Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans' morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

ChIP-SICAP: A New Tool to Explore Gene-Regulatory Networks in Candida albicans and Other Yeasts.

Chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) is a powerful method to identify protein interactions, and has long been used to gain insights into regulatory networks in relevant fungal species as well as many other organisms. In this chapter, we discuss a similar technique called ChIP-SICAP (chromatin immunoprecipitation with selective isolation of chromatin-associated proteins) that overcomes many of the traditional limitations of ChIP-MS, and describe a protocol that allows ChIP-SICAP to be applied to Candida albicans and other yeasts. Notably, the technique design permits stringent washing to remove contaminating proteins and antibodies before subsequent mass spectrometry processing, allows for genome-wide mapping of the bait protein by ChIP-seq after ChIP-SICAP from the same sample through a DNA recovery process, and specifically purifies and identifies proteins associating with chromatin. In the future, ChIP-SICAP will provide the yeast genomics research community an additional method to explore the complex dynamics of the gene-regulatory networks modulating morphology, metabolism and response to stress.

Overexpression approaches to advance understanding of Candida albicans.

Candida albicans is an opportunistic fungal pathogen that is responsible for infections linked to high mortality. Loss-of-function approaches, taking advantage of gene knockouts or inducible down-regulation, have been successfully used in this species in order to understand gene function. However, overexpression of a gene provides an alternative, powerful tool to elucidate gene function and identify novel phenotypes. Notably, overexpression can identify pathway components that might remain undetected using loss-of-function approaches. Several repressible or inducible promoters have been developed which allow to shut off or turn on the expression of a gene in C. albicans upon growth in the presence of a repressor or inducer. In this review, we summarize recent overexpression approaches used to study different aspects of C. albicans biology, including morphogenesis, biofilm formation, drug tolerance, and commensalism.

Commensalism instead of sex? Adapting mating to live in the gut.

In this issue of Cell Host & Microbe, Witchley et al. (2021) describe a rewired transcriptional network that reveals how the human fungal pathogen Candida albicans favors commensalism over sexual reproduction in the host environment.

A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans.

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species.

Ifu5, a WW domain-containing protein interacts with Efg1 to achieve coordination of normoxic and hypoxic functions to influence pathogenicity traits in Candida albicans.

Hypoxic adaptation pathways, essential for Candida albicans pathogenesis, are tied to its transition from a commensal to a pathogen. Herein, we identify a WW domain-containing protein, Ifu5, as a determinant of hypoxic adaptation that also impacts normoxic responses in this fungus. Ifu5 activity supports glycosylation homeostasis via the Cek1 mitogen-activated protein kinase-dependent up-regulation of PMT1, under normoxia. Transcriptome analysis of ifu5Δ/Δ under normoxia shows a significant up-regulation of the hypoxic regulator EFG1 and EFG1-dependent genes. We demonstrate physical interaction between Ifu5 by virtue of its WW domain and Efg1 that represses EFG1 expression under normoxia. This interaction is lost under hypoxic growth conditions, relieving EFG1 repression. Hypoxic adaptation processes such as filamentation and biofilm formation are affected in ifu5Δ/Δ cells revealing the role of Ifu5 in hypoxic signalling and modulating pathogenicity traits of C. albicans under varied oxygen conditions. Additionally, the WW domain of Ifu5 facilitates its role in hypoxic adaptation, revealing the importance of this domain in providing a platform to integrate various cellular processes. These data forge a relationship between Efg1 and Ifu5 that fosters the role of Ifu5 in hypoxic adaptation thus illuminating novel strategies to undermine the growth of C. albicans.

Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Dom34 Links Translation to Protein O-mannosylation.

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Candida albicans responds to glycostructure damage by Ace2-mediated feedback regulation of Cek1 signaling.

Candida albicans uses the Cek1 MAPK pathway to restore cells from damage of its cell wall glycostructures. Defective protein N- or O-glycosylation activates Cek1 and the transcription factor Ace2 as its downstream target, to upregulate genes encoding protein O-mannosyltransferases (Pmt proteins). In unstressed cells, Cek1-Ace2 activity blocks expression of PMT1, which is de-repressed by tunicamycin. Genomic binding targets of Ace2 included ZCF21, which was upregulated by Ace2 and found to repress PMT1 transcription in unstressed cells. Surprisingly, genes encoding components of the Cek1 pathway including MSB2, CST20, HST7, CEK1 and ACE2 were also identified as Ace2 targets indicating Ace2-mediated transcriptional amplification of pathway genes under N-glycosylation stress. In this condition, physical interaction of the Ace2 protein with the upstream MAPKKK Cst20 was detected. Cst20-GFP showed stress-induced import from the cytoplasm into the nucleus and phosphorylation of Ace2. Interestingly, forced nuclear localization of Cst20 inhibited Cek1-Ace2 signaling, while forced cytoplasmic localization of Cst20 retained full signaling activity, suggesting that nuclear Cst20 downregulates the Cek1 pathway. Collectively, the results indicate that Ace2 is a versatile multifunctional transcriptional regulator, which activates glycostress responses of C. albicans by both positive forward and negative feedback regulation of Cek1 signaling.

Hypoxia and Temperature Regulated Morphogenesis in Candida albicans.

Candida albicans is a common commensal in the human gut but in predisposed patients it can become an important human fungal pathogen. As a commensal, C. albicans adapts to low-oxygen conditions and represses its hyphal development by the transcription factor Efg1, which under normoxia activates filamentation. The repressive hypoxic but not the normoxic function of Efg1 required its unmodified N-terminus, was prevented by phosphomimetic residues at normoxic phosphorylation sites T179 and T206 and occurred only at temperatures ≤35°C. Genome-wide binding sites for native Efg1 identified 300 hypoxia-specific target genes, which overlapped partially with hypoxic binding sites for Ace2, a known positive regulator of hypoxic filamentation. Transcriptional analyses revealed that EFG1, ACE2 and their identified targets BCR1 and BRG1 encode an interconnected regulatory hub, in which Efg1/Bcr1 act as negative and Ace2/Brg1 act as positive regulators of gene expression under hypoxia. In this circuit, the hypoxic function of Ace2 was stimulated by elevated CO2 levels. The hyperfilamentous phenotype of efg1 and bcr1 mutants depended on Ace2/Brg1 regulators and required increased expression of genes encoding Cek1 MAP kinase and its downstream target Cph1. The intricate temperature-dependent regulatory mechanisms under hypoxia suggest that C. albicans restricts hyphal morphogenesis in oxygen-poor body niches, possibly to persist as a commensal in the human host.

Signaling domains of mucin Msb2 in Candida albicans.

Candida albicans adapts to the human host by environmental sensing using the Msb2 signal mucin, which regulates fungal morphogenesis and resistance characteristics. Msb2 is anchored within the cytoplasmic membrane by a single transmembrane (TM) region dividing it into a large N-terminal exodomain, which is shed, and a small cytoplasmic domain. Analyses of strains carrying deleted Msb2 variants revealed an exodomain segment required for cleavage, shedding, and all functions of Msb2. Phosphorylation of the mitogen-activated protein kinase (MAP kinase) Cek1 was regulated by three distinct regions in Msb2: in unstressed cells, N-terminal sequences repressed phosphorylation, while its induction under cell wall stress required the cytoplasmic tail (C-tail) and sequences N-terminally flanking the TM region, downstream of the proposed cleavage site. Within the latter Msb2 region, overlapping but not identical sequences were also required for hyphal morphogenesis, basal resistance to antifungals, and, in unstressed cells, downregulation of the PMT1 transcript, encoding protein O-mannosyltransferase-1. Deletion of two-thirds of the exodomain generated a truncated Msb2 variant with a striking ability to induce hyperfilamentous growth, which depended on the presence of the Msb2-interacting protein Sho1, the MAP kinase Cek1, and the Efg1 transcription factor. Under cell wall stress, the cytoplasmic tail relocalized partially to the nucleus and contributed to regulation of 117 genes, as revealed by transcriptomic analyses. Genes regulated by the C-tail contained binding sites for the Ace2 and Azf1 transcription factors and included the ALS cell wall genes. We concluded that Msb2 fulfills its numerous functions by employing functional domains that are distributed over its entire length.

A surprising role for the Sch9 protein kinase in chromosome segregation in Candida albicans.

The AGC kinase Sch9 regulates filamentation in Candida albicans. Here, we show that Sch9 binding is most enriched at the centromeres in C. albicans, but not in Saccharomyces cerevisiae. Deletion of CaSch9 leads to a 150- to 750-fold increase in chromosome loss. Thus, we report a previously unknown role of Sch9 in chromosome segregation.