Aaron P. Mitchell

Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo.

The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1.

Regulation of Cell-Surface Genes and Biofilm Formation by the C. albicans Transcription Factor Bcr1p

The impact of many microorganisms on their environment depends upon their ability to form surface bound communities called biofilms [1]. Biofilm formation on implanted medical devices has severe consequences for human health by providing both a portal of entry and a sanctuary for invasive bacterial and fungal pathogens [1 and 2]. Biofilm regulators and adherence molecules are extensively defined for many bacterial pathogens [3, 4, and 5], but not for fungal pathogens such as Candida albicans. Elongated filaments called hyphae are a prominent feature of C. albicans biofilms, and known genes that promote biofilm formation are required for hyphal development [2, 6, 7 and 8]. From a new library of transcription-factor mutants, we identify Bcr1p, a zinc finger protein required for formation of biofilms but not hyphae. Expression analysis shows that Bcr1p activates cell-surface protein and adhesin genes, including several induced during hyphal development. BCR1 expression depends upon the hyphal regulator Tec1p. Thus, BCR1 is a downstream component of the hyphal regulatory network that couples expression of cell-surface genes to hyphal differentiation. Our results indicate that hyphal cells are specialized to present adherence molecules that support biofilm integrity.

Genetic control of Candida albicans biofilm development

Candida species cause frequent infections owing to their ability to form biofilms — surface-associated microbial communities — primarily on implanted medical devices. Increasingly, mechanistic studies have identified the gene products that participate directly in the development of Candida albicans biofilms, as well as the regulatory circuitry and networks that control their expression and activity. These studies have uncovered new mechanisms and signals that govern C. albicans biofilm development and associated drug resistance, thus providing biological insight and therapeutic foresight.

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Candida albicans RIM101 pH Response Pathway Is Required for Host-Pathogen Interactions

ABSTRACT The ability of Candida albicans to respond to diverse environments is critical for its success as a pathogen. TheRIM101 pathway controls gene expression and the yeast-to-hyphal transition in C. albicans in response to changes in environmental pH in vitro. In this study, we found that theRIM101 pathway is necessary in vivo for pathogenesis. First, we show thatrim101−/rim101− andrim8−/rim8− mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, therim101−/rim101− andrim8−/rim8− mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of RIM101, RIM101-405, is a suppressor of the rim8− mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the rim8−/rim8−mutant. These results demonstrate that the RIM101 pathway is required for C. albicans virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.

Function of Candida albicans adhesin Hwp1 in biofilm formation.

ABSTRACT Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

Complementary adhesin function in C. albicans biofilm formation.

BACKGROUND Biofilms are surface-associated microbial communities with significant environmental and medical impact. Here, we focus on an adherence mechanism that permits biofilm formation by Candida albicans, the major invasive fungal pathogen of humans. RESULTS The Als surface-protein family has been implicated in biofilm formation, and we show that Als1 and Als3 have critical but redundant roles. Overexpression of several other Als proteins permits biofilm formation in a biofilm-defective als1/als1 als3/als3 strain, thus arguing that the function of Als proteins in this process is governed by their respective expression levels. The surface protein Hwp1 is also required for biofilm formation, and we find that a mixture of biofilm-defective hwp1/hwp1 and als1/als1 als3/als3 strains can form a hybrid biofilm both in vitro and in vivo in a catheter infection model. Complementary function of Hwp1 and Als1 and 3 seems to reflect their interaction because expression of Hwp1 in the heterologous host S. cerevisiae permits adherence to wild-type C. albicans, but not to an als1/als1 als3/als3 strain. CONCLUSIONS The complementary roles of Hwp1 and Als1 and Als3 in biofilm formation are analogous to the roles of sexual agglutinins in mating reactions. This analogy suggests that biofilm-adhesin complementarity may promote formation of monospecies biofilms.

The Transcription Factor Rim101p Governs Ion Tolerance and Cell Differentiation by Direct Repression of the Regulatory Genes NRG1 and SMP1 in Saccharomyces cerevisiae

ABSTRACT Environmental pH changes have broad consequences for growth and differentiation. The best-understood eukaryotic pH response pathway acts through the zinc-finger transcription factor PacC of Aspergillus nidulans, which activates alkaline pH-induced genes directly. We show here that Saccharomyces cerevisiae Rim101p, the pH response regulator homologous to PacC, functions as a repressor in vivo. Chromatin immunoprecipitation assays show that Rim101p is associated in vivo with the promoters of seven Rim101p-repressed genes. A reporter gene containing deduced Rim101p binding sites is negatively regulated by Rim101p and is associated with Rim101p in vivo. Deletion mutations of the Rim101p repression targets NRG1 and SMP1 suppress rim101Δ mutant defects in ion tolerance, haploid invasive growth, and sporulation. Therefore, transcriptional repression is the main biological function of Rim101p. The Rim101p repression target Nrg1p is in turn required for repression of two alkaline pH-inducible genes, including the Na+ pump gene ENA1, which is required for ion tolerance. Thus, Nrg1p, a known transcriptional repressor, functions as an inhibitor of alkaline pH responses. Our findings stand in contrast to the well-characterized function of PacC as a direct activator of alkaline pH-induced genes yet explain many aspects of Rim101p and PacC function in other organisms.

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.

GENETICS AND GENOMICS OF CANDIDA ALBICANS BIOFILM FORMATION

Biofilm formation by the opportunistic fungal pathogen Candida albicans is a complex process with significant consequences for human health: it contributes to implanted medical device‐associated infections. Recent advances in gene expression profiling and genetic analysis have begun to clarify the mechanisms that govern C. albicans biofilm development and acquisition of unique biofilm phenotypes. Such studies have identified candidate adhesin genes, and have revealed that biofilm drug resistance is multifactorial. Newly defined cell–cell communication pathways also have profound effects on biofilm formation. Future challenges include the elucidation of the structure and function of the extracellular exopolymeric substance that surrounds biofilm cells, and the extension of in vitro biofilm observations to newly developed in vivo biofilm models.