Titration of C-5 Sterol Desaturase Activity Reveals Its Relationship to Candida albicans Virulence and Antifungal Susceptibility Is Dependent upon Host Immune Status.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), which depletes cellular ergosterol and promotes synthesis of the dysfunctional lipid 14α-methylergosta-8,24(28)-dien-3ß,6α-diol, ultimately arresting growth. Mutations that inactivate sterol Δ5,6-desaturase (Erg3p), the enzyme that produces the sterol-diol upon S14DM inhibition, enhances Candida albicans growth in the presence of the azoles. However, erg3 null mutants are sensitive to some physiological stresses and can be less virulent than the wild type. These fitness defects may disfavor the selection of null mutants within patients. The objective of this study was to investigate the relationship between Erg3p activity, C. albicans pathogenicity, and the efficacy of azole therapy. An isogenic panel of strains was constructed that produce various levels of the ERG3 transcript. Analysis of the sterol composition confirmed a correspondingly wide range of Erg3p activity. Phenotypic analysis revealed that even moderate reductions in Erg3p activity are sufficient to greatly enhance C. albicans growth in the presence of fluconazole in vitro without impacting fitness. Moreover, even low levels of Erg3p activity are sufficient to support full virulence of C. albicans in the mouse model of disseminated infection. Finally, while the antifungal efficacy of fluconazole was similar for all strains in immunocompetent mice, there was an inverse correlation between Erg3p activity and the capacity of C. albicans to endure treatment in leukopenic mice. Collectively, these results establish that relative levels of Erg3p activity determine the antifungal efficacy of the azoles upon C. albicans and reveal the critical importance of host immunity in determining the clinical impact of this resistance mechanism. IMPORTANCE Mutations that completely inactivate Erg3p enable the prevalent human pathogen C. albicans to endure the azole antifungals in vitro. However, such null mutants are less frequently identified in azole-resistant clinical isolates than other resistance mechanisms, and previous studies have reported conflicting outcomes regarding antifungal resistance of these mutants in animal models of infection. The results of this study clearly establish a direct correlation between the level of Erg3p activity and the antifungal efficacy of fluconazole within a susceptible mammalian host. In addition, low levels of Erg3p activity are apparently more advantageous for C. albicans survival of azole therapy than complete loss of function. These findings suggest a more nuanced but more important role for Erg3p as a determinant of the clinical efficacy of the azole antifungals than previously appreciated. A revised model of the relationship between Erg3p activity, host immunity, and the antifungal susceptibility of C. albicans is proposed.

EBP-Colombia and the bioeconomy: Genomics in the service of biodiversity conservation and sustainable development.

The 2016 Peace Agreement has increased access to Colombia's unique ecosystems, which remain understudied and increasingly under threat. The Colombian government has recently announced its National Bioeconomic Strategy (NBS), founded on the sustainable characterization, management, and conservation of the nation's biodiversity as a means to achieve sustainability and peace. Molecular tools will accelerate such endeavors, but capacity remains limited in Colombia. The Earth Biogenome Project's (EBP) objective is to characterize the genomes of all eukaryotic life on Earth through networks of partner institutions focused on sequencing either specific taxa or eukaryotic communities at regional or national scales. Colombia's immense biodiversity and emerging network of stakeholders have inspired the creation of the national partnership "EBP-Colombia." Here, we discuss how this Colombian-driven collaboration between government, academia, and the private sector is integrating research with sustainable, environmentally focused strategies to develop Colombia's postconflict bioeconomy and conserve biological and cultural diversity. EBP-Colombia will accelerate the uptake of technology and promote partnership and exchange of knowledge among Colombian stakeholders and the EBP's global network of experts; assist with conservation strategies to preserve Colombia's vast biological wealth; and promote innovative approaches among public and private institutions in sectors such as agriculture, tourism, recycling, and medicine. EBP-Colombia can thus support Colombia's NBS with the objective of sustainable and inclusive development to address the many social, environmental, and economic challenges, including conflict, inequality, poverty, and low agricultural productivity, and so, offer an alternative model for economic development that similarly placed countries can adopt.

Species-Specific Differences in C-5 Sterol Desaturase Function Influence the Outcome of Azole Antifungal Exposure.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol diol that disrupts membrane function. In Candida albicans, sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3. Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensities of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans (CaERG3), Candida glabrata (CgERG3), Candida auris (CaurERG3), Cryptococcus neoformans (CnERG3), Aspergillus fumigatus (AfERG3A-C) and Rhizopus delemar (RdERG3A/B) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored C-5 sterol desaturase activity and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant, confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the MIC and residual growth observed at supra-MICs. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced various levels of 14α-methylergosta-8,24(28)-dien-3ß,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C-5 sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans.

While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase (DHFR), the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1 strain), we were able to demonstrate that Dfr1p is essential for growth in vitro Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when Dfr1p expression is suppressed or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase and, by inference, other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.IMPORTANCE The folate biosynthetic pathway is a promising and understudied source for novel antifungals. Even dihydrofolate reductase (DHFR), a well-characterized and historically important drug target, has not been conclusively validated as an antifungal target. Here, we demonstrate that repression of DHFR inhibits growth of Candida albicans, a major human fungal pathogen. Methotrexate, an antifolate, also inhibits growth but through pH-dependent activity. In addition, we show that C. albicans has a limited ability to take up or utilize exogenous folates as only the addition of high concentrations of folinic acid restored growth in the presence of methotrexate. Finally, we show that repression of DHFR in a mouse model of infection was sufficient to eliminate host mortality. Our work conclusively establishes DHFR as a valid antifungal target in C. albicans.

The Vacuolar Ca2+ ATPase Pump Pmc1p Is Required for Candida albicans Pathogenesis.

Calcium is a critically important secondary messenger of intracellular signal transduction in eukaryotes but must be maintained at low levels in the cytoplasm of resting cells to avoid toxicity. This is achieved by several pumps that actively transport excess cytoplasmic Ca2+ out of the cell across the plasma membrane and into other intracellular compartments. In fungi, the vacuole serves as the major storage site for excess Ca2+, with two systems actively transporting cytoplasmic calcium ions into the vacuole. The H+/Ca2+ exchanger, Vcx1p, harnesses the proton-motive force across the vacuolar membrane (generated by the V-ATPase) to drive Ca2+ transport, while the P-type ATPase Pmc1p uses ATP hydrolysis to translocate Ca2+ into the vacuole. Ca2+-dependent signaling is required for the prevalent human fungal pathogen Candida albicans to endure exposure to the azole antifungals and to cause disease within the mammalian host. The purpose of this study was to determine if the Pmc1p or Vcx1p Ca2+ pumps are required for C. albicans pathogenicity and if these pumps impact antifungal resistance. Our results indicate that Pmc1p is required by C. albicans to transition from yeast to hyphal growth, to form biofilms in vitro, and to cause disease in a mouse model of disseminated infection. Moreover, loss of Pmc1p function appears to enhance C. albicans azole tolerance in a temperature-dependent manner.IMPORTANCE Maintenance of Ca2+ homeostasis is important for fungal cells to respond to a multitude of stresses, as well as antifungal treatment, and for virulence in animal models. Here, we demonstrate that a P-type ATPase, Pmc1p, is required for Candidaalbicans to respond to a variety of stresses, affects azole susceptibility, and is required to sustain tissue invasive hyphal growth and to cause disease in a mouse model of disseminated infection. Defining the mechanisms responsible for maintaining proper Ca2+ homeostasis in this important human pathogen can ultimately provide opportunities to devise new chemotherapeutic interventions that dysregulate intracellular signaling and induce Ca2+ toxicity.

Loss of C-5 Sterol Desaturase Activity in Candida albicans: Azole Resistance or Merely Trailing Growth?

Increased expression of drug efflux pumps and changes in the target enzyme Erg11p are known to contribute to azole resistance in Candida albicans, one of the most prevalent fungal pathogens. Mutations that inactivate ERG3, which encodes sterol Δ5,6-desaturase, also confer in vitro azole resistance. However, it is unclear whether the loss of Erg3p activity is sufficient to confer resistance within the mammalian host, and relatively few erg3 mutants have been reported among azole-resistant clinical isolates. Trailing growth (residual growth in the presence of the azoles) is a phenotype observed with many C. albicans isolates and, in its extreme form, can be mistaken for resistance. The purpose of this study was to determine whether the growth of Erg3p-deficient C. albicans mutants in the presence of the azoles possesses the characteristics of azole resistance or of an exaggerated form of trailing growth. Our results demonstrate that, similar to trailing isolates, the capacity of an erg3Δ/Δ mutant to endure the consequences of azole exposure is at least partly dependent on both temperature and pH. This contrasts with true azole resistance that results from enhanced drug efflux and/or changes in the target enzyme. The erg3Δ/Δ mutant and trailing isolates also appear to sustain significant membrane damage upon azole treatment, further distinguishing them from resistant isolates. However, the insensitivity of the erg3Δ/Δ mutant to azoles is unaffected by the calcineurin inhibitor cyclosporin A, distinguishing it from trailing isolates. In conclusion, the erg3 mutant phenotype is qualitatively and quantitatively distinct from both azole resistance and trailing growth.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity.

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicanserg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance.IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans.

Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCECandida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis.

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis.

We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans.

The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that a Candida albicans vps21Δ/Δ mutant, deficient in membrane trafficking through the late endosome/prevacuolar compartment (PVC), continues to grow in the presence of the azoles despite the depletion of cellular ergosterol. Here, we report that the vps21Δ/Δ mutant exhibits less plasma membrane damage upon azole treatment than the wild type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has abnormal levels of intracellular Ca2+ and, in the presence of fluconazole, enhanced expression of a calcineurin-responsive RTA2-GFP reporter. Furthermore, the azole tolerance phenotype of the vps21Δ/Δ mutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment and indicate that this may occur through modulation of calcium- and calcineurin-dependent responses.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.

Trafficking through the late endosome significantly impacts Candida albicans tolerance of the azole antifungals.

The azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of "toxic" sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, a C. albicans vps21Δ/Δ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21Δ/Δ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of "trailing growth" that has been described for some clinical isolates. In contrast, the vps21Δ/Δ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular "redistribution" of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.