addition, BOP6 exhibited antifungal activity on planktonic and biofilm cultures of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Candida parapsisolis and reduced in vitro yeast cell adhesion to human keratinocytes at sub-inhibitory concentrations. BOP demonstrated significantly low toxicity in Galleria melonella larvae at antifungal doses. Lastly, a chemical analysis revealed the presence of caffeoyltartaric acid, 3,4-dicaffeoylquinic acid, quercetin, and gibberellins A7, A9, and A20, which may be responsible for the biological properties observed. Thus, our data indicate that BOP is a promising source of anti-inflammatory and antifungal molecules that may be used as a functional food.

Critical issues in the performance of yeast based microbial fuel cell

AbstractBACKGROUNDYeast, Saccharomyces cerevisiae, is quite safe, easily available, rapid‐growing and one of few microorganisms that can metabolize complex organic materials. The effect of different anode materials such as carbon cloth (CC), carbon paper (CP), Teflon‐treated carbon paper (CP‐T), and porous carbon plate (PCP) on the performance of mediatorless yeast, S. cerevisiae‐based MFC was investigated to explore some issues that are affecting the performance.RESULTSThe variation in the open circuit voltage (OCV), and power generation for CC, CP and CP‐T, were explained by the different areal densities of the yeast cells that adhered to the anode surface. The highest power was obtained using the CP anode. On the other hand, the PCP anode with dense adhesion of the yeast cells, which was expected to give the highest performance; showed the lowest performance. Modifying the PCP surface with a thin nanolayer of cobalt significantly increased the performance over fifty times.CONCLUSIONThe performance of a mediatorless yeast‐based microbial fuel cell, i.e. OCV and current density, was affected by the adhesion density of the yeast cell on the electrode surface. However, it was not the only factor affecting the cell performance. The yeast cell adhesion on the anode surface was dependent on the carbon materials. The improvement of the interfacial electron transfer between the anode and yeast cells is key for the development of yeast based mediatorless MFCs. © 2017 Society of Chemical Industry

Interaction between Cork and Yeast: Application to Sparkling Wine Processing

An innovative method has been developed to observe yeasts at the surface of cork using two-photon microscopy and to investigate if a removable crown cap substitution by a two-disc cork stopper may affect the bottle fermentation during sparkling wine processing. While much of the Champagne wine industry is using crown seals for tirage and aging, some producers are choosing cork closures, particularly for Champagne wines subject to longer aging periods. The developed technique is based on cork autofluorescence and on DAPI (4′6-diamidino-2-phenylindole) labeling of yeasts with a different emission wavelength range. This study was performed for cork in contact with model wine and in the bottle with Champagne wine, while following fermentation and maturation over one year. For both cases, neither biofilm nor strong adhesion of yeast cells was detected on the cork surface. However, in the case of Champagne wine, a small amount of yeast was observed in the empty cavities of the cork cell structure. The development of yeasts on the cork surface remains very limited, thus cork stoppers with discs can be used during Champagne wine processing without concern for cork as a potential fermentation support.

Amyloid-Like β-Aggregates as Force-Sensitive Switches in Fungal Biofilms and Infections.

Cellular aggregation is an essential step in the formation of biofilms, which promote fungal survival and persistence in hosts. In many of the known yeast cell adhesion proteins, there are amino acid sequences predicted to form amyloid-like β-aggregates. These sequences mediate amyloid formation in vitro. In vivo, these sequences mediate a phase transition from a disordered state to a partially ordered state to create patches of adhesins on the cell surface. These β-aggregated protein patches are called adhesin nanodomains, and their presence greatly increases and strengthens cell-cell interactions in fungal cell aggregation. Nanodomain formation is slow (with molecular response in minutes and the consequences being evident for hours), and strong interactions lead to enhanced biofilm formation. Unique among functional amyloids, fungal adhesin β-aggregation can be triggered by the application of physical shear force, leading to cellular responses to flow-induced stress and the formation of robust biofilms that persist under flow. Bioinformatics analysis suggests that this phenomenon may be widespread. Analysis of fungal abscesses shows the presence of surface amyloids in situ, a finding which supports the idea that phase changes to an amyloid-like state occur in vivo. The amyloid-coated fungi bind the damage-associated molecular pattern receptor serum amyloid P component, and there may be a consequential modulation of innate immune responses to the fungi. Structural data now suggest mechanisms for the force-mediated induction of the phase change. We summarize and discuss evidence that the sequences function as triggers for protein aggregation and subsequent cellular aggregation, both in vitro and in vivo.

Secretory component mediates Candida albicans binding to epithelial cells.

Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

In vitro modulation of probiotic bacteria on the biofilm of Candida glabrata

A conspicuous new concept of pathogens living as the microbial societies in the human host rather than free planktonic cells has raised considerable concerns among scientists and clinicians. Fungal biofilms are communities of cells that possess distinct characteristic such as increased resistance to the immune defence and antimycotic agents in comparison to their planktonic cells counterpart. Therefore, inhibition of the biofilm may represent a new paradigm for antifungal development. In this study, we aim to evaluate the in vitro modulation of vulvovaginal candidiasis (VVC)-causing Candida glabrata biofilms using probiotic lactobacilli strains. Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 were shown to have completely inhibited C. glabrata biofilms and the results were corroborated by scanning electron microscopy (SEM), which revealed scanty structures of the mixed biofilms of C. glabrata and probiotic lactobacilli strains. In addition, biofilm-related C. glabrata genes EPA6 and YAK1 were downregulated in response to the probiotic lactobacilli challenges. The present study suggested that probiotic L. rhamnosus GR-1 and L. reuteri RC-14 strains inhibited C. glabrata biofilm by partially impeding the adherence of yeast cells and the effect might be contributed by the secretory compounds produced by these probiotic lactobacilli strains. Further investigations are required to examine and identify the biofilm inhibitory compounds and the mechanism of probiotic actions of these lactobacilli strains.

Yeast Extract as an Effective and Safe Mediator for the Baker’s-Yeast-Based Microbial Fuel Cell

Although utilizing the exogenous mediators distinctly enhances the microbial fuel cell (MFC) performance, possibility of microorganisms’ toxicity, environmental aspect and cost are the main dilemmas facing wide applications. Therefore, successful applying of the yeast extract as a mediator in the baker’s-yeast-based (Saccharomyces cerevisiae-based) MFCs would be of great interest as it will overcome all the aforementioned problems. The influence of the yeast extract addition was investigated based on the yeast cell adhesion on the surface of plain and gold-sputtered carbon paper anodes. In the case of plain carbon paper, the addition of the yeast extract considerably enhanced the performance of the yeast-based MFC, which can be attributed to the yeast extract role as growth media or as a mediator; the current and power densities increased from 94 to 190 mA/cm2 and from 12.9 to 32.6 mW/cm2, respectively. However, compared with the plain carbon paper, in the case of gold-sputtered anode the performance sign...

Physicochemical aspects of Trichosporon cutaneum CCY 30-5-10 adhesion and biofilm formation potential on cellophane

AbstractThis paper focuses on the adhesion and biofilm formation potential of cellulolytic yeast Trichosporon cutaneum CCY 30-5-10 on solid cellophane from a novel perspective. First, physicochemical characterisation of the cells and carrier (cellophane) was performed to evaluate the effect of different culture media (complex vs mineral) on yeast cell adhesion. (Un)favourable adhesion conditions were predicted using the thermodynamic approach. Next, the ability of the cells to colonise the carrier under the above conditions was quantified and the biofilm structure was characterised using image analysis. The approaches described were found suitable to predict and experimentally verify favourable (cell-solid) adhesion, i.e. the hydrophobic and low electron-donor nature of cellophane together with hydrophobic cells (obtained when cultivated in a complex culture medium) were found to have a major impact in defining successful yeast adhesion with subsequent biofilm formation.

Isoconductivity method to study adhesion of yeast cells to gold electrode

Abstract In this paper, we used impedance spectroscopy and gold electrodes to detect the presence of yeast cells and monitor the attachment of these cells to the electrodes. We analyzed the effect of conductivity changes of the medium and the attachment on the electrode-electrolyte interface impedance. A three-electrode cell was designed to produce a uniform electric field distribution on the working electrode and to minimize the counter electrode impedance. Moreover, we used a small AC overpotential (10 mV) to keep the system within the linear impedance limits of the electrode-electrolyte interface. This study proposes a new method to differentiate the impedance changes due to the attachment of yeast cells from those due to conductivity changes of the medium. The experiments showed that when the difference between the cell suspension and base solution conductivities is within the experimental error, the impedance changes are only due to the attachment of yeast cells to the electrodes. The experiments also showed a strong dependence (decrease) of the parallel capacity of the electrode electrolyte interface with the yeast cell concentration of suspension. We suggest that this decrease is due to an asymmetrical redistribution of surface charges on both sides of cell, which can be modeled as a biologic capacity connected in series with the double layer capacity of the interface. Our results could help to explain the rate of biofilm formation through the determination of the rate of cell adhesion.

Time-lapse video microscopy and image analysis of adherence and growth patterns of Candida albicans strains

Digital image analysis of high time resolution video microscopy was used to investigate hyphal growth dynamics in different Candida albicans strains. The effects of the quorum sensing molecules tyrosol and farnesol, the deletion of the fungus specific protein phosphatase Z1 CaPPZ1), and the hypha-specific cyclin (HGC1) genes were analyzed by this method. Our system monitored cell growth in a CO2 incubator under near-physiological conditions and measured three major parameters under the following stringent conditions: (a) the time of yeast cell adherence, (b) the time of hyphal outgrowth, and (c) the rate of hyphal growth. This method showed that hyphal extension of wild-type SC5314 cells was accelerated by tyrosol and inhibited by farnesol. Hyphal growth rate was moderately lower in cappz1 and strongly reduced in hgc1 mutants. In addition, tyrosol treatment caused a firm adherence, while farnesol treatment and hgc1 mutation prevented the adherence of yeast cells to the surface of the culture flask. Transition from yeast-to-hyphal state was faster after tyrosol treatment, while it was reduced in farnesol-treated cells as well as in the cappz1 and hgc1 mutants. Our data confirm the notion that the attachment of yeast cells, the yeast-to-hyphal transition, and hyphal growth rate are closely related processes. Time-lapse video microscopy combined with image analysis offers a convenient and reliable method of testing chemicals, including potential drug candidates, and genetic manipulations on the dynamic morphological changes in C. albicans strains.

Role of Force-Sensitive Amyloid-Like Interactions in Fungal Catch Bonding and Biofilms

The Candida albicans Als adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. Shearing Saccharomyces cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm(2) increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm(2). Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly, S. cerevisiae cells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.

The Wsc1p Cell Wall Signaling Protein Controls Biofilm (Mat) Formation Independently of Flo11p in Saccharomyces cerevisiae

Saccharomyces cerevisiae strains of the ∑1278b background generate biofilms, referred to as mats, on low-density agar (0.3%) plates made with rich media (YPD). Mat formation involves adhesion of yeast cells to the surface of the agar substrate and each other as the biofilm matures, resulting in elaborate water channels that create filigreed patterns of cells. The cell wall adhesion protein Flo11p is required for mat formation; however, genetic data indicate that other unknown effectors are also required. For example, mutations in vacuolar protein sorting genes that affect the multivesicular body pathway, such as vps27∆, cause mat formation defects independently of Flo11p, presumably by affecting an unidentified signaling pathway. A cell wall signaling protein, Wsc1p, found at the plasma membrane is affected for localization and function by vps27∆. We found that a wsc1∆ mutation disrupted mat formation in a Flo11p-independent manner. Wsc1p appears to impact mat formation through the Rom2p-Rho1p signaling module, by which Wsc1p also regulates the cell wall. The Bck1p, Mkk1/Mkk2, Mpk1p MAP kinase signaling cascade is known to regulate the cell wall downstream of Wsc1p-Rom2p-Rho1p but, surprisingly, these kinases do not affect mat formation. In contrast, Wsc1p may impact mat formation by affecting Skn7p instead. Skn7p can also receive signaling inputs from the Sln1p histidine kinase; however, mutational analysis of specific histidine kinase receiver residues in Skn7p indicate that Sln1p does not play an important role in mat formation, suggesting that Skn7p primarily acts downstream of Wsc1p to regulate mat formation.