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.

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins.

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

Role of the inositol pyrophosphate multikinase Kcs1 in Cryptococcus inositol metabolism.

Cryptococcus neoformans is the most common cause of deadly fungal meningitis. This fungus has a complex inositol acquisition and utilization system, and our previous studies have shown the importance of inositol utilization in cryptococcal development and virulence. However, how inositol utilization is regulated in this fungus remains unknown. In this study, we found that inositol, irrespective of the presence of glucose in the media, represses the expression of C. neoformans genes involved in inositol pyrophosphate biosynthesis, including the gene encoding inositol hexakisphosphate kinase Kcs1. Kcs1 was recently reported to regulate inositol metabolism in Saccharomyces cerevisiae and to impact virulence in C. neoformans. To examine the potential role of Kcs1 in inositol regulation in C. neoformans, we generated the kcs1Δ mutant and compared its phenotype with the wild type strain. We found that Kcs1 negatively regulates inositol uptake and catabolism in C. neoformans, but, in contrast to Kcs1 function in S. cerevisiae, does not appear to regulate inositol biosynthesis. Together, these results show that Kcs1 functions to fine-tune inositol acquisition to maintain inositol homeostasis in C. neoformans.

The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin.

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.

The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast.

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as 'chromatin-dependent' when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at 'chromatin-dependent' origins. Finally, the 'chromatin-dependent' class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.

Structural and Biophysical Dynamics of Fungal Plasma Membrane Proteins and Implications for Echinocandin Action in Candida glabrata.

Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly characterized. Herein, we employ an integrative multimodal approach to elucidate the structural and functional organization of plasma membrane protein complexes in Candida glabrata , focusing on prominent and essential membrane proteins, the polysaccharide synthase ß-(1,3)-glucan synthase (GS) and the proton pump Pma1. Cryo-electron tomography (cryo-ET) and live cell imaging reveal that GS and Pma1 are heterogeneously distributed into distinct plasma membrane microdomains. Treatment with caspofungin, an echinocandin antifungal that targets GS, alters the plasma membrane and disrupts the native distribution of GS and Pma1. Based on these findings, we propose a model for echinocandin action that considers how drug interactions with the plasma membrane environment lead to inhibition of GS. Our work underscores the importance of interrogating the structural and dynamic characteristics of fungal plasma membrane proteins in situ to understand function and facilitate precisely targeted development of novel antifungal therapies.

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair.

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund-Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation-of-function alleles of SGS1 that suppress the slow growth of top3Delta and rmi1Delta cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild-type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1-D664Delta, unlike sgs1Delta, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication-associated X-shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1-D664Delta allele exhibits increased spontaneous RPA foci, suggesting that the persistent X-structures may contain single-stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.

Reactive oxidant species induced by antifungal drugs: identity, origins, functions, and connection to stress-induced cell death.

Reactive oxidant species (ROS) are unstable, highly reactive molecules that are produced by cells either as byproducts of metabolism or synthesized by specialized enzymes. ROS can be detrimental, e.g., by damaging cellular macromolecules, or beneficial, e.g., by participating in signaling. An increasing body of evidence shows that various fungal species, including both yeasts and molds, increase ROS production upon exposure to the antifungal drugs currently used in the clinic: azoles, polyenes, and echinocandins. However, the implications of these findings are still largely unclear due to gaps in knowledge regarding the chemical nature, molecular origins, and functional consequences of these ROS. Because the detection of ROS in fungal cells has largely relied on fluorescent probes that lack specificity, the chemical nature of the ROS is not known, and it may vary depending on the specific fungus-drug combination. In several instances, the origin of antifungal drug-induced ROS has been identified as the mitochondria, but further experiments are necessary to strengthen this conclusion and to investigate other potential cellular ROS sources, such as the ER, peroxisomes, and ROS-producing enzymes. With respect to the function of the ROS, several studies have shown that they contribute to the drugs' fungicidal activities and may be part of drug-induced programmed cell death (PCD). However, whether these "pro-death" ROS are a primary consequence of the antifungal mechanism of action or a secondary consequence of drug-induced PCD remains unclear. Finally, several recent studies have raised the possibility that ROS induction can serve an adaptive role, promoting antifungal drug tolerance and the evolution of drug resistance. Filling these gaps in knowledge will reveal a new aspect of fungal biology and may identify new ways to potentiate antifungal drug activity or prevent the evolution of antifungal drug resistance.

Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance.

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

The origin recognition complex interacts with a subset of metabolic genes tightly linked to origins of replication.

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC "affinity" genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.

Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU.

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1L518F and FKS1E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Determinants of fluconazole resistance and the efficacy of fluconazole and milbemycin oxim combination against Candida parapsilosis clinical isolates from Brazil and Turkey.

Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C. glabrata and C. albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators (TAC1, MRR1, and UPC2) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1, MDR1, and ERG11, examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C. parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1L518F mutation. Of these three isolates, one showed increased basal expression of CDR1, while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C. parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy.

Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells.

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target ß-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida sp., a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single-cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, of which the latter was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypotolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance. IMPORTANCE Echinocandin drugs are a first-line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen, and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here, we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypotolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.

Critical Assessment of Cell Wall Integrity Factors Contributing to in vivo Echinocandin Tolerance and Resistance in Candida glabrata.

Fungal infections are on the rise, and emergence of drug-resistant Candida strains refractory to treatment is particularly alarming. Resistance to azole class antifungals, which have been extensively used worldwide for several decades, is so high in several prevalent fungal pathogens, that another drug class, the echinocandins, is now recommended as a first line antifungal treatment. However, resistance to echinocandins is also prominent, particularly in certain species, such as Candida glabrata. The echinocandins target 1,3-ß-glucan synthase (GS), the enzyme responsible for producing 1,3-ß-glucans, a major component of the fungal cell wall. Although echinocandins are considered fungicidal, C. glabrata exhibits echinocandin tolerance both in vitro and in vivo, where a subset of the cells survives and facilitates the emergence of echinocandin-resistant mutants, which are responsible for clinical failure. Despite this critical role of echinocandin tolerance, its mechanisms are still not well understood. Additionally, most studies of tolerance are conducted in vitro and are thus not able to recapitulate the fungal-host interaction. In this study, we focused on the role of cell wall integrity factors in echinocandin tolerance in C. glabrata. We identified three genes involved in the maintenance of cell wall integrity - YPS1, YPK2, and SLT2 - that promote echinocandin tolerance both in vitro and in a mouse model of gastrointestinal (GI) colonization. In particular, we show that mice colonized with strains carrying deletions of these genes were more effectively sterilized by daily caspofungin treatment relative to mice colonized with the wild-type parental strain. Furthermore, consistent with a role of tolerant cells serving as a reservoir for generating resistant mutations, a reduction in tolerance was associated with a reduction in the emergence of resistant strains. Finally, reduced susceptibility in these strains was due both to the well described FKS-dependent mechanisms and as yet unknown, FKS-independent mechanisms. Together, these results shed light on the importance of cell wall integrity maintenance in echinocandin tolerance and emergence of resistance and lay the foundation for future studies of the factors described herein.

A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen.

DNA damage checkpoints are key guardians of genome integrity. Eukaryotic cells respond to DNA damage by triggering extensive phosphorylation of Rad53/CHK2 effector kinase, whereupon activated Rad53/CHK2 mediates further aspects of checkpoint activation, including cell cycle arrest and transcriptional changes. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug-resistant mutants. However, the mechanisms underlying this genetic variability are unclear. We used Western blotting and mass spectrometry to show that, unlike S. cerevisiae, C. glabrata cells exposed to DNA damage did not induce C. glabrata Rad53 (CgRad53) phosphorylation. Furthermore, flow cytometry analysis showed that, unlike S. cerevisiae, C. glabrata cells did not accumulate in S phase upon DNA damage. Consistent with these observations, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, transcriptome sequencing (RNAseq) analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including proliferating cell nuclear antigen (PCNA). Together, our results reveal a noncanonical fungal DNA damage response in C. glabrata, which may contribute to rapidly generating genetic change and drug resistance.IMPORTANCE In order to preserve genome integrity, all cells must mount appropriate responses to DNA damage, including slowing down or arresting the cell cycle to give the cells time to repair the damage and changing gene expression, for example to induce genes involved in DNA repair. The Rad53 protein kinase is a conserved central mediator of these responses in eukaryotic cells, and its extensive phosphorylation upon DNA damage is necessary for its activation and subsequent activity. Interestingly, here we show that in the opportunistic fungal pathogen Candida glabrata, Rad53 phosphorylation is not induced by DNA damage, nor do these cells arrest in S phase under these conditions, in contrast to the closely related yeast Saccharomyces cerevisiae Instead, C. glabrata cells continue to divide in the presence of DNA damage, resulting in significant cell lethality. Finally, we show that a number of genes involved in DNA repair are strongly induced by DNA damage in S. cerevisiae but repressed in C. glabrata Together, these findings shed new light on mechanisms regulating genome stability in fungal pathogens.