A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.     

Structure-based rational design,synthesis and antifungal activity of oxime-containing azole derivatives

In an attempt to find novel azole antifungal agents with improved activity and broader spectrum, computer modeling was used to design a series of new azoles with piperidin-4-one O-substituted oxime side chains. Molecular docking studies revealed that they formed hydrophobic and hydrogen-bonding interactions with lanosterol 14α-demethylase of Candida albicans (CACYP51). In vitro antifungal assay indicates that most of the synthesized compounds showed good activity against tested fungal pathogens. In comparison with fluconazole, itraconazole and voriconazole, several compounds (such as 10c, 10e, and 10i) show more potent antifungal activity and broader spectrum, suggesting that they are promising leads for the development of novel antifungal agents.     

Synergistic Antifungal Activity of Berberine Derivative B-7b and Fluconazole

Our previous study demonstrated berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. We also suggested BBR played a major antifungal role in the synergism of FLC and BBR, while FLC increased intracellular BBR concentrations. Our following systematic structural modification and reconstruction of BBR core identified the novel scaffold of N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(substituted phenyl)acet-amide derivatives 7a-i, including B-7b and B-7d exhibiting remarkable synergistic antifungal activity and low cytotoxicity. Here, the study mainly investigated the synergistic activity of FLC and B-7b and the underlying mechanism. In vitro interaction of FLC and B-7b was investigated against 30 FLC-resistant clinical isolates of C. albicans and non-C. albicans species, including Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The potent synergistic activity of B-7b in combination with FLC against FLC-resistant C. albicans was found through the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed its better synergism with FLC. And as expected, B-7b exhibited much lower cytotoxicity than BBR to human umbilical vein endothelial cells. In contrast to BBR, we found that endogenous ROS augmentation was not involved in the synergism of FLC and B-7b. According to the results from our present comparative proteomic study, it seemed that the disruption of protein folding and processing and the weakening of cells’ self-defensive ability contributed to the synergism of FLC and B-7b. Together, these results suggested novel scaffold BBR derivative B-7b could be a promising synergist in combination with FLC for the treatment of invasive fungal infections.     

Synergistic Antifungal Effect of Glabridin and Fluconazole

The incidence of invasive fungal infections is increasing in recent years. The present study mainly investigated glabridin (Gla) alone and especially in combination with fluconazole (FLC) against Cryptococcus neoformans and Candida species (Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida Glabratas) by different methods. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) indicated that Gla possessed a broad-spectrum antifungal activity at relatively high concentrations. After combining with FLC, Gla exerted a potent synergistic effect against drug-resistant C. albicans and C. tropicalis at lower concentrations when interpreted by fractional inhibitory concentration index (FICI). Disk diffusion test and time-killing test confirming the synergistic fungicidal effect. Cell growth tests suggested that the synergistic effect of the two drugs depended more on the concentration of Gla. The cell envelop damage including a significant decrease of cell size and membrane permeability increasing were found after Gla treatment. Together, our results suggested that Gla possessed a synergistic effect with FLC and the cell envelope damage maybe contributed to the synergistic effect, which providing new information for developing novel antifungal agents.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a d-octapeptide derivative inhibitor

Overexpression of the Candida albicans ATP‐binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ～ 1.89 × 10⁶ member d ‐octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4‐methoxy‐2,3,6‐trimethylbenzenesulphonyl derivative of the d ‐octapeptide d ‐NH₂‐FFKWQRRR‐CONH₂, as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization‐resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug‐like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.     

Synthesis and biological activity investigation of azole and quinone hybridized phosphonates

Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were synthesized in 3–4 steps with good yield. Antifungal assay of these compounds showed that ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus Fusarium graminearum, and the free-base phosphates have good activity against human pathogenic fungi Aspergillus flavus and Candida albicans. Structure- activity relationship (SAR) studies showed activity increases with longer carbon chain length between phosphonate and anthraquinone analogs consisting of azole and quinone moieties. These newly synthesized compounds also have mild antibacterial activities to Gram positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity analysis of these compounds against HeLa cells reveals that the phosphoric acid analogs are less toxic compared to ethyl protected phosphonates. Three leads compounds have been identified with prominent antifungal activity and low cytotoxicity.     

Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.     

Diversity-oriented synthesis of pyrazoles derivatives from flavones and isoflavones leads to the discovery of promising reversal agents of fluconazole resistance in Candida albicans

Diversity-oriented synthesis of derivatives of natural products is an important approach for the discovery of novel drugs. In this paper, a series of novel 3,4-diaryl-1H-pyrazoles and 3,5-diaryl-1H-pyrazoles derivatives were synthesized through the one-pot reaction of flavones and isoflavones with the hydrazine hydrate and substituted hydrazine hydrate. Some of these novel compounds exhibited antifungal effects against Candida albicans SC5314, and displayed more potent inhibitory activities against the efflux-pump-deficient strain DSY654. In addition, compounds 25, 28 and 32a displayed outstanding reversal activity of azole resistance against clinical azole-resistant Candida albicans in combination with fluconazole (FLC), with FICI values ranging from 0.012 to 0.141. The preliminary structure-activity relationship (SAR) of these compounds was also discussed. In conclusion, this study provides several novel agents that displayed potent antifungal activities alone or together with fluconazole, which makes progress for development of antifungal drugs.     

Calcineurin Controls Hyphal Growth,Virulence, and Drug Tolerance of Candida tropicalis

Candida tropicalis, a species closely related to Candida albicans, is an emerging fungal pathogen associated with high mortality rates of 40 to 70%. Like C. albicans and Candida dubliniensis, C. tropicalis is able to form germ tubes, pseudohyphae, and hyphae, but the genes involved in hyphal growth machinery and virulence remain unclear in C. tropicalis. Recently, echinocandin- and azole-resistant C. tropicalis isolates have frequently been isolated from various patients around the world, making treatment difficult. However, studies of the C. tropicalis genes involved in drug tolerance are limited. Here, we investigated the roles of calcineurin and its potential target, Crz1, for core stress responses and pathogenesis in C. tropicalis. We demonstrate that calcineurin and Crz1 are required for hyphal growth, micafungin tolerance, and virulence in a murine systemic infection model, while calcineurin but not Crz1 is essential for tolerance of azoles, caspofungin, anidulafungin, and cell wall-perturbing agents, suggesting that calcineurin has both Crz1-dependent and -independent functions in C. tropicalis. In addition, we found that calcineurin and Crz1 have opposite roles in controlling calcium tolerance. Calcineurin serves as a negative regulator, while Crz1 plays a positive role for calcium tolerance in C. tropicalis.     

Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin

Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90''s role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90''s role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.     

Plagiochin E, a botanic-derived phenolic compound, reverses fungal resistance to fluconazole relating to the efflux pump

Aim: In this study, we investigated the effect of plagiochin E (PLE), a botanic‐derived phenolic natural product, on reversal of fungal resistance to fluconazole (FLC) in vitro and the related mechanism. Methods and Results: A synergistic action of PLE and FLC was observed in the FLC‐resistant Candida albicans strains and was evaluated using the fractional inhibited concentration index. The effect of PLE on FLC intracellular uptake was investigated in FLC‐resistant C. albicans cells by liquid chromatography–tandem mass spectrometry, and the effect on efflux drug pump was assessed by measuring the efflux of Rhodamine 123 (Rh123). PLE significantly inhibited the efflux, but not the absorption, of Rh123 in FLC‐resistant strains in phosphate‐buffered saline with 5% glucose. Overexpression of the multidrug‐resistance gene CDR1 in FLC‐resistant C. albicans isolates was detected, and the introduction of PLE to the cells showed a significant reduction of the CDR1 expression in those FLC‐resistant isolates. Conclusions: These findings indicate that PLE could reverse the fungal resistant to FLC by inhibiting the efflux of FLC from C. albicans, and this effect may be related to the efflux pump. Significance and Impact of the Study: These results indicate that the combination of PLE and FLC may provide an approach for the clinical therapy of fungus infection induced by FLC‐resistant strains.     

PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin,and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.     

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90‐dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.     

The synergy of honokiol and fluconazole against clinical isolates of azole‐resistant Candida albicans

Aims: To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole‐resistant (azole‐R) clinical isolates of Candida albicans. Methods and Results: A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole‐R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC‐resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time‐killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Conclusions: Synergistic activity of HNK and FLC against clinical isolates of FLC‐resistant C. albicans was observed in vitro and in vivo. Significance and Impact of the Study: This report might provide a potential therapeutic method to overcome the problem of drug‐resistance in C. albicans.     

Phosphatidylserine decarboxylase governs plasma membrane fluidity and impacts drug susceptibilities of Candida albicans cells

Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C. albicans cells. Genetic manipulation of PSD1 and PSD2 genes is known to impact virulence, cell wall thickness and mitochondrial function in C. albicans. In the present study, we have examined the impact of PSD1 and PSD2 deletion on physiochemical properties of PM. Our fluorescence recovery after photobleaching (FRAP) experiments point that the PM of psd1Δ/Δ psd2Δ/Δ mutant strain displays increased membrane fluidity and reduced PM dipole potential. Further, the result of PSD1 and PSD2 deletion on the thermotropic phase behavior monitored by differential scanning calorimetry (DSC) showed that in comparison to WT, the apparent phase transition temperature is reduced by ~3 °C in the mutant strain. The functional consequence of altered physical state of PM of psd1Δ/Δ psd2Δ/Δ mutant strain was evident from observed high diffusion of fluorescent dye rhodamine 6G and radiolabelled fluconazole (FLC). The higher diffusion of FLC resulted in an increased drug accumulation in psd1Δ/Δ psd2Δ/Δ mutant cells, which was manifested in an increased susceptibility to azoles. To the best of our knowledge, these results constitute the first report on the effect of the levels of phospholipid biosynthesis enzyme on physiochemical properties of membranes and drug susceptibilities of Candida cells.     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.