Comparative Ploidy Proteomics of Candida albicans Biofilms Unraveled the Role of the AHP1 Gene in the Biofilm Persistence Against Amphotericin B.

Candida albicans is a major fungal pathogen causing lethal infections in immunocompromised patients. C. albicans forms antifungal tolerant biofilms contributing significantly to therapeutic failure. The recently established haploid C. albicans biofilm model provides a new toolbox to uncover the mechanism governing the higher antifungal tolerance of biofilms. Here, we comprehensively examined the proteomics and antifungal susceptibility of standard diploid (SC5314 and BWP17) and stable haploid (GZY792 and GZY803) strains of C. albicans biofilms. Subsequent downstream analyses identified alkyl hydroperoxide reductase 1 (AHP1) as a critical determinant of C. albicans biofilm's tolerance of amphotericin B. At 32 µg/ml of amphotericin B, GZY803 haploid biofilms showed 0.1% of persister population as compared with 1% of the diploid biofilms. AHP1 expression was found to be lower in GZY803 biofilms, and AHP1 overexpression in GZY803 restored the percentage of persister population. Consistently, deleting AHP1 in the diploid strain BWP17 caused a similar increase in amphotericin B susceptibility. AHP1 expression was also positively correlated with the antioxidant potential. Furthermore, C. albicans ira2Δ/Δ biofilms were susceptible to amphotericin B and had a diminished antioxidant capacity. Interestingly, AHP1 overexpression in the ira2Δ/Δ strain restored the antioxidant potential and enhanced the persister population against amphotericin B, and shutting down the AHP1 expression in ira2Δ/Δ biofilms reversed the effect. In conclusion, we provide evidence that the AHP1 gene critically determines the amphotericin B tolerance of C. albicans biofilms possibly by maintaining the persisters' antioxidant capacity. This finding will open up new avenues for developing therapies targeting the persister population of C. albicans biofilms. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD004274.

Characterization of a hyperactive Cyr1 mutant reveals new regulatory mechanisms for cellular cAMP levels in Candida albicans.

The adenylyl cyclase Cyr1 plays a pivotal role in regulating virulence traits in the human fungal pathogen Candida albicans. Although a diverse range of signals are known to activate Cyr1, it remains unclear how low activity is maintained in the absence of stimuli. To uncover negative regulatory elements, we designed a genetic screen to identify mutations in Cyr1 that increase its catalytic activity. We found such a mutant carrying a single Glu1541 to Lys substitution in a conserved motif C-terminal to the catalytic domain. This E1541K mutation caused constitutive filamentous growth, hypersensitivity to stress, resistance to farnesol and overproduction of riboflavin. The mutant phenotype depends on Cap1 and Ras1, two known positive regulators of Cyr1, and the filamentous growth requires Hgc1, a key promoter of hyphal growth. Strikingly, expressing a truncated version of the mutant protein lacking the entire region N-terminal to the catalytic domain in cyr1Δ cells caused a fivefold increase in the cellular cAMP level. Such cells exhibited dramatic enlargement, cytokinetic defects, G1 arrest and impaired hyphal development. Thus, our studies have revealed novel regulatory elements in Cyr1 that normally repress Cyr1 activity to prevent the toxicity of unregulated high cAMP levels.

Candida albicans Ubiquitin and Heat Shock Factor-Type Transcriptional Factors Are Involved in 2-Dodecenoic Acid-Mediated Inhibition of Hyphal Growth.

Cis-2-dodecenoic acid (i.e., Burkholderia cenocepacia Diffusible Signal Factor, BDSF), a signaling molecule produced by Burkholderia cenocepacia but not by Candida albicans, can prevent Candida albicans hyphal formation. The mechanism by which BDSF controls the morphological switch of C. albicans is still unknown. To address this issue, we used the cDNA microarray method to investigate the differential expression of genes in C. albicans in the presence and absence of BDSF. The microarray result indicated that 305 genes were significantly different in the expression level. This included the downregulation of 75 genes and the upregulation of 230 genes. Based on the microarray data, a mutant library was screened to search for genes, once mutated, conferred insensitivity to BDSF. The results showed that the repressors (Ubi4 and Sfl1 proteins) and the activator (Sfl2 protein) of filamentous growth are involved in the BDSF regulation of hyphal morphogenesis. Ubi4, an ubiquitin polypeptide that participates in ubiquitin-mediated protein turnover, is the protein required for the degradation of Sfl2. Sfl1 and Sfl2 proteins antagonistically control C. albicans morphogenesis. In the hyphal induction condition, the amount of Ubi4 and Sfl1 protein increased rapidly with the exogenous addition of BDSF. As a result, the protein level of the activator of filamentous growth, Sfl2, decreased correspondingly, thereby facilitating the C. albicans cells to remain in the yeast form.

Sac7 and Rho1 regulate the white-to-opaque switching in Candida albicans.

Candida albicans cells homozygous at the mating-type locus stochastically undergo the white-to-opaque switching to become mating-competent. This switching is regulated by a core circuit of transcription factors organized through interlocking feedback loops around the master regulator Wor1. Although a range of distinct environmental cues is known to induce the switching, the pathways linking the external stimuli to the central control mechanism remains largely unknown. By screening a C. albicans haploid gene-deletion library, we found that SAC7 encoding a GTPase-activating protein of Rho1 is required for the white-to-opaque switching. We demonstrate that Sac7 physically associates with Rho1-GTP and the constitutively active Rho1G18V mutant impairs the white-to-opaque switching while the inactive Rho1D124A mutant promotes it. Overexpressing WOR1 in both sac7Δ/Δ and rho1 G18V cells suppresses the switching defect, indicating that the Sac7/Rho1 module acts upstream of Wor1. Furthermore, we provide evidence that Sac7/Rho1 functions in a pathway independent of the Ras/cAMP pathway which has previously been positioned upstream of Wor1. Taken together, we have discovered new regulators and a signaling pathway that regulate the white-to-opaque switching in the most prevalent human fungal pathogen C. albicans.

Identification and functional characterization of a novel Candida albicans gene CaMNN5 that suppresses the iron-dependent growth defect of Saccharomyces cerevisiae aft1Delta mutant.

In Saccharomyces cerevisiae, the transcription factor Aft1p plays a central role in regulating many genes involved in iron acquisition and utilization. An aft1Delta mutant exhibits severely retarded growth under iron starvation. To identify the functional counterpart of AFT1 in Candida albicans, we transformed a C. albicans genomic DNA library into aft1Delta to isolate genes that could allow the mutant to grow under iron-limiting conditions. In the present paper, we describe the unexpected discovery in this screen of CaMNN5. CaMnn5p is an alpha-1,2-mannosyltransferease, but its growth-promoting function in iron-limiting conditions does not require this enzymatic activity. Its function is also independent of the high-affinity iron transport systems that are mediated by Ftr1p and Fth1p. We obtained evidence suggesting that CaMnn5p may function along the endocytic pathway, because it cannot promote the growth of end4Delta and vps4Delta mutants, where the endocytic pathway is blocked at an early and late step respectively. Neither can it promote the growth of a fth1Delta smf3Delta mutant, where the vacuole-cytosol iron transport is blocked. Expression of CaMNN5 in S. cerevisiae specifically enhances an endocytosis-dependent mechanism of iron uptake without increasing the uptake of Lucifer Yellow, a marker for fluid-phase endocytosis. CaMnn5p contains three putative Lys-Glu-Xaa-Xaa-Glu iron-binding sites and co-immunoprecipitates with 55Fe. We propose that CaMnn5p promotes iron uptake and usage along the endocytosis pathway under iron-limiting conditions, a novel function that might have evolved in C. albicans.

New "haploid biofilm model" unravels IRA2 as a novel regulator of Candida albicans biofilm formation.

Clinical isolates of the fungal human pathogen Candida albicans are invariably diploid and heterozygous, impeding genetic study. Recent isolation of C. albicans haploids opens opportunities to apply technologies unfeasible in diploids. However, doubts remain on whether the haploids, derived from chromosome loss, can represent the diploids. Here, we use C. albicans haploids to investigate biofilm, a key virulence attribute. We conducted the first comprehensive characterization of biofilm formation of the haploids in comparison with the diploids. We demonstrate that the haploids form biofilms with essentially the same characteristics as the diploids. Screening a haploid mutant library has uncovered novel GTPase-related genes as biofilm regulators, including IRA2 that encodes an activator of the Ras GTPase. IRA2-deletion mutants develop poorly constructed biofilm in both haploid and diploid C. albicans. Our results demonstrate that the haploids are a valid model for C. albicans biofilm research and a powerful tool for uncovering novel regulators.

One-step targeted gene deletion in Candida albicans haploids.

The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in C. albicans haploids, which takes ∼11 d to complete (not including plasmid construction, which may take ∼2 weeks). A gene deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR before final confirmation by flow cytometry.

Predominance of Candida tropicalis bloodstream infections in a Singapore teaching hospital.

An 18-month epidemiologic investigation of Candida bloodstream infections in a Singapore hospital identified 52 candidemic patients: 36% of whose infections were caused by C. tropicalis, 29% were due to C. albicans, 10% with C. parapsilosis and 21% involved C. glabrata. A predominant clonal C. tropicalis strain was demonstrated. No association with ICU stay, prior exposure to fluconazole/broad-spectrum antibiotics or increased mortality was found in this apparent shift towards non-C. albicans Candida species as the primary agents of candidemia.

MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans.

The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans alpha-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both alpha-1,2- and alpha-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Delta mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Delta mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Delta mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.