RESUMEN
ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in Candida albicans, orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed CDR6/ROA1 for consistency with C. albicans nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a CDR6/ROA1 null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This in vitro finding was supported by in vivo data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in C. albicans, involving increased membrane rigidity and TOR signaling.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Proteínas Fúngicas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Candida albicans/metabolismo , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fúngicas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.
Asunto(s)
Evolución Molecular , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Esteroles/metabolismo , Dedos de Zinc/genética , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Candida albicans/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Yarrowia/genéticaRESUMEN
Candida albicans is a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginal C. albicans isolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginal C. albicans isolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginal C. albicans isolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine ß-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginal C. albicans isolates.
Asunto(s)
Azoles/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/fisiología , Vagina/microbiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antifúngicos/farmacología , Candida albicans/aislamiento & purificación , Candida albicans/fisiología , Candidiasis Vulvovaginal/microbiología , Farmacorresistencia Fúngica/efectos de los fármacos , Femenino , Fluorescencia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana/métodos , Técnicas Microbiológicas/métodosRESUMEN
Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation.
Asunto(s)
Arthrodermataceae/inmunología , Dermatomicosis/inmunología , Queratinocitos/microbiología , Sistema de Señalización de MAP Quinasas/inmunología , Trichophyton/inmunología , Arthrodermataceae/patogenicidad , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunidad Innata , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Trichophyton/patogenicidad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The fungal pathogen Aspergillus fumigatus causes serious illness and often death when it invades tissues, especially in immunocompromised individuals. The azole class of drugs is the most commonly prescribed treatment for many fungal infections and acts on the ergosterol biosynthesis pathway. One common mechanism of acquired azole drug resistance in fungi is the prevention of drug accumulation to toxic levels in the cell. While drug efflux is a well-known resistance strategy, reduced azole import would be another strategy to maintain low intracellular azole levels. Recently, azole uptake in Candida albicans and other yeasts was analyzed using [(3)H]fluconazole. Defective drug import was suggested to be a potential mechanism of drug resistance in several pathogenic fungi, including Cryptococcus neoformans, Candida krusei, and Saccharomyces cerevisiae. We have adapted and developed an assay to measure azole accumulation in A. fumigatus using radioactively labeled azole drugs, based on previous work done with C. albicans. We used this assay to study the differences in azole uptake in A. fumigatus isolates under a variety of drug treatment conditions, with different morphologies and with a select mutant strain with deficiencies in the sterol uptake and biosynthesis pathway. We conclude that azole drugs are specifically selected and imported into the fungal cell by a pH- and ATP-independent facilitated diffusion mechanism, not by passive diffusion. This method of drug transport is likely to be conserved across most fungal species.
Asunto(s)
Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Azoles/farmacocinética , Adenosina Trifosfato/metabolismo , Antifúngicos/farmacocinética , Antifúngicos/farmacología , Azoles/farmacología , Candida/efectos de los fármacos , Candida/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/metabolismo , Fluconazol/farmacocinética , Fluconazol/farmacología , Concentración de Iones de Hidrógeno , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMEN
Sterol import has been characterized under various conditions in three distinct fungal species, the model organism Saccharomyces cerevisiae and two human fungal pathogens Candida glabrata and Candida albicans, employing cholesterol, the sterol of higher eukaryotes, as well as its fungal equivalent, ergosterol. Import was confirmed by the detection of esterified cholesterol within the cells. Comparing the three fungal species, we observe sterol import under three different conditions. First, as previously well characterized, we observe sterol import under low oxygen levels in S. cerevisiae and C. glabrata, which is dependent on the transcription factor Upc2 and/or its orthologs or paralogs. Second, we observe sterol import under aerobic conditions exclusively in the two pathogenic fungi C. glabrata and C. albicans. Uptake emerges during post-exponential-growth phases, is independent of the characterized Upc2-pathway and is slower compared to the anaerobic uptake in S. cerevisiae and C. glabrata. Third, we observe under normoxic conditions in C. glabrata that Upc2-dependent sterol import can be induced in the presence of fetal bovine serum together with fluconazole. In summary, C. glabrata imports sterols both in aerobic and anaerobic conditions, and the limited aerobic uptake can be further stimulated by the presence of serum together with fluconazole. S. cerevisiae imports sterols only in anaerobic conditions, demonstrating aerobic sterol exclusion. Finally, C. albicans imports sterols exclusively aerobically in post-exponential-growth phases, independent of Upc2. For the first time, we provide direct evidence of sterol import into the human fungal pathogen C. albicans, which until now was believed to be incapable of active sterol import.
Asunto(s)
Candida albicans/metabolismo , Candida glabrata/metabolismo , Colesterol/metabolismo , Ergosterol/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiosis , Anaerobiosis , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Candida glabrata/efectos de los fármacos , Candida glabrata/genética , Candida glabrata/crecimiento & desarrollo , Medios de Cultivo , Esterificación , Fluconazol/farmacología , Técnicas de Inactivación de Genes , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
BACKGROUND: The antifungal posaconazole concentrates within host cells and protects against Aspergillus fumigatus. The specific subcellular location of posaconazole and the mechanism by which cell-associated posaconazole inhibits fungal growth remain uncharacterized. METHODS: Posaconazole was conjugated with the fluorophore boron-dipyrromethene (BDP-PCZ). A549 pulmonary epithelial cells and A. fumigatus were exposed to BDP-PCZ individually and in coculture. BDP-PCZ subcellular localization and trafficking were observed using confocal microscopy and flow cytometry. RESULTS: BDP-PCZ concentrated within A549 cell membranes, and in particular within the endoplasmic reticulum. Epithelial cell-associated BDP-PCZ rapidly transferred to and concentrated within A. fumigatus cell membranes on contact. BDP-PCZ transfer to conidia did not require phagocytosis and was markedly enhanced by the conidial hydrophobin RodA. Within AF, BDP-PCZ also concentrated in membranes including the endoplasmic reticulum and colocalized with the azole target enzyme CYP51a. Concentration of BDP-PCZ within host and fungal cell membranes persisted for >48 hours and could be competitively inhibited by posaconazole but not voriconazole. CONCLUSIONS: Posaconazole concentrates within host cell membranes and rapidly transfers to A. fumigatus, where it accumulates to high concentrations and persists at the site of its target enzyme. These intracellular and intercellular pharmacokinetic properties probably contribute to the efficacy of PCZ.
Asunto(s)
Antifúngicos/metabolismo , Células Epiteliales/metabolismo , Hongos/metabolismo , Triazoles/metabolismo , Profilaxis Antibiótica , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Epiteliales/efectos de los fármacos , Hongos/efectos de los fármacos , Humanos , Micosis/tratamiento farmacológico , Micosis/prevención & control , Unión Proteica , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/metabolismo , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Candida auris is an emerging human fungal pathogen, first described in Japan in 2009, and first detected in the United States in 2016. Here, we report the first-ever description of C. auris colonizing a human pet, the first identification of C. auris in a non-human mammal in the United States and the first C. auris isolate from the state of Kansas. While analyzing the oral mycobiome of dogs from a shelter in Kansas, the oral swab from one dog was found to contain C. auris as well as three other fungal species. The presence of C. auris in a dog suggests the possibility of zoonotic transmission to humans. The isolate is a member of Clade IV, which has been found in patients in Chicago and Florida, while Clades I and III are the most prevalent in the United States. The isolate is resistant to fluconazole, terbinafine, and amphotericin B but susceptible to caspofungin, consistent with the drug-resistant characteristics of many human C. auris isolates. The source of C. auris transient colonization in this dog is unknown, and there is no evidence that it was further transmitted to humans, other dogs in the shelter, or pets in its adopted household. Isolation of C. auris from a dog in Kansas has public health implications as a potential emerging source for the zoonotic spread of this pathogenic fungus, and for the development of antifungal resistance.IMPORTANCECandida auris is an emerging fungal infection of humans and is particularly problematic because it is multi-drug resistant and difficult to treat. It is also known to be spread from person to person by contact and can remain on surfaces for long periods of time. In this report, a dog in a shelter in Kansas is found to be colonized with Candida auris. This is the first study to document the presence of Candida auris on a pet, the first to document C. auris presence on a non-human mammal in the United States, and the first to report an isolate of C. auris within the state of Kansas. The presence of C. auris in a pet dog raises the possibility of zoonotic transmission from pets to human or vice versa.
Asunto(s)
Antifúngicos , Candidiasis , Perros , Humanos , Animales , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida , Candidiasis/microbiología , Candida auris , Kansas , Boca , Mamíferos , Pruebas de Sensibilidad MicrobianaRESUMEN
Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild-type (wt) strain when yeast cells were challenged by sub-inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Ergosterol/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Azoles/metabolismo , Transporte Biológico , Farmacorresistencia Fúngica , Prueba de Complementación Genética , Fenotipo , Proteínas de Transferencia de Fosfolípidos/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1Δ and tco2Δ) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1- and Swi4-like protein 1). Expression analysis revealed that MBS1 was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of MBS1 exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis.
Asunto(s)
Antifúngicos/farmacología , Criptococosis/microbiología , Cryptococcus neoformans/patogenicidad , Farmacorresistencia Fúngica , Flucitosina/farmacología , Proteínas Fúngicas/metabolismo , Estrés Oxidativo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Recuento de Colonia Microbiana , Secuencia Conservada , Criptococosis/inmunología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/fisiología , Daño del ADN , Ergosterol/biosíntesis , Femenino , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Pleiotropía Genética , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Proteínas Quinasas/metabolismo , Factores de Transcripción/genética , Virulencia , Factores de Virulencia/biosíntesisRESUMEN
One of the most prevalent mechanisms of antifungal drug resistance is export of the molecule from the fungal cells through the action of putative efflux pumps or transporters. Drug efflux is a particularly common mechanism of resistance to azole antifungals, one of the most widely used classes of antifungal drugs. Here, we provide detailed protocols for two assays of small-molecule efflux activity: rhodamine 6G efflux and alanine-naphthylamide accumulation. Protocols applicable to both yeast and filamentous fungi are provided.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Fúngicas , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Antifúngicos/farmacología , Transporte Biológico , Saccharomyces cerevisiae/metabolismo , Farmacorresistencia Fúngica , Candida albicans/metabolismoRESUMEN
The ability of many antifungal molecules to traverse the fungal cell wall and accumulate within the cell is crucial to its ability to have the desired biological activity. Altered accumulation of the drug is an important mechanism of antifungal drug resistance. The best characterized mechanism for altered accumulation is through the action of the drug efflux pump which actively transports the drugs out of the membrane, although this is not the only mechanism for this phenomenon. Here, we describe protocols for the measurement of uptake of tritiated fluconazole in both yeast and filamentous fungi.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Transporte de Membrana , Proteínas de Transporte de Membrana/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antifúngicos/farmacología , Antifúngicos/metabolismo , Fluconazol/farmacología , Fluconazol/metabolismo , Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Candida albicans/metabolismoRESUMEN
The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.
RESUMEN
Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a K(m) of 0.64 µM and V(max) of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.
Asunto(s)
Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Criptococosis/tratamiento farmacológico , Cryptococcus neoformans/efectos de los fármacos , Fluconazol/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Antifúngicos/farmacología , Candida albicans/aislamiento & purificación , Candida albicans/patogenicidad , Candidiasis/genética , Candidiasis/microbiología , Criptococosis/genética , Criptococosis/microbiología , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Difusión , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Eliminación de Gen , Humanos , Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/patogenicidadRESUMEN
In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although CgYOR1 has previously been reported to be upregulated in various azole-resistant clinical isolates of C. glabrata, deletion of this gene did not change the susceptibility to any of the tested azoles. Additionally, Cgyor1Δ showed no change in susceptibility toward oligomycin, which is otherwise a well-known substrate of Yor1 in other yeasts. The role of CgYor1 in azole susceptibility only became evident when the major transporter CgCDR1 gene was deleted. However, under nitrogen-depleted conditions, Cgyor1Δ demonstrated an azole-susceptible phenotype, independent of CgCdr1. Notably, Cgyor1Δ cells also showed increased susceptibility to target of rapamycin (TOR) and calcineurin inhibitors. Moreover, increased phytoceramide levels in Cgyor1Δ and the deletions of regulators downstream of TOR and the calcineurin signaling cascade (Cgypk1Δ, Cgypk2Δ, Cgckb1Δ, and Cgckb2Δ) in the Cgyor1Δ background and their associated fluconazole (FLC) susceptibility phenotypes confirmed their involvement. Collectively, our findings show that TOR and calcineurin signaling govern CgYor1-mediated azole susceptibility in C. glabrata. IMPORTANCE The increasing incidence of Candida glabrata infections in the last 40 years is a serious concern worldwide. These infections are usually associated with intrinsic azole resistance and increasing echinocandin resistance. Efflux pumps, especially ABC transporter upregulation, are one of the prominent mechanisms of azole resistance; however, only a few of them are characterized. In this study, we analyzed the mechanisms of azole resistance due to a multidrug resistance-associated protein (MRP) subfamily ABC transporter, CgYor1. We demonstrate for the first time that CgYor1 does not transport oligomycin but is involved in azole resistance. Under normal growing conditions its function is masked by major transporter CgCdr1; however, under nitrogen-depleted conditions, it displays its azole resistance function independently. Moreover, we propose that the azole susceptibility due to removal of CgYor1 is not due to its transport function but involves modulation of TOR and calcineurin cascades.
Asunto(s)
Azoles , Candidiasis , Antifúngicos/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Azoles/farmacología , Calcineurina/metabolismo , Candida glabrata/genética , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Nitrógeno/metabolismo , Oligomicinas/farmacología , Sirolimus/farmacología , Proteínas Fúngicas/metabolismoRESUMEN
In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in Candida glabrata by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with S. cerevisiae counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)2C), which accounts for the majority of complex SL structures in S. cerevisiae membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in ΔCgipt1 and ΔCgskn1 cells, albeit to lesser extent in the latter. Noticeably, ΔCgipt1 cells showed an increased susceptibility to azoles; however, ΔCgskn1 cells showed no significant changes in the drug susceptibility profiles. Interestingly, the azole susceptible phenotype of ΔCgipt1 cells seems to be independent of the ergosterol content. ΔCgipt1 cells displayed altered lipid homeostasis, increased membrane fluidity as well as high diffusion of radiolabeled fluconazole (3H-FLC), which could together influence the azole susceptibility of C. glabrata. Furthermore, in vivo experiments also confirmed compromised virulence of the ΔCgipt1 strain. Contrarily, specific functions of CgSkn1 remain unclear.
RESUMEN
The Candida albicans Upc2p transcription factor regulates ERG11, encoding the target of azole drugs. Gain-of-function mutations that contribute to resistance were recently identified in a series of sequential clinical isolates (N. Dunkel, T. T. Liu, K. S. Barker, R. Homayouni, J. Morschhauser, and P. D. Rogers, Eukaryot. Cell 7:1180-1190, 2008). In the present study, UPC2 was sequenced from a matched set of 17 isolates. An A643V substitution was present in all of the isolates in the series that overexpressed ERG11. Azole susceptibility, ergosterol levels, and expression of ERG genes were elevated in the A643V clinical isolates and in reconstructed strains.
Asunto(s)
Sustitución de Aminoácidos , Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Regulación Fúngica de la Expresión Génica , Transactivadores/genética , Candida albicans/genética , Candida albicans/metabolismo , Ergosterol/biosíntesis , Ergosterol/genética , Fluconazol/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de ADN , Transactivadores/metabolismoRESUMEN
Although dermatophytes are the most common cause of fungal infections in the world, their basic biology is not well understood. The recent sequencing and annotation of the genomes of five representative dermatophyte species allows for the creation of hypotheses as to how they cause disease and have adapted to their distinct environments. An understanding of the microbiology of these strains will be essential for testing these hypotheses. This study is the first to generally characterize these five sequenced strains of dermatophytes for their microbiological aspects. We measured the growth rate on solid medium and found differences between species, with Microsporum gypseum CBS118893 having the fastest growth and Trichophyton rubrum CBS118892 the slowest. We also compared different media for conidia production and found that the highest numbers of conidia were produced when dermatophytes were grown on MAT agar. We determined the Minimum Inhibitory Concentration (MIC) of nine antifungal agents and confirmed susceptibility to antifungals commonly used as selectable markers. Finally, we tested virulence in the Galleria mellonella (wax moth) larvae model but found the results variable. These results increase our understanding of the microbiology and molecular biology of these dermatophyte strains and will be of use in advancing hypothesis-driven research about dermatophytes.
Asunto(s)
Arthrodermataceae/crecimiento & desarrollo , Arthrodermataceae/patogenicidad , Esporas Fúngicas/crecimiento & desarrollo , Animales , Antifúngicos/farmacología , Arthrodermataceae/efectos de los fármacos , Arthrodermataceae/genética , Medios de Cultivo/química , Larva/microbiología , Lepidópteros/microbiología , Pruebas de Sensibilidad Microbiana , Análisis de Supervivencia , VirulenciaRESUMEN
RNA interference/silencing mechanisms triggered by double-stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well-conserved Argonaute and a non-canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down-regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans.
Asunto(s)
Candida albicans/genética , Interferencia de ARN , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Candida albicans/metabolismo , Regulación hacia Abajo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , MicroARNs/genética , ARN Bicatenario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransgenesRESUMEN
BACKGROUND: The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. RESULTS: The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. CONCLUSIONS: Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.