Extrathymic expression of Aire controls the induction of effective TH17 cell-mediated immune response to Candida albicans.

Patients with loss of function in the gene encoding the master regulator of central tolerance AIRE suffer from a devastating disorder called autoimmune polyendocrine syndrome type 1 (APS-1), characterized by a spectrum of autoimmune diseases and severe mucocutaneous candidiasis. Although the key mechanisms underlying the development of autoimmunity in patients with APS-1 are well established, the underlying cause of the increased susceptibility to Candida albicans infection remains less understood. Here, we show that Aire+MHCII+ type 3 innate lymphoid cells (ILC3s) could sense, internalize and present C. albicans and had a critical role in the induction of Candida-specific T helper 17 (TH17) cell clones. Extrathymic Rorc-Cre-mediated deletion of Aire resulted in impaired generation of Candida-specific TH17 cells and subsequent overgrowth of C. albicans in the mucosal tissues. Collectively, our observations identify a previously unrecognized regulatory mechanism for effective defense responses against fungal infections.

Transmission of trained immunity and heterologous resistance to infections across generations.

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.

Safeguard function of PU.1 shapes the inflammatory epigenome of neutrophils.

Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.

CARD9+ microglia promote antifungal immunity via IL-1ß- and CXCL1-mediated neutrophil recruitment.

The C-type lectin receptor-Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1ß served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1ß and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1ß, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1ß and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host-pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.

The Hog1 MAPK substrate governs Candida glabrata-epithelial cell adhesion via the histone H2A variant.

CgHog1, terminal kinase of the high-osmolarity glycerol signalling pathway, orchestrates cellular response to multiple external stimuli including surplus-environmental iron in the human fungal pathogen Candida glabrata (Cg). However, CgHog1 substrates remain unidentified. Here, we show that CgHog1 adversely affects Cg adherence to host stomach and kidney epithelial cells in vitro, but promotes Cg survival in the iron-rich gastrointestinal tract niche. Further, CgHog1 interactome and in vitro phosphorylation analysis revealed CgSub2 (putative RNA helicase) to be a CgHog1 substrate, with CgSub2 also governing iron homeostasis and host adhesion. CgSub2 positively regulated EPA1 (encodes a major adhesin) expression and host adherence via its interactor CgHtz1 (histone H2A variant). Notably, both CgHog1 and surplus environmental iron had a negative impact on CgSub2-CgHtz1 interaction, with CgHTZ1 or CgSUB2 deletion reversing the elevated adherence of Cghog1Δ to epithelial cells. Finally, the surplus-extracellular iron led to CgHog1 activation, increased CgSub2 phosphorylation, elevated CgSub2-CgHta (canonical histone H2A) interaction, and EPA1 transcriptional activation, thereby underscoring the iron-responsive, CgHog1-induced exchange of histone partners of CgSub2. Altogether, our work mechanistically defines how CgHog1 couples Epa1 adhesin expression with iron abundance, and point towards specific chromatin composition modification programs that probably aid fungal pathogens align their adherence to iron-rich (gut) and iron-poor (blood) host niches.

Nociceptive Sensory Fibers Drive Interleukin-23 Production from CD301b+ Dermal Dendritic Cells and Drive Protective Cutaneous Immunity.

Innate resistance to Candida albicans in mucosal tissues requires the production of interleukin-17A (IL-17A) by tissue-resident cells early during infection, but the mechanism of cytokine production has not been precisely defined. In the skin, we found that dermal γδ T cells were the dominant source of IL-17A during C. albicans infection and were required for pathogen resistance. Induction of IL-17A from dermal γδ T cells and resistance to C. albicans required IL-23 production from CD301b(+) dermal dendritic cells (dDCs). In addition, we found that sensory neurons were directly activated by C. albicans. Ablation of sensory neurons increased susceptibility to C. albicans infection, which could be rescued by exogenous addition of the neuropeptide CGRP. These data define a model in which nociceptive pathways in the skin drive production of IL-23 by CD301b(+) dDCs resulting in IL-17A production from γδ T cells and resistance to cutaneous candidiasis.

MCPIP1 Endoribonuclease Activity Negatively Regulates Interleukin-17-Mediated Signaling and Inflammation.

Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1's endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra(-/-) background. Conversely, IL-17-dependent pathology in Zc3h12a(+/-) mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3' UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.

Clonal Dissemination of Antifungal-Resistant Candida haemulonii, China.

Candida haemulonii, a relative of C. auris, frequently shows antifungal resistance and is transmissible. However, molecular tools for genotyping and investigating outbreaks are not yet established. We performed genome-based population analysis on 94 C. haemulonii strains, including 58 isolates from China and 36 other published strains. Phylogenetic analysis revealed that C. haemulonii can be divided into 4 clades. Clade 1 comprised strains from China and other global strains; clades 2-4 contained only isolates from China, were more recently evolved, and showed higher antifungal resistance. Four regional epidemic clusters (A, B, C, and D) were identified in China, each comprising ≥5 cases (largest intracluster pairwise single-nucleotide polymorphism differences <50 bp). Cluster A was identified in 2 hospitals located in the same city, suggesting potential intracity transmissions. Cluster D was resistant to 3 classes of antifungals. The emergence of more resistant phylogenetic clades and regional dissemination of antifungal-resistant C. haemulonii warrants further monitoring.

Isolated Chronic Mucocutaneous Candidiasis due to a Novel Duplication Variant of IL17RC.

PURPOSE: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Among inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a 7-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. METHODS: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. RESULTS: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of 3 months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. CONCLUSIONS: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for the diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36-49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

IL-17 regulates systemic fungal immunity by controlling the functional competence of NK cells.

Interleukin 17 (IL-17)-mediated immunity plays a key role in protection from fungal infections in mice and man. Here, we confirmed that mice deficient in the IL-17 receptor or lacking the ability to secrete IL-17 are highly susceptible to systemic candidiasis, but we found that temporary blockade of the IL-17 pathway during infection in wild-type mice did not impact fungal control. Rather, mice lacking IL-17 receptor signaling had a cell-intrinsic impairment in the development of functional NK cells, which accounted for the susceptibility of these mice to systemic fungal infection. NK cells promoted antifungal immunity by secreting GM-CSF, necessary for the fungicidal activity of neutrophils. These data reveal that NK cells are crucial for antifungal defense and indicate a role for IL-17 family cytokines in NK cell development. The IL-17-NK cell axis may impact immunity against not only fungi but also bacteria, viruses, and tumors.

Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor.

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.

Mrr1 regulation of methylglyoxal catabolism and methylglyoxal-induced fluconazole resistance in Candida lusitaniae.

Transcription factor Mrr1, best known for its regulation of Candida azole resistance genes such as MDR1, regulates other genes that are poorly characterized. Among the other Mrr1-regulated genes are putative methylglyoxal reductases. Methylglyoxal (MG) is a toxic metabolite that is elevated in diabetes, uremia, and sepsis, which are diseases that increase the risk for candidiasis, and MG serves as a regulatory signal in diverse organisms. Our studies in Clavispora lusitaniae, also known as Candida lusitaniae, showed that Mrr1 regulates expression of two paralogous MG reductases, MGD1 and MGD2, and that both participate in MG resistance and MG catabolism. Exogenous MG increased Mrr1-dependent expression of MGD1 and MGD2 as well as expression of MDR1, which encodes an efflux pump that exports fluconazole. MG improved growth in the presence of fluconazole and this was largely Mrr1-dependent with contributions from a secondary transcription factor, Cap1. Increased fluconazole resistance was also observed in mutants lacking Glo1, a Mrr1-independent MG catabolic enzyme. Isolates from other Candida species displayed heterogeneity in MG resistance and MG stimulation of azole resistance. We propose endogenous and host-derived MG can induce MDR1 and other Mrr1-regulated genes causing increased drug resistance, which may contribute to some instances of fungal treatment failure.

Integrating GWAS with bulk and single-cell RNA-sequencing reveals a role for LY86 in the anti-Candida host response.

Candida bloodstream infection, i.e. candidemia, is the most frequently encountered life-threatening fungal infection worldwide, with mortality rates up to almost 50%. In the majority of candidemia cases, Candida albicans is responsible. Worryingly, a global increase in the number of patients who are susceptible to infection (e.g. immunocompromised patients), has led to a rise in the incidence of candidemia in the last few decades. Therefore, a better understanding of the anti-Candida host response is essential to overcome this poor prognosis and to lower disease incidence. Here, we integrated genome-wide association studies with bulk and single-cell transcriptomic analyses of immune cells stimulated with Candida albicans to further our understanding of the anti-Candida host response. We show that differential expression analysis upon Candida stimulation in single-cell expression data can reveal the important cell types involved in the host response against Candida. This confirmed the known major role of monocytes, but more interestingly, also uncovered an important role for NK cells. Moreover, combining the power of bulk RNA-seq with the high resolution of single-cell RNA-seq data led to the identification of 27 Candida-response QTLs and revealed the cell types potentially involved herein. Integration of these response QTLs with a GWAS on candidemia susceptibility uncovered a potential new role for LY86 in candidemia susceptibility. Finally, experimental follow-up confirmed that LY86 knockdown results in reduced monocyte migration towards the chemokine MCP-1, thereby implying that this reduced migration may underlie the increased susceptibility to candidemia. Altogether, our integrative systems genetics approach identifies previously unknown mechanisms underlying the immune response to Candida infection.

ß-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

An IL-17F.S65L Knock-In Mouse Reveals Similarities and Differences in IL-17F Function in Oral Candidiasis: A New Tool to Understand IL-17F.

Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans IL-17R signaling is essential to prevent OPC in mice and humans, but the individual roles of its ligands, IL-17A, IL-17F, and IL-17AF, are less clear. A homozygous IL-17F deficiency in mice does not cause OPC susceptibility, whereas mice lacking IL-17A are moderately susceptible. In humans, a rare heterozygous mutation in IL-17F (IL-17F.S65L) was identified that causes chronic mucocutaneous candidiasis, suggesting the existence of essential antifungal pathways mediated by IL-17F and/or IL-17AF. To investigate the role of IL-17F and IL-17AF in more detail, we exploited this "experiment of nature" by creating a mouse line bearing the homologous mutation in IL-17F (Ser65Leu) by CRISPR/Cas9. Unlike Il17f-/- mice that are resistant to OPC, Il17fS65L/S65L mice showed increased oral fungal burdens similar to Il17a -/- mice. In contrast to humans, however, disease was only evident in homozygous, not heterozygous, mutant mice. The mutation was linked to modestly impaired CXC chemokine expression and neutrophil recruitment to the infected tongue but not to alterations in oral antimicrobial peptide expression. These findings suggest mechanisms by which the enigmatic cytokine IL-17F contributes to host defense against fungi. Moreover, because these mice do not phenocopy Il17f-/- mice, they may provide a valuable tool to interrogate IL-17F and IL-17AF function in vivo in other settings.

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

Plasma membrane architecture protects Candida albicans from killing by copper.

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

DNA damage response of major fungal pathogen Candida glabrata offers clues to explain its genetic diversity.

How cells respond to DNA damage is key to maintaining genome integrity or facilitating genetic change. In fungi, DNA damage responses have been extensively characterized in the model budding yeast Saccharomyces cerevisiae, which is generally not pathogenic. However, it is not clear how closely these responses resemble those in fungal pathogens, in which genetic change plays an important role in the evolutionary arms race between pathogen and host and the evolution of antifungal drug resistance. A close relative of S. cerevisiae, Candida glabrata, is an opportunistic pathogen that displays high variability in chromosome structure among clinical isolates and rapidly evolves antifungal drug resistance. The mechanisms facilitating such genomic flexibility and evolvability in this organism are unknown. Recently we characterized the DNA damage response of C. glabrata and identified several features that distinguish it from the well characterized DNA damage response of S. cerevisiae. First, we discovered that, in contrast to the established paradigm, C. glabrata effector kinase Rad53 is not hyperphosphorylated upon DNA damage. We also uncovered evidence of an attenuated DNA damage checkpoint response, wherein in the presence of DNA damage C. glabrata cells did not accumulate in S-phase and proceeded with cell division, leading to aberrant mitoses and cell death. Finally, we identified evidence of transcriptional rewiring of the DNA damage response of C. glabrata relative to S. cerevisiae, including an upregulation of genes involved in mating and meiosis-processes that have not been reported in C. glabrata. Together, these results open new possibilities and raise tantalizing questions of how this major fungal pathogen facilitates genetic change.

Circuit diversification in a biofilm regulatory network.

Genotype-phenotype relationships can vary extensively among members of a species. One cause of this variation is circuit diversification, the alteration of gene regulatory relationships among members of a species. Circuit diversification is thought to be a starting point for the circuit divergence or rewiring that occurs during speciation. How widespread is circuit diversification? Here we address this question with the fungal pathogen Candida albicans, which forms biofilms rich in distinctive hyphal cells as a prelude to infection. Our understanding of the biofilm/hyphal regulatory network comes primarily from studies of one clinical isolate, strain SC5314, and its marked derivatives. We used CRISPR-based methods to create mutations of four key biofilm transcription factor genes-BCR1, UME6, BRG1, and EFG1 -in SC5314 and four additional clinical isolates. Phenotypic analysis revealed that mutations in BCR1 or UME6 have variable impact across strains, while mutations in BRG1 or EFG1 had uniformly severe impact. Gene expression, sampled with Nanostring probes and examined comprehensively for EFG1 via RNA-Seq, indicates that regulatory relationships are highly variable among isolates. Our results suggest that genotype-phenotype relationships vary in this strain panel in part because of differences in control of BRG1 by BCR1, a hypothesis that is supported through engineered constitutive expression of BRG1. Overall, the data show that circuit diversification is the rule, not the exception, in this biofilm/hyphal regulatory network.