RESUMEN
Staphylococcus aureus is a major human pathogen associated with high mortality rates. The extensive use of antibiotics is associated with the rise of drug resistance, and exotoxins are not targeted by antibiotics. Therefore, monoclonal antibody (mAb) therapy has emerged as a promising solution to solve the clinical problems caused by refractory S aureus. Recent research suggests that the synergistic effects of several cytotoxins, including bicomponent toxins, are critical to the pathogenesis of S aureus. By comparing the amino acid sequences, researchers found that α-toxin and bicomponent toxins have high homology. Therefore, we aimed to screen an antibody, designated an all-in-one mAb, that could neutralize α-toxin and bicomponent toxins through hybridoma fusion. We found that this mAb has a significant pharmacodynamic effect within in vivo mouse models and in vitro experiments.
Asunto(s)
Toxinas Bacterianas , Infecciones Estafilocócicas , Humanos , Animales , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Staphylococcus aureus , Infecciones Estafilocócicas/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
C-type lectin receptors (CLRs) play critical roles as pattern-recognition receptors (PRRs) for sensing Candida albicans infection, which can be life-threatening for immunocompromised individuals. Here we have shown that Dectin-3 (also called CLECSF8, MCL, or Clec4d), a previously uncharacterized CLR, recognized α-mannans on the surfaces of C. albicans hyphae and induced NF-κB activation. Mice with either blockade or genetically deleted Dectin-3 were highly susceptible to C. albicans infection. Dectin-3 constantly formed heterodimers with Dectin-2, a well-characterized CLR, for recognizing C. albicans hyphae. Compared to their respective homodimers, Dectin-3 and Dectin-2 heterodimers bound α-mannans more effectively, leading to potent inflammatory responses against fungal infections. Together, our study demonstrates that Dectin-3 forms a heterodimeric PRR with Dectin-2 for sensing fungal infection and suggests that different CLRs may form different hetero- and homodimers, which provide different sensitivity and diversity for host cells to detect various microbial infections.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Activación Enzimática , Femenino , Humanos , Hifa/inmunología , Hifa/metabolismo , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Mananos/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Transducción de SeñalRESUMEN
Baicalein could inhibit the growth and biofilm formation of Candida albicans, the most common clinical fungal pathogen. However, the antifungal mechanism of baicalein has not been elucidated. In this study, isobaric tags for relative and absolute quantification (iTRAQ) was used to verify the mechanism of antifungal fluconazole and baicalein. A total of 58 common proteins were detected in cells treated with fluconazole. These proteins encompassed fluconazole-targeted sterol synthesis pathway, including Erg11p, Erg6p, Erg3p, Erg25p, Erg5p, Erg10p, and Ncp1p. Next, iTRAQ was applied to the comparison of baicalein-treated C. albicans proteins, which detected 16 common proteins. The putative NADH dehydrogenase Cpd2p and the ATP-binding cassette transporter Snq2p were the most upregulated proteins with the treatment of baicalein. Our results showed that CPD2 disruption elevated C. albicans resistance to baicalein significantly both in vitro and in vivo. Further in-depth studies revealed that CPD2 disruption reduced the activation of C. albicans metacaspase and partially restored the mitochondrial membrane potential reduction caused by the treatment of baicalein, which indicated that CPD2 was involved in the apoptosis induced by baicalein. Consistently, under the treatment of baicalein, CPD2Δ/Δ mutant produced lower reactive oxygen species that was critical in causing oxidative damage and apoptosis in C. albicans. These results indicated that baicalein could increase intracellular oxidative damage by upregulating the expression of Cpd2p so as to inhibit the growth of C. albicans, which provides new insights for investigating the antifungal target of baicalein.
In our study, isobaric tags for relative and absolute quantification (iTRAQ) was used to study the antifungal mechanisms of fluconazole and baicalein. Baicalein could enhance the oxidative stress of Candida albicans by upregulating CPD2 expression.
Asunto(s)
Candida albicans , Fluconazol , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Farmacorresistencia Fúngica , Flavanonas , Fluconazol/farmacología , Pruebas de Sensibilidad Microbiana/veterinaria , Estrés Oxidativo , ProteómicaRESUMEN
In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 µg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 µg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.
Asunto(s)
Antifúngicos/farmacología , Criptococosis/tratamiento farmacológico , Cryptococcus neoformans/efectos de los fármacos , Triazoles/farmacología , Animales , Criptococosis/microbiología , Femenino , Humanos , Ratones , Ratones Endogámicos ICRRESUMEN
There is currently a small number of classes of antifungal drugs, and these drugs are known to target a very limited set of cellular functions. We derived a set of approximately 900 nonessential, transactivator-defective disruption strains from the tetracycline-regulated GRACE collection of strains of the fungal pathogen Candida albicans This strain set was screened against classic antifungal drugs to identify gene inactivations that conferred either enhanced sensitivity or increased resistance to the compounds. We examined two azoles, fluconazole and posaconazole; two echinocandins, caspofungin and anidulafungin; and a polyene, amphotericin B. Overall, the chemogenomic profiles within drug classes were highly similar, but there was little overlap between classes, suggesting that the different drug classes interacted with discrete networks of genes in C. albicans We also tested two pyridine amides, designated GPI-LY7 and GPI-C107; these drugs gave very similar profiles that were distinct from those of the echinocandins, azoles, or polyenes, supporting the idea that they target a distinct cellular function. Intriguingly, in cases where these gene sets can be compared to genetic disruptions conferring drug sensitivity in other fungi, we find very little correspondence in genes. Thus, even though the drug targets are the same in the different species, the specific genetic profiles that can lead to drug sensitivity are distinct. This implies that chemogenomic screens of one organism may be poorly predictive of the profiles found in other organisms and that drug sensitivity and resistance profiles can differ significantly among organisms even when the apparent target of the drug is the same.
RESUMEN
The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface ß-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1ß and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding ß-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.
Asunto(s)
Candida albicans/patogenicidad , Candidiasis/inmunología , Evasión Inmune/inmunología , Lectinas Tipo C/inmunología , Manosiltransferasas/metabolismo , Virulencia/inmunología , Animales , Western Blotting , Candida albicans/inmunología , Línea Celular , Pared Celular/química , Pared Celular/inmunología , Pared Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/metabolismo , Humanos , Mananos/inmunología , Mananos/metabolismo , Manosa/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Cumarinas/farmacología , Fluconazol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Candida albicans/genética , Candidiasis/microbiología , Farmacorresistencia Fúngica/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo/efectos de los fármacosRESUMEN
Candida albicans biofilms show resistance to many clinical antifungal agents and play a considerable contributing role in the process of C. albicans infections. New antifungal agents against C. albicans biofilms are sorely needed. The aim of this study was to evaluate sanguinarine (SAN) for its activity against Candida albicans biofilms and explore the underlying mechanism. The MIC50 of SAN was 3.2 µg/ml, while ≥0.8 µg/ml of SAN could suppress C. albicans biofilms. Further study revealed that ≥0.8 µg/ml of SAN could decrease cellular surface hydrophobicity (CSH) and inhibited hypha formation. Real-time reverse transcription-PCR (RT-PCR) results indicated that the exposure of C. albicans to SAN suppressed the expression of some adhesion- and hypha-specific/essential genes related to the cyclic AMP (cAMP) pathway, including ALS3, HWP1, ECE1, HGC1, and CYR1 Consistently, the endogenous cAMP level of C. albicans was downregulated after SAN treatment, and the addition of cAMP rescued the SAN-induced filamentation defect. In addition, SAN showed relatively low toxicity to human umbilical vein endothelial cells, the 50% inhibitory concentration (IC50) being 7.8 µg/ml. Collectively, the results show that SAN exhibits strong activity against C. albicans biofilms, and the activity was associated with its inhibitory effect on adhesion and hypha formation due to cAMP pathway suppression.
Asunto(s)
Antifúngicos/farmacología , Benzofenantridinas/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Hifa/efectos de los fármacos , Isoquinolinas/farmacología , Células Cultivadas , AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Hifa/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Candida albicans biofilms contribute to invasive infections and dramatic drug resistance, and anti-biofilm agents are urgently needed in the clinic. Pterostilbene (PTE) is a natural plant product with potentials to be developed as an anti-biofilm agent. In this study, we evaluated the structure-activity relationship (SAR) of PTE analogues against C. albicans biofilms. XTT (Sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) reduction assay was used to evaluate the activity of the analogues against C. albicans biofilms. Knowing that hyphal formation is essential for C. albicans biofilms, anti-hyphal assay was further carried out. By comparing a series of compounds tested in this study, we found that compounds with para-hydroxy (-OH) in partition A exhibited better activity than those with other substituents in the para position, and the double bond in partition B and meta-dimethoxy (-OCH3) in partition C both contributed to the best activity. Consistent results were obtained by anti-hyphal assay. Collectively, para-hydroxy (-OH), double bond and meta-dimethoxy (-OCH3) are all needed for the best activity of PTE against C. albicans biofilms.
Asunto(s)
Antifúngicos/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/fisiología , Estilbenos/farmacología , Antifúngicos/química , Candida albicans/efectos de los fármacos , Hifa/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Estilbenos/química , Relación Estructura-ActividadRESUMEN
To identify effective and low toxicity synergistic antifungal compounds, 24 derivatives of chalcone were synthesized to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. The minimal inhibitory concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant Candida albicans. This was done via methods established by the clinical and laboratory standards institute (CLSI). Of the synthesized compounds, 2'-hydroxy-4'-methoxychalcone (8) exhibited the most potent in vitro (FICI=0.007) effects. The structure activity relationship of the compounds are then discussed.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Chalconas/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos , Antifúngicos/síntesis química , Antifúngicos/química , Chalconas/síntesis química , Chalconas/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This study aimed to investigate the antifungal activity of Rubus chingii extract in combination with fluconazole (FLC) against FLC-resistant Candida albicans 100 in vitro. A R. chingii extract and FLC-resistant C. albicans fungus suspension were prepared. The minimum inhibitory concentration and fractional inhibitory concentration index of R. chingii extract combined with FLC against C. albicans were determined, after which growth curves for C. albicans treated with R. chingii extract, FLC alone and a combination of these preparations were constructed. Additionally, the mechanisms of drug combination against C. albicans were explored by flow cytometry, gas chromatographic mass spectrometry and drug efflux pump function detection. R. chingii extract combined with FLC showed significant synergy. Flow cytometry suggested that C. albicans cells mainly arrest in G1 and S phases when they have been treated with the drug combination. The drug combination resulted in a marked decrease in the ergosterol content of the cell membrane. Additionally, efflux of Rhodamine 6G decreased with increasing concentrations of R. chingii extract. R. chingii extract combined with FLC has antifungal activity against FLC-resistant C. albicans.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Fluconazol/farmacología , Extractos Vegetales/farmacología , Rubus/química , Apoptosis/efectos de los fármacos , Candida albicans/citología , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Ciclo Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Farmacorresistencia Fúngica , Sinergismo Farmacológico , Ergosterol/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Rodaminas/metabolismoRESUMEN
Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield ß-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed ß-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition.
Asunto(s)
Antígenos Fúngicos/inmunología , Candida albicans/inmunología , Pared Celular/inmunología , Proteínas Fúngicas/inmunología , Glicosilfosfatidilinositoles/metabolismo , Lectinas Tipo C/metabolismo , Animales , Antígenos Fúngicos/metabolismo , Candida albicans/metabolismo , Pared Celular/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Macrófagos/inmunología , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , beta-Glucanos/inmunología , beta-Glucanos/metabolismoRESUMEN
Although ribosomal proteins (RPs) are components of the ribosome, and function centrally in protein synthesis, several lines of evidence suggest that S4 ribosomal proteins (Rps4ps) can function in other cellular roles. In Candida albicans, ribosomal protein S4 (Rps4p) is encoded by two distinct but highly similar genes, RPS41 (C2_10620W_A) and RPS42 (C1_01640W_A). Previous studies indicated that in Saccharomyces cerevisiae loss of one isoform generated distinct phenotypes. To probe this relationship in C. albicans, rps41Δ and rps42Δ homozygous null mutants were generated. The transcript levels of the RPS41 and RPS42 genes are asymmetric in C. albicans, RPS41 mRNA levels were similar in wild-type strains and rps42Δ null mutants, while RPS42 gene transcript levels were induced 20 fold relative to wild type in rps41Δ null mutants. We found that the rps41Δ homozygous null mutant showed a reduced growth rate, and had defects in filament formation in liquid media and on solid media, while these phenotypes were not observed in the rps42Δ mutant strain. Neither the rps41Δ nor rps42Δ mutant strains displayed differential sensitivity to azoles, although intriguingly ectopic expression of either RPS41 or RPS42 in a wild-type strain leads to decreased sensitivity to fluconazole (FLC). C. albicans cDNA microarray analysis experiments found that carbohydrate and nitrogen metabolic processes were repressed but transport-process-related genes were up-regulated in the rps41Δ mutant. Overall, our present study suggests that loss of the RPS41 gene but not its paralog the RPS42 gene can generate distinct phenotypes including effects on growth rate, morphological transitions, and susceptibility to osmotic stress due to the fact that mRNA levels of RPS41 is much higher than RPS42 in C. albicans.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Candida albicans/fisiología , Fluconazol/farmacología , Hongos/genética , Hongos/fisiología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Mutación , Presión Osmótica , Fenotipo , ARN Mensajero/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.
Asunto(s)
Candida albicans , Candidiasis/inmunología , Proteínas Fúngicas/inmunología , Manosiltransferasas/inmunología , Proteínas Recombinantes/inmunología , Vacunación , Vacunas/inmunología , Animales , Candida albicans/crecimiento & desarrollo , Candidiasis/microbiología , Pared Celular , Citocinas/sangre , Femenino , Inmunoglobulinas/sangre , Masculino , Ratones Endogámicos BALB C , Estructura Terciaria de ProteínaRESUMEN
Heterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. In the fungal pathogen Candida albicans, the STE18 gene (ORF19.6551.1) encodes a potential γ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX box characteristic of γ subunits and has sequence similarity to γ subunits implicated in the mating pathways of a variety of fungi. Disruption of this gene was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the Gα or the Gß subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the Gγ subunit and restore both pheromone-induced gene expression and morphology changes.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Regulación Fúngica de la Expresión Génica , Secuencia de Aminoácidos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Eliminación de Gen , Genes del Tipo Sexual de los Hongos , Datos de Secuencia Molecular , Feromonas/genética , Feromonas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 µg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 µg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 µg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway.
Asunto(s)
Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Estilbenos/farmacología , Estilbenos/uso terapéutico , Animales , Candida albicans/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Ratas , Ratas Sprague-DawleyRESUMEN
Zorro2 is a member of a non-long terminal repeat (LTR) retrotransposon family in Candida albicans, but as yet no clear evidence has been provided to establish either transcription or transposition activity for Zorro2. In this study, the relative expression changes of two open reading frames in Zorro2, ORF19.7274 and ORF19.7275, were examined in response to miconazole (MCZ), and were found to be increased by this treatment. As well, the copy number and the transcripts of Zorro2 in MCZ-induced resistant daughter strains were increased compared to the parental strain, indicating that transposition of Zorro2 occurred during long-term MCZ treatment. Intriguingly, the transcription activity of Zorro2 retrotransposons was significantly inhibited when the cells were treated with MCZ together with antioxidant N-acetyl-L-cysteine (NAC). As both the level of intracellular reactive oxygen species (ROS) and the expression of genes involving DNA repair activated by MCZ were reduced when combined with the treatment of NAC, we propose that the damage caused by accumulation of ROS under MCZ stress is a major reason for the transcription and transposition activation of the Zorro2 retrotransposon.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Miconazol/farmacología , Sistemas de Lectura Abierta , Especies Reactivas de Oxígeno/metabolismo , Retroelementos , Transcripción Genética , Acetilcisteína/farmacología , Antioxidantes/farmacología , Candida albicans/genética , Reparación del ADN , Genoma FúngicoRESUMEN
Over-expression of the Candida drug resistance gene CDR1 is a common mechanism generating azole-resistant Candida albicans in clinical isolates. CDR1 is transcriptionally activated through the binding of the transcription factor Tac1p to the cis-acting drug-responsive element (DRE) in its promoter. We previously demonstrated that the combination of fluconazole (FLC) and berberine (BBR) produced significant synergy when used against FLC-resistant C. albicans in vitro. In this study, we found that BBR inhibited both the up-regulation of CDR1 mRNA and the transport function of Cdr1p induced by fluphenazine (FNZ). Further, electrophoretic mobility shift assays suggested that the transcription activation complex of protein-DRE was disrupted by BBR, and electrospray ionization mass spectrometry analysis showed that BBR bound to the DRE of CDR1. Thus we propose that BBR inhibits the FNZ-induced transcriptional activation of CDR1 in C. albicans by blocking transcription factor binding to the DRE of CDR1. These results contribute to our understanding of the mechanism of synergistic effect of BBR and FLC.
Asunto(s)
Antifúngicos/farmacología , Berberina/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Flufenazina/efectos adversos , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Extractos Vegetales/farmacología , Candida albicans/metabolismo , Sinergismo Farmacológico , Flufenazina/uso terapéutico , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia ArribaRESUMEN
The incidence of systemic fungal infections have increased dramatically, moreover, drug resistance including either primary (intrinsic) or secondary (acquired) resistance, becomes one of the main reasons accounting for the failure of treating invasive fungal infections in the past decades. Nowadays, clinically available antifungal drugs are limited and their combination in antifungal therapy was not effective. It is expected to be a new strategy to synergistically sensitize antifungal drugs against drug-resistant fungi by using new small molecules. Based on the study in our research group and the reported work of others, we reviewed the research of the natural products which have synergistic effect with the antifungal agents against drug-resistant fungi. This review focused on the resource, structure, pharmacological activity, and action mechanism of the compounds, as well as somewhat in common, and would provide theoretical base for seeking new drug against drug-resistance fungi.
Asunto(s)
Antifúngicos/química , Productos Biológicos/química , Hongos/efectos de los fármacos , Antifúngicos/farmacología , Productos Biológicos/farmacología , Sinergismo FarmacológicoRESUMEN
Abstract: Our previous work revealed berberine can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, which suggested that berberine has synergistic antifungal activity with fluconazole. Preliminary SAR of berberine needs to be studied for the possibility of investigating its target and SAR, improving its drug-likeness, and exploring new scaffold. In this work, 13-substitutited benzyl berberine derivatives and N-benzyl isoquinoline analogues were synthesized and characterized by 1H NMR and MS. Their synergetic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The 13-substitutited benzyl berberine derivatives 1a-1e exhibited comparable activity to berberine, which suggested that the introduction of functional groups to C-13 can maintain its activity. The N-benzyl isoquinolines, which were designed as analogues of berberine with its D ring opened, exhibited lower activity than berberine. However, compound 2b, 2c, and 4b showed moderate activity, which indicated that berberine may be deconstructed to new scaffold with synergistic antifungal activity with fluconazole. The results of our research may be helpful to the SAR studies on its other biological activities.