Dataset Information

Quantitative Analyses of Force-Induced Amyloid Formation in Candida albicans Als5p: Activation by Standard Laboratory Procedures.

ABSTRACT: Candida albicans adhesins have amyloid-forming sequences. In Als5p, these amyloid sequences cluster cell surface adhesins to create high avidity surface adhesion nanodomains. Such nanodomains form after force is applied to the cell surface by atomic force microscopy or laminar flow. Here we report centrifuging and resuspending S. cerevisiae cells expressing Als5p led to 1.7-fold increase in initial rate of adhesion to ligand coated beads. Furthermore, mechanical stress from vortex-mixing of Als5p cells or C. albicans cells also induced additional formation of amyloid nanodomains and consequent activation of adhesion. Vortex-mixing for 60 seconds increased the initial rate of adhesion 1.6-fold. The effects of vortex-mixing were replicated in heat-killed cells as well. Activation was accompanied by increases in thioflavin T cell surface fluorescence measured by flow cytometry or by confocal microscopy. There was no adhesion activation in cells expressing amyloid-impaired Als5pV326N or in cells incubated with inhibitory concentrations of anti-amyloid dyes. Together these results demonstrated the activation of cell surface amyloid nanodomains in yeast expressing Als adhesins, and further delineate the forces that can activate adhesion in vivo. Consequently there is quantitative support for the hypothesis that amyloid forming adhesins act as both force sensors and effectors.

SUBMITTER: Chan CX

PROVIDER: S-EPMC4457901 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Quantitative Analyses of Force-Induced Amyloid Formation in Candida albicans Als5p: Activation by Standard Laboratory Procedures.

Chan Cho X J CX Joseph Ivor G IG Huang Andy A Jackson Desmond N DN Lipke Peter N PN

PloS one 20150605 6

Candida albicans adhesins have amyloid-forming sequences. In Als5p, these amyloid sequences cluster cell surface adhesins to create high avidity surface adhesion nanodomains. Such nanodomains form after force is applied to the cell surface by atomic force microscopy or laminar flow. Here we report centrifuging and resuspending S. cerevisiae cells expressing Als5p led to 1.7-fold increase in initial rate of adhesion to ligand coated beads. Furthermore, mechanical stress from vortex-mixing of Als5 ...[more]

PMID: 26047318

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Project description:UnlabelledBiofilm formation by Candida albicans on medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and an rhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Under in vitro conditions, the rhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2Δ/Δ mutant is also severely defective in biofilm formation in vivo in a rat catheter infection model. Expression profiling indicates that the rhr2Δ/Δ mutant has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of the rhr2Δ/Δ mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes.ImportanceCandida albicans is a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.

Project description:Candida albicans cells are often detected with Streptococcus mutans in plaque biofilms from children affected with early childhood caries. The coadhesion between these 2 organisms appears to be largely mediated by the S. mutans-derived exoenzyme glucosyltransferase B (GtfB); GtfB readily binds to C. albicans cells in an active form, producing glucans locally that provide enhanced binding sites for S. mutans. However, knowledge is limited about the mechanisms by which the bacterial exoenzyme binds to and functions on the fungal surface to promote this unique cross-kingdom interaction. In this study, we use atomic force microscopy to understand the strength and binding dynamics modulating GtfB-C. albicans adhesive interactions in situ. Single-molecule force spectroscopy with GtfB-functionalized atomic force microscopy tips demonstrated that the enzyme binds with remarkable strength to the C. albicans cell surface (~2 nN) and showed a low dissociation rate, suggesting a highly stable bond. Strikingly, the binding strength of GtfB to the C. albicans surface was ~2.5-fold higher and the binding stability, ~20 times higher, as compared with the enzyme adhesion to S. mutans. Furthermore, adhesion force maps showed an intriguing pattern of GtfB binding. GtfB adhered heterogeneously on the surface of C. albicans, showing a higher frequency of adhesion failure but large sections of remarkably strong binding forces, suggesting the presence of GtfB binding domains unevenly distributed on the fungal surface. In contrast, GtfB bound uniformly across the S. mutans cell surface with less adhesion failure and a narrower range of binding forces (vs. the C. albicans surface). The data provide the first insights into the mechanisms underlying the adhesive and mechanical properties governing GtfB interactions with C. albicans. The strong and highly stable GtfB binding to C. albicans could explain, at least in part, why this bacterially derived exoenzyme effectively modulates this virulent cross-kingdom interaction.

Dataset Information

Quantitative Analyses of Force-Induced Amyloid Formation in Candida albicans Als5p: Activation by Standard Laboratory Procedures.

Publications

Quantitative Analyses of Force-Induced Amyloid Formation in Candida albicans Als5p: Activation by Standard Laboratory Procedures.

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