Fungal Biofilms, Drug Resistance, and Recurrent Infection.

<p>A biofilm is a surface-associated microbial community. Diverse fungi are capable of biofilm growth. The significance of this growth form for infection biology is that biofilm formation on implanted devices is a major cause of recurrent infection. Biofilms also have limited drug susceptibility, making device-associated infection extremely difficult to treat. Biofilm-like growth can occur during many kinds of infection, even when an implanted device is not present. Here we summarize the current understanding of fungal biofilm formation, its genetic control, and the basis for biofilm drug resistance.</p

cis- and trans-acting localization determinants of pH response regulator Rim13 in Saccharomyces cerevisiae.

<p>The Rim101/PacC pathway governs adaptation to alkaline pH in many fungi. Output of the pathway is mediated by transcription factors of the Rim101/PacC family, which are activated by proteolytic cleavage. The proteolytic complex includes scaffold protein Rim20 and endosome-associated subunits of the endosomal sorting complex required for transport (ESCRT). We provide here evidence that Saccharomyces cerevisiae Rim13, the protease that is implicated in Rim101 cleavage, is associated with the Rim20-ESCRT complex, and we investigate its regulation. Rim13-GFP is dispersed in cells grown in acidic medium but forms punctate foci when cells encounter alkaline conditions. A vps4Δ mutant, which accumulates elevated levels of endosomal ESCRT, also accumulates elevated levels of Rim13-GFP foci, independently of external pH. In the vps4Δ background, mutation of ESCRT subunit Snf7 or of Rim20 blocks the formation of Rim13 foci, and we found that Rim13 and Rim20 are colocalized. The Rim13 ortholog PalB of Aspergillus nidulans has been shown to undergo ESCRT and membrane association through an N-terminal MIT domain, but Rim13 orthologs in the Saccharomyces clade lack homology to this N-terminal region. Instead, there is a clade-limited C-terminal region, and we show that point mutations in this region prevent punctate localization and impair Rim13 function. We suggest that RIM13 arose from its ancestral gene through two genome rearrangements. The ancestor lost the coding region for its MIT domain through a 5' rearrangement and acquired the coding region for the Saccharomyces-specific functional equivalent through a 3' rearrangement.</p

Portrait of Candida albicans adherence regulators.

<p>Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans.</p

Regulatory role of glycerol in Candida albicans biofilm formation.

<p>UNLABELLED: Biofilm 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.</p> <p>IMPORTANCE: Candida 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.</p