clarifying-and-imaging-candida-albicans-biofilms

Clarifying and Imaging <em>Candida albicans</em> Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Carnegie Mellon University

Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the cak1 DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one.

Bypass of Candida albicans Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1.

We focus this article on turning a biofilm inside out. The "inside" of the biofilm comprises the individual biofilm-related phenotypes, their environmental drivers and genetic determinants, and the coordination of gene functions through transcriptional regulators. Investigators have viewed the inside of the biofilm through diverse approaches, and this article will attempt to capture the essence of many. The ultimate goal is to connect the inside to the "outside," which we view as biofilm structure, development, pharmacological attributes, and medical impact.

Fungal Biofilms: Inside Out.

Fungal biofilms are heterogeneous, surface-associated colonies comprised of filamentous hyphae (chains of elongated cells), pseudohyphal cells, yeast-form cells, and various forms of extracellular matrix. When grown on a substratum under liquid culture medium, the microbial fungus Candida albicans forms dense biofilms that range in thickness from 100 to 600μm. Apical hyphae in the medium and invasive hyphae in the substratum may add greatly to the thickness and complexity of the biofilm. Because of the heterogeneity of the structure, and the large refractive index differences between cell walls, cytoplasm, and medium, fungal biofilms appear optically opaque. For fixed specimens that can be transferred out of an aqueous medium, refractive index matching methods provide a high degree of clarification. Confocal scanning, 2-photon scanning, or selective-plane illumination microscopy then can be used to obtain high-quality image data spanning the full thickness of the biofilm. Using refractive index matching and confocal microscopy, we have imaged many interesting features within wild-type, mutant, and engineered biofilms, including cellular phenotypes that vary with position, the effect of growth conditions, and gene expression through reporter constructs. This approach greatly expands the range of microscopical studies, allowing researchers to observe and quantify specific phenomena within medically or industrially relevant forms of microbial growth.

Microscopy of fungal biofilms.

Candida albicans is a fungal pathogen that causes serious biofilm-based infections. Here we have asked whether surface topography may affect C. albicans biofilm formation. We tested biofilm growth of the prototypical wild-type strain SC5314 on a series of polydimethylsiloxane (PDMS) solids. The surfaces were prepared with monolayer coatings of monodisperse spherical silica particles that were fused together into a film using silica menisci. The surface topography was varied by varying the diameter of the silica particles that were used to form the film. Biofilm formation was observed to be a strong function of particle size. In the particle size range 4.0-8.0 μm, there was much more biofilm than in the size range 0.5-2.0 μm. The behavior of a clinical isolate from a clade separate from SC5314, strain p76067, showed results similar to that of SC5314. Our results suggest that topographic coatings may be a promising approach to reduce C. albicans biofilm infections.

Impact of surface topography on biofilm formation by Candida albicans.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Article	Year
Bypass of Candida albicans Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1. PLoS genetics\| PubMed ID: 27935965	2016
Fungal Biofilms: Inside Out. Microbiology spectrum\| PubMed ID: 28387175	2017
Microscopy of fungal biofilms. Current opinion in microbiology\| PubMed ID: 29414442	2018
Impact of surface topography on biofilm formation by Candida albicans. PloS one\| PubMed ID: 29912894	2018
Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality. PLoS genetics\| PubMed ID: 31961865	2020

Katherine Lagree

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