RESUMO
The scarce antifungal arsenal, changes in the susceptibility profile of fungal agents, and lack of adherence to treatment have contributed to the increase of cases of dermatomycoses. In this context, new antimicrobial substances have gained importance. Chalcones are precursors of the flavonoid family that have multiple biological activities, have high tolerability by humans, and easy synthesis. In this study, we evaluated the in vitro antifungal activity, alone and in combination with conventional antifungal drugs, of the VS02-4'ethyl chalcone-derived compound against dermatophytes and Candida spp. Susceptibility testing was carried out by broth microdilution. Experiments for determination of the target of the compound on the fungal cell, time-kill kinetics, and toxicity tests in Galleria mellonella model were also performed. Combinatory effects were evaluated by the checkerboard method. Results showed high activity of the compound VS02-4'ethyl against dermatophytes (MIC of 7.81-31.25 µg/ml). The compound targeted the cell membrane, and the time-kill test showed the compound continues to exert gradual activity after 5 days on dermatophytes, but no significant activity on Candida. Low toxicity was observed at 250 mg/kg. Excellent results were observed in the combinatory test, where VS02-4'ethyl showed synergistic interactions with itraconazole, fluconazole, terbinafine, and griseofulvin, against all isolates tested. Although further investigation is needed, these results revealed the great potential of chalcone-derived compounds against fungal infections for which treatments are long and laborious.
RESUMO
Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn5393m transposon and a qnrB19-bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like ß-lactamases were detected and expression of OXA-931 demonstrated a 2-64-fold increase in the minimum inhibitory concentration for ß-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.
Assuntos
Acinetobacter , Saúde Única , Animais , Genômica , Acinetobacter/genética , BrasilRESUMO
Interest in Antarctic fungi has grown due to their resilience in harsh environments, suggesting the presence of valuable compounds from its organisms, such as those presenting photoprotective potential, since this environment suffers the most dangerous UV exposure in the world. Therefore, this research aimed to assess the photoprotective potential of compounds from sustainable marine sources, specifically seaweed-derived fungi from Antarctic continent. These studies led to discovery of photoprotective and antioxidant properties of metabolites from Arthrinium sp., an endophytic fungus from Antarctic brown algae Phaeurus antarcticus. From crude extract, fractions A-I were obtained and compounds 1-6 isolated from E and F fractions, namely 3-Hydroxybenzyl alcohol (1), (-)-orthosporin (2), norlichexanthone (3), anomalin B (4), anomalin A (5), and agonodepside B (6). Compounds 1, 2, and 6 were not previously reported in Arthrinium. Fraction F demonstrated excellent absorbance in both UVA and UVB regions, while compound 6 exhibited lower UVB absorbance, possibly due to synergistic effects. Fraction F and compound 6 displayed photostability and were non-phototoxic to HaCaT cells. They also exhibited antioxidant activity by reducing intracellular ROS production induced by UVA in keratinocyte monolayers and reconstructed human skin models (resulting in 34.6% and 30.2% fluorescence reduction) and did not show irritation potential in HET-CAM assay. Thus, both are promising candidates for use in sunscreens. It is noted that Fraction F does not require further purification, making it advantageous, although clinical studies are necessary to confirm its potential applicability for sunscreen formulations.
Assuntos
Raios Ultravioleta , Xylariales , Humanos , Protetores Solares/farmacologia , Protetores Solares/química , Pele , Antioxidantes/farmacologia , Antioxidantes/metabolismoRESUMO
The fast-emerging and multidrug-resistant Candida auris is the first fungal pathogen to be considered a threat to global public health. Thus, there is a high unmet medical need to develop new therapeutic strategies to control this species. Antimicrobial photodynamic therapy (APDT) is a promising alternative that simultaneously targets and damages numerous microbial biomolecules. Here, we investigated the in vitro and in vivo effects of APDT with four phenothiazinium photosensitizers: (i) methylene blue (MB), (ii) toluidine blue (TBO), and two MB derivatives, (iii) new methylene blue (NMBN) and (iv) the pentacyclic derivative S137, against C. auris. To measure the in vitro efficacy of each PS, minimal inhibitory concentrations (MICs) and survival fraction were determined. Also, the efficiency of APDT was evaluated in vivo with the Galleria mellonella insect model for infection and treatment. Although the C. auris strain used in our study was shown to be resistant to the most-commonly used clinical antifungals, it could not withstand the damages imposed by APDT with any of the four photosensitizers. However, for the in vivo model, only APDT performed with S137 allowed survival of infected G. mellonella larvae. Our results show that structural and chemical properties of the photosensitizers play a major role on the outcomes of in vivo APDT and underscore the need to synthesize and develop novel photosensitizing molecules against multidrug-resistant microorganisms.
Assuntos
Anti-Infecciosos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Azul de Metileno/farmacologia , Candida auris , Antifúngicos/farmacologia , Cloreto de Tolônio , Fotoquimioterapia/métodos , Anti-Infecciosos/farmacologiaRESUMO
BACKGROUND: Cryptococcus neoformans/ Cryptococcus gattii species complex is composed of encapsulated yeast species that are causative agents of cryptococcosis. The characterisation of pathogenic Cryptococcus species provides useful data for epidemiological studies as well as the clinical diagnosis and treatment of patients. OBJECTIVES: This study aimed to characterise the epidemiology, antifungal susceptibility and virulence of 72 clinical strains isolated from cryptococcosis cases between 2012 and 2017 in a tertiary reference hospital in south-eastern Brazil. METHODS: Species and molecular types were molecularly assessed by PCR and PCR-restriction fragment length polymorphism (RFLP) of the URA5 gene. Antifungal susceptibility testing was performed according to the CLSI protocols. The virulence was studied in a Galleria mellonella infection model. RESULTS: The most frequently isolated strain was C. neoformans molecular type VNI (61/72; 84.7%), although C. neoformans molecular type VNII (3/72; 4.2%) was also isolated. Additionally, C. deuterogattii molecular type VGII (8/72; 11.1%) was present, but most frequently from non-HIV-infected patients. Non-wild-type phenotype to the antifungals was observed in 26.4% (19/72) of the C. neoformans and C. deuterogattii clinical isolates, and the latter demonstrated higher MIC to fluconazole and itraconazole than C. neoformans clinical isolates. Finally, the virulence of C. neoformans and C. deuterogattii clinical isolates was diverse in G mellonella larvae and uncorrelated with the virulence factors of melanin and capsule. CONCLUSIONS: The assessment of the spread of cryptococcal species and molecular types as well as the pattern of corresponding antifungal susceptibility and virulence aids in surveil the emergence of resistant strains, ensuring more accurate management of the cryptococcal infection.
Assuntos
Antifúngicos/farmacologia , Criptococose/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Brasil , Criança , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/patogenicidade , Farmacorresistência Fúngica , Feminino , Humanos , Larva , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem Molecular , Mariposas , Técnicas de Tipagem Micológica , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos , Virulência , Adulto JovemRESUMO
Arsenic (As) is a non-threshold human carcinogenic. This element can be volatilized either by nature or anthropogenic sources. In the present study, the analytical performance of an As volatile species trapping system was evaluated to assess the As volatilization promoted by Penicillium sp. and Aspergillus sp., both isolated from rice rhizosphere, and Aspergillus niger sp. considered as a reference. The study was conducted for 60 days (sampling of volatile As species from 1st to 30th day and from 31st to 60th day). The efficiency of As-volatilization was associated with the fungal growth. The highest As volatilization occurred from 31st to 60th day. Penicillium sp., Aspergillus sp. and A. niger were capable of producing 57.8, 46.4, and 5.2% of volatile arsenic species, respectively. The speciation analysis has shown trimethylarsine (TMAs) as the main volatilized As-form, followed by mono- and dimethylarsine (MMAs and DMAs). The results are following the "Challenger pathway". Therefore, the tested fungi isolated from rice rhizosphere have shown promising properties concerning bio-volatilization with potential use for As-mitigation in paddy soils.
Assuntos
Arsênio , Oryza , Penicillium , Aspergillus , Humanos , Rizosfera , VolatilizaçãoRESUMO
Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.
Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Transcriptoma , Animais , Aspergillus fumigatus/genética , Parede Celular , Feminino , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Ontologia Genética , Genoma Fúngico , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pressão Osmótica , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/fisiologiaRESUMO
This study evaluated the morphological changes produced by LLLT on the initial stages of bone healing and also studied the pathways that stimulate the expression of genes related to bone cell proliferation and differentiation. One hundred Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 94 s, 2.8 J) was performed for 1, 2, 3, 5, and 7 sessions. Histopathology revealed that treated animals produced increased amount of newly formed bone at the site of the injury. Moreover, microarray analysis evidenced that LLLT produced a significant increase in the expression TGF-ß, BMP, FGF, and RUNX-2 that could stimulate osteoblast proliferation and differentiation, which may be related to improving the deposition of newly formed bone at the site of the injury. Thus, it is possible to conclude that LLLT improves bone healing by producing a significant increase in the expression of osteogenic genes.
Assuntos
Terapia com Luz de Baixa Intensidade , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese/genética , Osteogênese/efeitos da radiação , Tíbia/fisiologia , Tíbia/efeitos da radiação , Cicatrização/efeitos da radiação , Animais , Regeneração Óssea/genética , Regeneração Óssea/efeitos da radiação , Masculino , Ratos , Ratos Wistar , Cicatrização/genéticaRESUMO
Primary Cutaneous Cryptococcosis is an uncommon infection caused by the yeast Cryptococcus neoformans and C. gattii. Few case reports are available in the literature describing in detail primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Herein, we present a case of a 68-year-old immunocompetent male patient with erythematous nodular lesions on the right forearm due to C. gattii mating-type α and molecular type VGI. The virulence factors test was performed for capsule diameter, melanin production and phospholipase activity. In vitro fluconazole testing showed the sensitivity profile of this clinical isolate. In addition, a review of the literature on this subject was carried out and verified that this is the first reported case of VGI in the south-east region of Brazil.
Assuntos
Criptococose/microbiologia , Cryptococcus gattii/isolamento & purificação , Idoso , Brasil , Criptococose/imunologia , Cryptococcus gattii/classificação , Humanos , Imunocompetência , MasculinoRESUMO
Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas Culina/metabolismo , Proteínas Fúngicas/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Aspergillus nidulans/genética , Complexo do Signalossomo COP9 , Proteínas Culina/genética , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/genética , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.
RESUMO
Disseminated fusariosis is treated with amphotericin B and voriconazole. To determine adequate therapy, the minimal inhibitory concentration (MIC) is used. However, MIC analysis is based on visual observation and requires a long period of fungal incubation. The measure of the minimal profile change concentration (MPCC) using MALDI-TOF MS is a quick spectral method that has presented good results in determining the antimicrobial resistance of yeasts. However, there is a lack of information on filamentous fungi. In the present work, 13 Fusarium spp. clinical isolates and two reference strains were used. MIC was obtained according to the M38-A2 protocol of the Clinical Laboratory Standards Institute, while MPPC was obtained following the initial steps of the M38-A2 protocol. Both Biotyper and the Rstudio environment were used to analyze mass spectra. For some fungal strains, the data obtained from the software MALDI Biotyper Compass 4.1 led to fuzzy heatmaps resulting in difficult interpretation, while heatmaps obtained using Rstudio tools generated better MPCC resolutions. Herein, 86.6% of the AMB MPCC values were highly correlated with the gold-standard AMB MIC. MALDI-TOF MS is a prominent tool used to determine MPCCs quicker, cost-effectively, and more accurately for Fusarium spp. strains. However, better statistical analyses could help measure the technique's limit detection.
RESUMO
Antimicrobial photodynamic therapy (APDT) and silver nanoparticles (AgNPs) are known as promising alternatives for the control of microorganisms. This study aims to evaluate the antifungal activity of APDT, particularly by using the association of low concentrations of phenothiazinium photosensitizers (PS) methylene blue (MB), new methylene blue N (NMBN), and new methylene blue N Zinc (NMBN-Zn) in association with biosynthesized AgNPs. The AgNPs were characterized by UV-Vis spectrophotometry, transmission electron microscopy, and the dynamic light scattering method. The minimum inhibitory concentration of compounds in APDT against Candida albicans and Fusarium keratoplasticum was obtained and the Fractional Inhibitory Concentration Index determined the antifungal effect. The toxicity of compounds and associations in APDT were evaluated in Galleria mellonella. The AgNPs presented a surface plasmon band peak at 420 nm, hydrodynamic diameter of 86.72 nm, and zeta potential of -28.6 mV. AgNPs-PS showed a wider and displaced plasmon band peak due to PS ligands on the surface and decreased zeta potential. AgNPs-NMBN and AgNPs-NMBN-Zn associations presented synergistic effect in APDT with 15 J cm-2 against both fungi and did not show toxicity to G. mellonella. Hence, the enhancement of antifungal activity with low concentrations of compounds and absence of toxicity makes APDT with AgNPs-PS a promising therapeutic alternative for fungal infections.
RESUMO
The rapid dissemination of extended-spectrum ß-lactamases (ESBLs)-producing Enterobacterales from different spheres worldwide over recent years has become a serious problem in both human and veterinary medicine. CTX-M-3-type ESBL has only been reported on few occasions, and in Brazil the blaCTX-M-3 gene has been identified only once in clinical strains. In this study, we aimed to molecularly characterize a hypermucoviscous (hm), hypervirulent (hv), and extensively drug-resistant (XDR) Klebsiella pneumoniae strain isolated from a lung tissue culture of an infected elephant. The A246 strain belonged to ST2121 and presented hm phenotype, hypervirulence-associated genes, and carried blaCTX-M-3 and plasmid-mediated quinolone resistance genes (qnrB2 and qnrS1) on an IncFII-IncQ1-IncM1 multireplicon plasmid (pA246-CTX-M-3, â¼ 162 kb). A novel genetic context of blaCTX-M-3, in which a 482-bp ISEcp1 was truncated by an IS26, was also harbored by pA246-CTX-M-3. Furthermore, in vivo experiments revealed that the hm/hv A246 strain killed 100 % of the Galleria mellonella larvae at 72 h post-infection. Our findings evidence the intercontinental dissemination of a rare K. pneumoniae ST2121 and the multidrug resistance IncFII-IncQ1-IncM1 plasmid. Therefore, to the best of our knowledge, this is the first report of an XDR K. pneumoniae coproducing CTX-M-3, QnrB2, and QnrS1 isolated from captive wild animals.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Elefantes/microbiologia , Infecções por Klebsiella/veterinária , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/patogenicidade , beta-Lactamases/biossíntese , Animais , Animais de Zoológico/microbiologia , Técnicas de Tipagem Bacteriana , Brasil , Escherichia coli/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/microbiologia , Tipagem de Sequências Multilocus , Plasmídeos/genética , Quinolonas/farmacologia , Virulência , beta-Lactamases/genética , beta-Lactamas/farmacologiaRESUMO
Microbial communities infiltrate the respiratory tract of cystic fibrosis patients, where chronic colonization and infection lead to clinical decline. This report aims to provide an overview of the diversity of bacterial and fungal species from the airway secretion of three young CF patients with severe pulmonary disease. The bacterial and fungal microbiomes were investigated by culture isolation, metataxonomics, and metagenomics shotgun. Virulence factors and antibiotic resistance genes were also explored. A. fumigatus was isolated from cultures and identified in high incidence from patient sputum samples. Candida albicans, Penicillium sp., Hanseniaspora sp., Torulaspora delbrueckii, and Talaromyces amestolkiae were isolated sporadically. Metataxonomics and metagenomics detected fungal reads (Saccharomyces cerevisiae, A. fumigatus, and Schizophyllum sp.) in one sputum sample. The main pathogenic bacteria identified were Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, and Achromobacter xylosoxidans. The canonical core CF microbiome is composed of species from the genera Streptococcus, Neisseria, Rothia, Prevotella, and Haemophilus. Thus, the airways of the three young CF patients presented dominant bacterial genera and interindividual variability in microbial community composition and diversity. Additionally, a wide diversity of virulence factors and antibiotic resistance genes were identified in the CF lung microbiomes, which may be linked to the clinical condition of the CF patients. Understanding the microbial community is crucial to improve therapy because it may have the opposite effect, restructuring the pathogenic microbiota. Future studies focusing on the influence of fungi on bacterial diversity and microbial interactions in CF microbiomes will be welcome to fulfill this huge gap of fungal influence on CF physiopathology.
Assuntos
Fibrose Cística , Microbiota , Brasil , Fibrose Cística/complicações , Humanos , Pulmão , Escarro , TalaromycesRESUMO
The protein phosphatase calcineurin is an important mediator connecting calcium-dependent signalling to various cellular responses in multiple organisms. In fungi calcineurin acts largely through regulating Crz1p-like transcription factors. Here we characterize an Aspergillus fumigatus CRZ1 homologue, CrzA and demonstrate its mediation of cellular tolerance to increased concentrations of calcium and manganese. In addition to acute sensitivity to these ions, and decreased conidiation, the crzA null mutant suffers altered expression of calcium transporter mRNAs under high concentrations of calcium, and loss of virulence when compared with the corresponding complemented and wild-type strains. We use multiple expression analyses to probe the transcriptional basis of A. fumigatus calcium tolerance identifying several genes having calA and/or crzA dependent mRNA accumulation patterns. We also demonstrate that contrary to previous findings, the gene encoding the Aspergillus nidulans calcineurin subunit homologue, cnaA, is not essential and that the cnaA deletion mutant shares the morphological phenotypes observed in the corresponding A. fumigatus mutant, DeltacalA. Exploiting the A. nidulans model system, we have linked calcineurin activity with asexual developmental induction, finding that CrzA supports appropriate developmental induction in a calcineurin and brlA-dependent manner in both species.
Assuntos
Aspergillus fumigatus/genética , Calcineurina/genética , Proteínas Fúngicas/genética , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Calcineurina/metabolismo , Cloreto de Cálcio/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Manganês/farmacologia , Microscopia Confocal , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fusarium keratoplasticum and Fusarium moniliforme are filamentous fungi common in the environment and cause mycosis in both animals and plants. Human infections include mycetoma, keratitis and onychomycosis, while deeper mycosis occurs in immunocompromised patients. Most of the Fusarium spp. are frequently resistant to treatment with currently used antifungals. The frequent occurrence of antifungal resistance has motivated the study of antimicrobial photodynamic therapy as an alternative treatment for fungal infections. Many studies have investigated the in vitro use of antimicrobial photodynamic therapy to kill fungi, but rarely in animal models of infection. Thus, here we employed the invertebrate wax moth Galleria mellonella to study the in vivo effects of antimicrobial photodynamic therapy with three different phenothiazinium photosensitizers, methylene blue, new methylene blue N and the pentacyclic S137 against infection with microconidia of Fusarium keratoplasticum and Fusarium moniliforme. The effect of antimicrobial photodynamic therapy using these photosensitizers and light-emitting diodes with an emission peak at 635 nm and an integrated irradiance from 570 to 670 nm of 9.8 mW cm-2 was investigated regarding the toxicity, fungal burden, larval survival and cellular immune response. The results from this model indicate that antimicrobial photodynamic therapy with methylene blue, new methylene blue N and S137 is efficient for the treatment of infection with F. keratoplasticum and F. moniliforme. The efficiency can be attributed to the fungal cell damage caused by antimicrobial photodynamic therapy which facilitates the action of the host immune response.
Assuntos
Fusarium/efeitos dos fármacos , Fenotiazinas/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Antifúngicos/farmacologia , Dipeptídeos/farmacologia , Farmacorresistência Fúngica , Larva/efeitos dos fármacos , Lasers Semicondutores/uso terapêutico , Azul de Metileno/análogos & derivados , Azul de Metileno/farmacologia , Mariposas , Pirimidinas/farmacologiaRESUMO
Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the "AGC kinases" Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkA Y PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the ΔypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways.
RESUMO
The process of bone healing as well as the expression of inflammatory and angiogenic genes after low level laser therapy (LLLT) were investigated in an experimental model of bone defects. Sixty Wistar rats were distributed into control group and laser group (830nm, 30mW, 2,8J, 94seg). Histopathological analysis showed that LLLT was able to modulate the inflammatory process in the area of the bone defect and also to produce an earlier deposition of granulation tissue and newly formed bone tissue. Microarray analysis demonstrated that LLLT produced an up-regulation of the genes related to the inflammatory process (MMD, PTGIR, PTGS2, Ptger2, IL1, 1IL6, IL8, IL18) and the angiogenic genes (FGF14, FGF2, ANGPT2, ANGPT4 and PDGFD) at 36h and 3days, followed by the decrease of the gene expression on day 7. Immunohistochemical analysis revealed that the subjects that were treated presented a higher expression of COX-2 at 36h after surgery and an increased VEGF expression on days 3 and 7 after surgery. Our findings indicate that LLLT was efficient on accelerating the development of newly formed bone probably by modulating the inflammatory and angiogenic gene expression as well as COX2 and VEGF immunoexpression during the initial phase of bone healing.
Assuntos
Doenças Ósseas/radioterapia , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia com Luz de Baixa Intensidade , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Regeneração Óssea/efeitos da radiação , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Masculino , Análise em Microsséries , Ratos , Ratos Wistar , Receptores de Epoprostenol , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Regulação para Cima/efeitos da radiação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The search for alternatives to control microorganisms is necessary both in clinical and agricultural areas. Antimicrobial photodynamic treatment (APDT) is a promising light-based approach that can be used to control both human and plant pathogenic fungi. In the present study, we evaluated the effects of photodynamic treatment with red light and four phenothiazinium photosensitizers (PS): methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN) and the phenothiazinium derivative S137 on ungerminated and germinated microconidia of Fusarium oxysporum, F. moniliforme, and F. solani. APDT with each PS killed efficiently both the quiescent ungerminated microconidia and metabolically active germinated microconidia of the three Fusarium species. Washing away the unbound PS from the microconidia (both ungerminated and germinated) before red light exposure reduced but did not prevent the effect of APDT. Subcelullar localization of PS in ungerminated and germinated microconidia and the effects of photodynamic treatment on cell membranes were also evaluated in the three Fusarium species. APDT with MB, TBO, NMBN or S137 increased the membrane permeability in microconidia and APDT with NMBN or S137 increased the lipids peroxidation in microconidia of the three Fusarium species. These findings expand the understanding of photodynamic inactivation of filamentous fungi with phenothiazinium PS.