Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence

ABSTRACT Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes. IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

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A systemic analysis of amino acid transporters identifies Gnp2 as the main proline permease in Candida albicans

Access Microbiology ◽

10.1099/acmi.cc2021.po0080 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Enrico Garbe ◽

Pedro Miramon ◽

Franziska Gerwien ◽

Michael Lorenz ◽

Slavena Vylkova

Keyword(s):

Amino Acids ◽

Candida Albicans ◽

Amino Acid ◽

Fungal Growth ◽

Free Form ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Fungal Virulence ◽

Proline Utilization

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in free form or peptide bound, are an abundant carbon and nitrogen source in many host niches. Further,the capacity to sense and utilize certain amino acids, like proline, is directly linked to virulence. The C. albicans genome encodes for at least 24 amino acid permeases (AAPs), highlighting the importance of flexible amino acid uptake for fungal growth and virulence. Although the substrate specificity and role of certain AAPs has been investigated, a comprehensive characterization was missing. Therefore, we assembled a library of AAP deletion strains, which was tested for resistance to toxic amino acid analogs. Most striking was the specific resistance of gnp2Δ to the proline analog 3,4-dehydroproline. Subsequent tests validated that Gnp2 is a specific proline permease in C. albicans, which is contrary to the model yeast Saccharomyces cerevisiae where proline transport is mediated by four permeases. Furthermore, the induction of GNP2 appears to be independent of the SPS (Ssy1-Ptr3-Ssy5) regulatory pathway that controls proline utilization in the model yeast, pointing towards rewired proline uptake in C. albicans. Additionally, strains lacking GNP2were unable to respond to proline-induced filamentation, displayed decreased cytotoxicity to macrophages and showed increased sensitivity to oxidative stress, underlining the importance of proline uptake for fungal virulence. Taken together, the role of Gnp2-mediated proline uptake illustrates the importance of metabolism-driven virulence in C. albicans.

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Candida albicans Activation of Human Monocytes Toward M2 Profile is Reversed by Amphotericin B and Fluconazole

Journal of Bacteriology and Mycology ◽

10.26420/jbacteriolmycol.2021.1168 ◽

2021 ◽

Vol 8 (2) ◽

Author(s):

Icely PA ◽

◽

Vigezzi C ◽

Rodriguez E ◽

Miró MS ◽

...

Keyword(s):

Nitric Oxide ◽

Candida Albicans ◽

Amphotericin B ◽

Immune Cells ◽

Fungal Growth ◽

Antifungal Drugs ◽

Arginase Activity ◽

Intrinsic Activity ◽

No Production ◽

Human Monocytes

Phagocytes, including monocytes/macrophages, play an important role in the host defense during Candida albicans infections. In the L-arginine metabolism, the balance between the activation of two enzymes, inducible Nitric Oxide Synthase (iNOS) and arginase, promotes in the macrophages two alternative metabolic states, while M1 profile is related with host protection, M2 favored the fungal growth and evasion. Our aim was to evaluate the effect of Amphotericin B (AMB) and Fluconazole (FLC) on polarization of human monocytes to M2 profile induced by C. albicans. The human monocytic (Mo) cell line U937 was co-cultured with viable yeast of C. albicans, or Lipopolysaccharides (LPS) or Phorbol-12-myristate-13-acetate (PMA). Nitric Oxide (NO), cytokines production and arginase activity were evaluated. The effect of AMB or FLC on these metabolic pathways in immune cells and on fungus intrinsic arginase activity was studied. C. albicans inhibits NO production in human-monocyte and induces strong host arginase activity (p<0.0001). AMB and FLC inhibited C. albicansinduced arginase activity in immune cells (p<0.001), reaching a percentage of inhibition of 90% for AMB and 78% for FLC. Arginase intrinsic activity of the fungus was blocked by nor-NOHA (arginase inhibitor) and AMB (p<0.05). These results show that C. albicans drives human Mo toward M2 profile and that both antifungal drugs evaluated have the ability to revert C. albicans-induced M2 profile. In a relevant manner, it also provides data about additional effect of AMB as inhibitor of C. albicans endogenous arginase activity. Here in we provide new evidence for the effect of these drugs over the immune cells and the yeast.

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The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.05060-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1034-1042 ◽

Cited By ~ 79

Author(s):

Rebecca A. Hall ◽

Kara J. Turner ◽

James Chaloupka ◽

Fabien Cottier ◽

Luisa De Sordi ◽

...

Keyword(s):

Candida Albicans ◽

Quorum Sensing ◽

Signaling Pathway ◽

Homoserine Lactone ◽

Burkholderia Cenocepacia ◽

Camp Signaling ◽

Mammalian Host ◽

Content Type ◽

Hyphal Development ◽

Natural Flora

ABSTRACTLiving as a commensal,Candida albicansmust adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects onC. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C12-homoserine lactone, a quorum-sensing molecule secreted byPseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of theCandidaadenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving theC. albicanshyphal repressor, Sfl1p. Deletion ofSFL1did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing inC. albicansis mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that theBurkholderia cenocepaciadiffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C12-homoserine lactone, may be used by other quorum-sensing molecules.

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Metabolic Reprogramming in the Opportunistic Yeast Candida albicans in Response to Hypoxia

mSphere ◽

10.1128/msphere.00913-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Anaïs Burgain ◽

Faiza Tebbji ◽

Inès Khemiri ◽

Adnane Sellam

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Metabolic Pathways ◽

Membrane Lipids ◽

Oxygen Depletion ◽

Metabolic Reprogramming ◽

Pathogenic Yeast ◽

Fungal Virulence ◽

Content Type ◽

The Impact

ABSTRACT Hypoxia is the predominant condition that the human opportunistic fungus Candida albicans encounters in the majority of the colonized niches within the host. So far, the impact of such a condition on the overall metabolism of this important human-pathogenic yeast has not been investigated. Here, we have undertaken a time-resolved metabolomics analysis to uncover the metabolic landscape of fungal cells experiencing hypoxia. Our data showed a dynamic reprogramming of many fundamental metabolic pathways, such as glycolysis, the pentose phosphate pathway, and different metabolic routes related to fungal cell wall biogenesis. The C. albicans lipidome was highly affected by oxygen depletion, with an increased level of free fatty acids and biochemical intermediates of membrane lipids, including phospholipids, lysophospholipids, sphingolipids, and mevalonate. The depletion of oxygen-dependent lipids such as ergosterol or phosphatidylcholine with longer and polyunsaturated lateral fatty acid chains was observed only at the later hypoxic time point (180 min). Transcriptomics data supported the main metabolic response to hypoxia when matched to our metabolomic profiles. The hypoxic metabolome reflected different physiological alterations of the cell wall and plasma membrane of C. albicans under an oxygen-limiting environment that were confirmed by different approaches. This study provided a framework for future in vivo investigations to examine relevant hypoxic metabolic trajectories in fungal virulence and fitness within the host. IMPORTANCE A critical aspect of cell fitness is the ability to sense and adapt to variations in oxygen levels in their local environment. Candida albicans is an opportunistic yeast that is the most prevalent human fungal pathogen. While hypoxia is the predominant condition that C. albicans encounters in most of its niches, its impact on fungal metabolism remains unexplored so far. Here, we provided a detailed landscape of the C. albicans metabolome that emphasized the importance of many metabolic routes for the adaptation of this yeast to oxygen depletion. The fungal hypoxic metabolome identified in this work provides a framework for future investigations to assess the contribution of relevant metabolic pathways in the fitness of C. albicans and other human eukaryotic pathogens with similar colonized human niches. As hypoxia is present at most of the fungal infection foci in the host, hypoxic metabolic pathways are thus an attractive target for antifungal therapy.

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The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction

Infection and Immunity ◽

10.1128/iai.00763-19 ◽

2020 ◽

Vol 88 (5) ◽

Cited By ~ 2

Author(s):

Pedro Miramón ◽

Andrew W. Pountain ◽

Ambro van Hoof ◽

Michael C. Lorenz

Keyword(s):

Candida Albicans ◽

Transcription Factors ◽

Amino Acid ◽

Cell Damage ◽

Nutrient Acquisition ◽

Acid Uptake ◽

Content Type ◽

Host Interaction ◽

Amino Acid Utilization

ABSTRACT Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

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Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00215-12 ◽

2012 ◽

Vol 11 (10) ◽

pp. 1289-1299 ◽

Cited By ~ 105

Author(s):

Stephanie A. Flowers ◽

Katherine S. Barker ◽

Elizabeth L. Berkow ◽

Geoffrey Toner ◽

Sean G. Chadwick ◽

...

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Clinical Isolates ◽

Transcriptional Analysis ◽

Single Amino Acid ◽

Ergosterol Biosynthesis ◽

Amino Acid Substitutions ◽

Gain Of Function ◽

Content Type ◽

Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increasedERG11expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

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Physiologic Expression of the Candida albicans Pescadillo Homolog Is Required for Virulence in a Murine Model of Hematogenously Disseminated Candidiasis

Eukaryotic Cell ◽

10.1128/ec.00171-12 ◽

2012 ◽

Vol 11 (12) ◽

pp. 1552-1556 ◽

Cited By ~ 5

Author(s):

Priya Uppuluri ◽

Ashok K. Chaturvedi ◽

Niketa Jani ◽

Read Pukkila-Worley ◽

Carlos Monteagudo ◽

...

Keyword(s):

Candida Albicans ◽

Murine Model ◽

Critical Role ◽

Growth Defect ◽

Mammalian Host ◽

Developmental Cycle ◽

Yeast Growth ◽

Content Type ◽

Disseminated Candidiasis

ABSTRACT Morphogenetic conversions contribute to the pathogenesis of Candida albicans invasive infections. Many studies to date have convincingly demonstrated a link between filamentation and virulence; however, relatively little is known regarding the role of the filament-to-yeast transition during the pathogenesis of invasive candidiasis. We previously identified the C. albicans pescadillo homolog ( PES1 ) as essential during yeast growth and growth of lateral yeast on hyphae but not during hyphal growth. Furthermore, we demonstrated that PES1 is required for virulence in vivo in a Galleria mellonella larva model of candidiasis. Here, we have used a regulatable tetO-PES1 / pes1 strain to assess the contribution of C. albicans PES1 to pathogenesis in the commonly used and clinically relevant murine model of hematogenously disseminated candidiasis. Our results indicate that a physiologically controlled level of PES1 expression is required for full virulence in this animal model, with virulence defects observed both when PES1 is overexpressed and and when it is depleted. The pathogenetic defect of cells depleted of PES1 is not due to a general growth defect, as demonstrated by the fact that PES1 -depleted cells still kill Caenorhabditis elegans as efficiently as the wild type due to hyphal outgrowth through worm tissues. Our results suggest a critical role of lateral yeast growth in the ability of C. albicans to normally proliferate within tissues, as well as a pivotal role for Pes1 in the normal developmental cycle of C. albicans within the mammalian host during infection.

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Protection from Systemic Candida albicans Infection by Inactivation of the Sts Phosphatases

Infection and Immunity ◽

10.1128/iai.02789-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 637-645 ◽

Cited By ~ 19

Author(s):

Shamoon Naseem ◽

David Frank ◽

James B. Konopka ◽

Nick Carpino

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Fungal Growth ◽

Systemic Infection ◽

Content Type ◽

Host Immune Responses ◽

Human Fungal Pathogen ◽

Rapid Induction ◽

Inflammatory Damage ◽

Functional Inactivation

The human fungal pathogenCandida albicanscauses invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. Thesuppressor ofTCRsignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection byC. albicans, such that greater than 80% of mice lacking Sts-1 and -2 survive a dose ofC. albicans(2.5 × 105CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

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Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance

mSphere ◽

10.1128/msphere.00547-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 2

Author(s):

Csaba Papp ◽

Katica Kocsis ◽

Renáta Tóth ◽

László Bodai ◽

Jesse R. Willis ◽

...

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Candida Parapsilosis ◽

Hot Spot ◽

Abiotic Stress Tolerance ◽

Acquired Resistance ◽

Cross Resistance ◽

First Line ◽

Content Type

ABSTRACT Candida species are a major cause of life-threatening bloodstream infections worldwide. Although Candida albicans is responsible for the vast majority of infections, the clinical relevance of other Candida species has also emerged over the last twenty years. This shift might be due in part to changes in clinical guidelines, as echinocandins became the first line of therapeutics for the treatment. Candida parapsilosis is an emerging non-albicans Candida species that exhibits lower susceptibility levels to these drugs. Candida species frequently display resistance to echinocandins, and the mechanism for this is well-known in C. albicans and Candida glabrata, where it is mediated by amino acid substitutions at defined locations of the β-1,3-glucan synthase, Fks1p. In C. parapsilosis isolates, Fks1p harbors an intrinsic amino acid change at position 660 of the hot spot 1 (HS1) region, which is thought to be responsible for the high MIC values. Less is known about acquired substitutions in this species. In this study, we used directed evolution experiments to generate C. parapsilosis strains with acquired resistance to caspofungin, anidulafungin, and micafungin. We showed that cross-resistance was dependent on the type of echinocandin used to generate the evolved strains. During their characterization, all mutant strains showed attenuated virulence in vivo and also displayed alterations in the exposure of inner cell wall components. The evolved strains harbored 251 amino acid changes, including three in the HS1, HS2, and HS3 regions of Fks1p. Altogether, our results demonstrate a direct connection between acquired antifungal resistance and virulence of C. parapsilosis. IMPORTANCE Candida parapsilosis is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all Candida species. Resistance of Candida albicans to this drug type is well characterized. C. parapsilosis strains have the lowest in vitro susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in C. parapsilosis and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of C. parapsilosis and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.

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Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽

10.1128/msphere.00385-16 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Henry Ng ◽

Neta Dean

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Virulence Factors ◽

Fungal Pathogen ◽

Fungal Virulence ◽

Important Conclusion ◽

Content Type ◽

Guide Rna ◽

Improve Efficiency ◽

Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

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