RESUMO
Plant infections caused by fungi lead to significant crop losses worldwide every year. This study aims to better understand the plant defence mechanisms regulated by red light, in particular, the effects of red light at night when most phytopathogens are highly infectious. Our results showed that superoxide production significantly increased immediately after red light exposure and, together with hydrogen peroxide levels, was highest at dawn after 30 min of nocturnal red-light treatment. In parallel, red-light-induced expression and increased the activities of several antioxidant enzymes. The nocturnal red light did not affect salicylic acid but increased jasmonic acid levels immediately after illumination, whereas abscisic acid levels increased 3 h after nocturnal red-light exposure at dawn. Based on the RNAseq data, red light immediately increased the transcription of several chloroplastic chlorophyll a-b binding protein and circadian rhythm-related genes, such as Constans 1, CONSTANS interacting protein 1 and zinc finger protein CONSTANS-LIKE 10. In addition, the levels of several transcription factors were also increased after red light exposure, such as the DOF zinc finger protein and a MYB transcription factor involved in the regulation of circadian rhythms and defence responses in tomato. In addition to identifying these key transcription factors in tomato, the application of red light at night for one week not only reactivated key antioxidant enzymes at the gene and enzyme activity level at dawn but also contributed to a more efficient and successful defence against Botrytis cinerea infection.
Assuntos
Botrytis , Regulação da Expressão Gênica de Plantas , Luz , Doenças das Plantas , Solanum lycopersicum , Botrytis/fisiologia , Solanum lycopersicum/microbiologia , Solanum lycopersicum/genética , Solanum lycopersicum/efeitos da radiação , Solanum lycopersicum/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ácido Salicílico/metabolismo , Ritmo Circadiano/fisiologia , Ritmo Circadiano/efeitos da radiação , Reguladores de Crescimento de Plantas/metabolismo , Peróxido de Hidrogênio/metabolismo , Luz VermelhaRESUMO
Fungal infections with high mortality rates represent an increasing health risk. The Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2) is a small, cysteine-rich, cationic protein exhibiting potent anti-Candida activity. As the underlying mechanism, pore formation has been demonstrated; however, molecular level details on its membrane disruption action are lacking. Herein, we addressed the lipid binding of NFAP2 using a combined computational and experimental approach to simple lipid compositions with various surface charge properties. Simulation results revealed binding preferences for negatively charged model membranes, where selectivity is mediated by anionic lipid components enriched at the protein binding site but also assisted by zwitterionic lipid species. Several potential binding routes initiated by various anchoring contacts were observed, which resulted in one main binding mode and a few variants, with NFAP2 residing on the membrane surface. Region 10NCPNNCKHKKG20 of the flexible N-terminal part of the protein showed potency to insert into the lipid bilayer, where the disulfide bond-stabilized short motif 11CPNNC15 could play a key role. In addition, several areas, including the beginning of the N-terminal (residues 1-8), played roles in facilitating initial membrane contacts. Besides, individual roles of residues such as Lys24, Lys32, Lys34, and Trp42 were also revealed by the simulations. Combined data demonstrated that the solution conformation was not perturbed markedly upon membrane interaction, and the folded part of the protein also contributed to stabilizing the bound state. Data also highlighted that the binding of NFAP2 to lipid vesicles is sensitively affected by environmental factors such as ionic strength. Electrostatic interactions driven by anionic lipids were found pivotal, explaining the reduced membrane activity observed under high salt conditions. Experimental data supported the lipid-selective binding mechanisms and pointed to salt-dependent effects, particularly to protein-assisted vesicle aggregation at low ionic strength. Our findings can contribute to the development of NFAP2-based anti-Candida agents and studies aiming at future medical use of peptide-based natural antifungal compounds.
Assuntos
Antifúngicos , Proteínas Fúngicas , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Sítios de Ligação , Ligação Proteica , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Modelos MolecularesRESUMO
Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity.
Assuntos
Antifúngicos , Proteínas Fúngicas , Antifúngicos/química , Proteínas Fúngicas/metabolismo , alfa-Fetoproteínas , DissulfetosRESUMO
The introduction of the first antibiotic (penicillin) by Sir Alexander Fleming in 1928 was a huge milestone in the treatment of infectious diseases [...].
Assuntos
Peptídeos Antimicrobianos , Penicilinas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias , FungosRESUMO
Light is essential for plant life. It provides a source of energy through photosynthesis and regulates plant growth and development and other cellular processes, such as by controlling the endogenous circadian clock. Light intensity, quality, duration and timing are all important determinants of plant responses, especially to biotic stress. Red light can positively influence plant defence mechanisms against different pathogens, but the molecular mechanism behind this phenomenon is not fully understood. Therefore, we reviewed the impact of red light on plant biotic stress responses against viruses, bacteria, fungi and nematodes, with a focus on the physiological effects of red light treatment and hormonal crosstalk under biotic stress in plants. We found evidence suggesting that exposing plants to red light increases levels of salicylic acid (SA) and induces SA signalling mediating the production of reactive oxygen species, with substantial differences between species and plant organs. Such changes in SA levels could be vital for plants to survive infections. Therefore, the application of red light provides a multidimensional aspect to developing innovative and environmentally friendly approaches to plant and crop disease management.
Assuntos
Controle de Insetos/métodos , Luz , Doenças das Plantas/prevenção & controle , Reguladores de Crescimento de Plantas/metabolismo , Estresse Fisiológico , Animais , Bactérias/efeitos da radiação , Fungos/efeitos da radiação , Nematoides/efeitos da radiação , Vírus de Plantas/efeitos da radiaçãoRESUMO
The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Conformação Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Motivos de Aminoácidos , Sequência de Aminoácidos , Testes de Sensibilidade Microbiana , Penicillium , Penicillium chrysogenum , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.
Assuntos
Antifúngicos/metabolismo , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Neosartorya/metabolismo , Antifúngicos/farmacologia , Candida/fisiologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas Fúngicas/farmacologia , HumanosRESUMO
As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.
Assuntos
Antifúngicos/metabolismo , Antifúngicos/uso terapêutico , Candidíase Vulvovaginal/tratamento farmacológico , Neosartorya/metabolismo , Animais , Candidíase Vulvovaginal/microbiologia , Farmacorresistência Fúngica , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade MicrobianaRESUMO
Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the ß-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Retículo Endoplasmático/enzimologia , Hidroximetilglutaril-CoA Redutases/genética , Mucor/enzimologia , Mucor/genética , Deleção de Genes , Ácido Mevalônico/metabolismo , Microscopia de Fluorescência , MutaçãoRESUMO
The emerging number of life-threatening invasive fungal infections caused by drug-resistant Candida strains urges the need for the development and application of fundamentally new and safe antifungal strategies in the clinical treatment. Recent studies demonstrated that the extracellular cysteine-rich and cationic antifungal proteins (crAFPs) originating from filamentous fungi, and de novo designed synthetic peptide derivatives of these crAFPs provide a feasible basis for this approach. This mini-review focuses on the global challenges of the anti-Canidia therapy and on the crAFPs as potential drug candidates to overcome existing problems. The advantages and limitations in the use of crAFPs and peptide derivatives compared to those of conventional antifungal drugs will also be critically discussed.
RESUMO
We report a case of a 59-year-old male patient with a postoperative fungal infection of the left eye. A dark-pigmented yeast, Exophiala dermatitidis (previously known as Wangiella dermatitidis), was identified from the culture of the biopsy taken from the posterior capsule. The infection was successfully eradicated by a combination of surgical and medical (i.e., voriconazole and fluconazole) treatment. This is the first report of successfully treated E. dermatitidis endophthalmitis, which demonstrates that a prompt and aggressive antifungal therapy combined with surgical intervention is necessary to prevent vision loss in cases of endophthalmitis due to Exophiala species. Beside the case description, we also aim to provide a literature review of previously reported eye infections caused by Exophiala species in order to help the future diagnosis and management of the disease.
Assuntos
Endoftalmite/diagnóstico , Endoftalmite/patologia , Exophiala/isolamento & purificação , Feoifomicose/diagnóstico , Feoifomicose/patologia , Infecção da Ferida Cirúrgica/diagnóstico , Infecção da Ferida Cirúrgica/patologia , Antifúngicos/administração & dosagem , Biópsia , Desbridamento , Endoftalmite/microbiologia , Endoftalmite/terapia , Humanos , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Feoifomicose/microbiologia , Feoifomicose/terapia , Pigmentos Biológicos/análise , Infecção da Ferida Cirúrgica/microbiologia , Infecção da Ferida Cirúrgica/terapia , Resultado do TratamentoRESUMO
Despite the current therapeutic options, filamentous fungal infections are associated with high mortality rate especially in immunocompromised patients. In order to find a new potential therapeutic approach, the in vitro inhibitory effect of two antiarrhythmic agents, diltiazem and verapamil hydrochloride were tested against different clinical isolates of ascomycetous and mucoralean filamentous fungi. The in vitro combinations of these non-antifungal drugs with azole and polyene antifungal agents were also examined. Susceptibility tests were carried out using the broth microdilution method according to the instructions of the Clinical and Laboratory Standards Institute document M38-A2. Checkerboard microdilution assay was used to assess the interactions between antifungal and non-antifungal drugs. Compared to antifungal agents, diltiazem and verapamil hydrochloride exerted a relatively low antifungal activity with high minimal inhibitory concentration values (853-2731 µg/ml). Although in combination they could increase the antifungal activity of amphotericin B, itraconazole and voriconazole. Indifferent and synergistic interactions were registered in 33 and 17 cases, respectively. Antagonistic interactions were not revealed between the investigated compounds. However, the observed high MICs suggest that these agents could not be considered as alternative systemic antifungal agents.
Assuntos
Antifúngicos/administração & dosagem , Bloqueadores dos Canais de Cálcio/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Fungos/fisiologia , Relação Dose-Resposta a Droga , Combinação de MedicamentosRESUMO
BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Cisteína/metabolismo , Penicillium chrysogenum/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular/métodos , Cisteína/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Espectroscopia de Ressonância Magnética/métodos , Mutagênese Sítio-Dirigida , Penicillium chrysogenum/genéticaRESUMO
In recent years, Scedosporium species have been more commonly recognized from severe, difficult-to-treat human infections, such as upper respiratory tract and pulmonary infections. To select an appropriate therapeutic approach for these infections is challenging, because of the commonly observed resistance of the causative agents to several antifungal drugs. Therefore, to find a novel strategy for the treatment of pulmonary Scedosporium infections the in vitro antifungal effect of a mucolytic agent, N-acetyl-L-cysteine and its in vitro combinations with conventional antifungals were investigated. Synergistic and indifferent interactions were registered in 23 and 13 cases, respectively. Antagonism was not revealed between the compounds.
Assuntos
Acetilcisteína/farmacologia , Antifúngicos/farmacologia , Interações Medicamentosas , Scedosporium/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Micoses/microbiologia , Scedosporium/isolamento & purificaçãoRESUMO
In the present study, in vitro antifungal activities of five antipsychotic drugs (i.e., chlorpromazine hydrochloride, CPZ; trifluoperazine hydrochloride, TPZ; amantadine hydrochloride; R-(-)-deprenyl hydrochloride, and valproic acid sodium salt) and five conventional antifungal drugs (i.e., amphotericin B, AMB; caspofungin, CSP; itraconazole; terbinafine, TRB and voriconazole, VRC) were investigated in broth microdilution tests against four clinical and five environmental Scedosporium and Pseudallescheria isolates. When used alone, phenothiazines CPZ and TPZ exerted remarkable antifungal effects. Thus, their in vitro combinations with AMB, CSP, VRC, and TRB were also examined against the clinical isolates. In combination with antifungal agents, CPZ was able to act synergistically with AMB and TRB in cases of one and two isolates, respectively. In all other cases, indifferent interactions were revealed. Antagonism was not observed between the tested agents. These combinations may establish a more effective and less toxic therapy after further in vitro and in vivo studies for Scedosporium and Pseudallescheria infections.
Assuntos
Antifúngicos/farmacologia , Antipsicóticos/farmacologia , Interações Medicamentosas , Pseudallescheria/efeitos dos fármacos , Scedosporium/efeitos dos fármacos , Microbiologia Ambiental , Humanos , Testes de Sensibilidade Microbiana , Micoses/microbiologia , Pseudallescheria/isolamento & purificação , Scedosporium/isolamento & purificaçãoRESUMO
The present study was carried out to investigate the antifungal effects of Cinnamomum zeylanicum, Citrus limon, Juniperus communis, Eucalyptus citriodora, Gaultheria procumbens, Melaleuca alternifolia, Origanum majorana, Salvia sclarea, and Thymus vulgaris essential oils against Fusarium species, the most common etiologic agents of filamentous fungal keratitis in South India. C. zeylanicum essential oil showed strong anti-Fusarium activity, whereas all the other tested essential oils proved to be less effective. The main component of C. zeylanicum essential oil, trans-cinnamaldehyde, was also tested and showed a similar effect as the oil. The in vitro interaction between trans-cinnamaldehyde and natamycin, the first-line therapeutic agent of Fusarium keratitis, was also investigated; an enhanced fungal growth inhibition was observed when these agents were applied in combination. Light and fluorescent microscopic observations revealed that C. zeylanicum essential oil/trans-cinnamaldehyde reduces the cellular metabolism and inhibits the conidia germination. Furthermore, necrotic events were significantly more frequent in the presence of these two compounds. According to our results, C. zeylanicum essential oil/trans-cinnamaldehyde provides a promising basis to develop a novel strategy for the treatment of Fusarium keratitis.
Assuntos
Antifúngicos/farmacologia , Fusarium/efeitos dos fármacos , Ceratite/microbiologia , Óleos Voláteis/farmacologia , Acroleína/análogos & derivados , Cinnamomum zeylanicum/química , Citrus/química , Interações Medicamentosas , Eucalyptus/química , Fusarium/patogenicidade , Gaultheria , Humanos , Índia , Juniperus/química , Testes de Sensibilidade Microbiana , Natamicina/farmacologia , Óleos Voláteis/química , Origanum/químicaRESUMO
A reliable identification method was developed for three closely related Curvularia species, which are frequently isolated from human keratomycoses. Since the traditionally used morphological method and the increasingly used internal transcribed spacer (ITS)-based molecular method proved to be insufficient to discern C. australiensis, C. hawaiiensis and C. spicifera, other molecular targets, such as ß-tubulin, translation elongation factor 1-α and the nuclear ribosomal intergenic spacer (IGS), were tested. Among them, the use of the highly divergent IGS sequence is suggested and the species-specific discriminating characters were determined in appropriate reference strains. It was also concluded that C. hawaiiensis and C. spicifera can be predominantly isolated from eye infections among the three species. The in vitro antifungal susceptibility of 10 currently used antifungal agents against 32 Curvularia isolates was also investigated. MICs were determined in each case. Isolates of C. spicifera proved to be less susceptible to the tested antifungals than those of C. hawaiiensis, which underline the importance of the correct identification of these species.
Assuntos
Antifúngicos/farmacologia , Ascomicetos/classificação , Ascomicetos/efeitos dos fármacos , DNA Fúngico/genética , Infecções Oculares Fúngicas/microbiologia , Tipagem Molecular , Técnicas de Tipagem Micológica , Ascomicetos/genética , Ascomicetos/isolamento & purificação , DNA Intergênico , DNA Espaçador Ribossômico/genética , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Análise de Sequência de DNA , Tubulina (Proteína)/genéticaRESUMO
Neosartorya fischeri NRRL 181 isolate secretes a defensin-like antifungal protein (NFAP) which has a remarkable antifungal effect against ascomycetous filamentous fungi. This protein is a promising antifungal agent of biotechnological value; however in spite of the available knowledge of the nature of its 5'-upstream transcriptional regulation elements, the bulk production of NFAP has not been resolved yet. In this study we carried out its heterologous expression in the yeast Pichia pastoris and investigated the growth inhibition effect exerted by the heterologous NFAP (hNFAP) on filamentous fungal isolates from human infections compared with what was caused by the native NFAP. P. pastoris KM71H transformant strain harboring the pPICZαA plasmid with the mature NFAP encoding gene produced the protein. The final yield of the hNFAP was sixfold compared to the NFAP produced by N. fischeri NRRL 181. Based on the signal dispersion of the amide region, it was proven that the hNFAP exists in folded state. The purified hNFAP effectively inhibited the growth of fungal isolates belonging to the Aspergillus and to the Fusarium genus, but all investigated zygomycetous strain proved to be insusceptible. There was no significant difference between the growth inhibition effect exerted by the native and the heterologous NFAP. These data indicated that P. pastoris KM71H can produce the NFAP in an antifungally active folded state. Our results provide a base for further research, e.g., investigation the connection between the protein structure and the antifungal activity using site directed mutagenesis.
Assuntos
Antifúngicos/farmacologia , Defensinas/biossíntese , Proteínas Fúngicas/biossíntese , Fungos/efeitos dos fármacos , Sequência de Aminoácidos , Defensinas/genética , Defensinas/isolamento & purificação , Defensinas/farmacologia , Dermatomicoses/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Fungos/patogenicidade , Humanos , Neosartorya/química , Neosartorya/genética , Neosartorya/metabolismo , Pichia/genéticaRESUMO
The investigation of the antifungal activities of drugs whose primary activities are not related to their antimicrobial potential is in the current forefront of research. Statin compounds, which are routinely used as cholesterol-lowering drugs, may also exert direct antimicrobial effects. In this study, the in vitro antifungal activities of various statins (lovastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin and pravastatin) were examined against one isolate each of four dermatophyte species (Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum). Basically, statins were effective in inhibiting all dermatophyte studied, but were particularly active against M. canis and T. mentagrophytes. Fluvastatin and simvastatin were active against all of the tested fungi causing a complete inhibition of their growth at very low concentrations (6.25-12.5 µg/ml). Lovastatin and rosuvastatin had inhibitory effects at higher concentrations (25-128 µg/ml), while atorvastatin and pravastatin proved the less effective. The in vitro interactions between statins and different antifungals (ketoconazole, itraconazole, fluconazole, amphotericin B, nystatin, griseofulvin, terbinafine and primycin) were also investigated using a standard chequerboard broth microdilution method. Synergetic interactions were observed in several cases, most of them were noticed when statins were combined with terbinafine and the different azoles. Some combinations were particularly active (ketoconazole-simvastatin or terbinafine-simvastatin), as they were found to exert synergistic effect against all of the investigated isolates. The other antifungals showed synergistic interactions with statins in only certain cases. These results suggest that statins exert substantial antifungal effects against dermatophyte fungi and they should be promising components in a combination therapy as they can act synergistically with a number of clinically used antifungal agents.
Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Microsporum/efeitos dos fármacos , Trichophyton/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.