RESUMO
Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.
Assuntos
Aspergilose , Gliotoxina , Humanos , Aspergillus fumigatus/metabolismo , Gliotoxina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aspergilose/microbiologiaRESUMO
Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.
RESUMO
Aspergillus fumigatus is the main etiological agent of aspergillosis. The antifungal drug caspofungin (CSP) can be used against A. fumigatus, and CSP tolerance is observed. We have previously shown that the transcription factor FhdA is important for mitochondrial activity. Here, we show that FhdA regulates genes transcribed by RNA polymerase II and III. FhdA influences the expression of tRNAs that are important for mitochondrial function upon CSP. Our results show a completely novel mechanism that is impacted by CSP.
Assuntos
Antifúngicos , Aspergillus fumigatus , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Caspofungina/farmacologia , Uso do Códon , Equinocandinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipopeptídeos/farmacologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/genéticaRESUMO
Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.
Assuntos
Aspergillus fumigatus , Águias , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Caspofungina/farmacologia , Águias/metabolismo , Equinocandinas/metabolismo , Equinocandinas/farmacologia , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , Esporos Fúngicos/metabolismoRESUMO
Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.
Assuntos
Aspergillus fumigatus/classificação , Cromossomos Fúngicos/genética , Heterogeneidade Genética , Histonas/metabolismo , Aspergilose Pulmonar/microbiologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Cromatina , Elementos de DNA Transponíveis , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Código das Histonas , Humanos , Regiões Promotoras Genéticas , Metabolismo Secundário , VirulênciaRESUMO
Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion. This work shows that A. fumigatus metabolizes acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterizes acetate utilization in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.
Assuntos
Acetatos/metabolismo , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/genética , Proteínas Fúngicas/metabolismo , Humanos , Larva/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Neutrófilos/microbiologia , Fenótipo , Metabolismo Secundário , VirulênciaRESUMO
The genomes of a large number of Cryptococcus neoformans isolates have been sequenced and analyzed in recent years. These genomes have been used to understand the global population structure of this opportunistic pathogen. However, only a small number of South American isolates have been considered in these studies, and the population structure of C. neoformans in this part of the world remains elusive. Here, we analyzed the genomic sequences of 53 Brazilian Cryptococcus isolates and deciphered the C. neoformans population structure in this country. Our data reveal an African-like structure that suggested repeated intercontinental transports from Africa to South America. We also identified a mutator phenotype in one VNBII Brazilian isolate, exemplifying how fast-evolving isolates can shape the Cryptococcus population structure. Finally, phenotypic analyses revealed wide diversity but not lineage specificity in the expression of classical virulence traits within the set of isolates.
Assuntos
Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Brasil , Metagenômica , Cryptococcus neoformans/genética , Genômica , Cryptococcus gattii/genéticaRESUMO
The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.IMPORTANCEAspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.
Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/patogenicidade , Parede Celular , Modelos Animais de Doenças , Feminino , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Fenótipo , Reprodução Assexuada , Transdução de Sinais , Transcriptoma , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Invasive pulmonary aspergillosis is a life-threatening fungal infection especially in the immunocompromised patients. The low diversity of available antifungal drugs coupled with the emergence of antifungal resistance has become a worldwide clinical concern. The echinocandin Caspofungin (CSP) is recommended as a second-line therapy but resistance and tolerance mechanisms have been reported. However, how the fungal cell articulates the response to CSP is not completely understood. This work provides a detailed characterization of ZnfA, a transcription factor (TF) identified in previous screening studies that is involved in the A. fumigatus responses to calcium and CSP. This TF plays an important role in the regulation of iron homeostasis and cell wall organization in response to high CSP concentrations as revealed by Chromatin Immunoprecipitation coupled to DNA sequencing (ChIP-seq) analysis. Furthermore, ZnfA acts collaboratively with the key TF CrzA in modulating the response to calcium as well as cell wall and osmotic stresses. This study therefore describes the existence of an additional, previously unknown TF that bridges calcium signaling and the CSP cellular response and further exposes the complex connections that exist among different pathways which govern stress sensing and signaling in A. fumigatus.
RESUMO
G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis.IMPORTANCEA. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.
Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Receptores Acoplados a Proteínas G/genética , Metabolismo Secundário , Animais , Aspergillus fumigatus/química , Regulação Fúngica da Expressão Gênica , Larva/microbiologia , Macrófagos/microbiologia , Masculino , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fagocitose , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug ß-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance.IMPORTANCEAspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive ß-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.
Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Caspofungina/farmacologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antifúngicos/farmacologia , Aspergilose/microbiologia , Feminino , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Transdução de SinaisRESUMO
Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulanssensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity.IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.
Assuntos
Aspergilose/microbiologia , Aspergillus nidulans/genética , Aspergillus nidulans/patogenicidade , Carbono/metabolismo , Metabolômica , Adulto , Animais , Parede Celular/genética , Feminino , Genômica , Doença Granulomatosa Crônica/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutropenia , Fagocitose , Virulência , Peixe-Zebra/microbiologiaRESUMO
The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.IMPORTANCEAspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkCA. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Caspofungina/farmacologia , Parede Celular/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aspergillus fumigatus/genética , Parede Celular/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Concentração Osmolar , Pressão Osmótica , Fosforilação , Proteoma , Transdução de SinaisRESUMO
There are only few drugs available to treat fungal infections, and the lack of new antifungals, along with the emergence of drug-resistant strains, results in millions of deaths/year. An unconventional approach to fight microbial infection is to exploit nutritional vulnerabilities of microorganism metabolism. The metal gallium can disrupt iron metabolism in bacteria and cancer cells, but it has not been tested against fungal pathogens such as Aspergillus and Candida. Here, we investigate in vitro activity of gallium nitrate III [Ga(NO3)3] against these human pathogens, to reveal the gallium mechanism of action and understand the interaction between gallium and clinical antifungal drugs. Ga(NO3)3 presented a fungistatic effect against azole-sensitive and -resistant A. fumigatus strains (MIC50/90 = 32.0 mg/L) and also had a synergistic effect with caspofungin, but not with azoles and amphotericin B. Its antifungal activity seems to be reliant on iron-limiting conditions, as the presence of iron increases its MIC value and because we observed a synergistic interaction between gallium and iron chelators against A. fumigatus. We also show that an A. fumigatus mutant (ΔhapX) unable to grow in the absence of iron is more susceptible to gallium, reinforcing that gallium could act by disrupting iron homeostasis. Furthermore, we demonstrate that gallium has a fungistatic effect against different species of Candida ranging from 16.0 to 256.0 mg/L, including multidrug-resistant Candida auris, C. haemulonii, C. duobushaemulonii, and C. glabrata. Our findings indicate that gallium can inhibit fungal pathogens in vitro under iron-limiting conditions, showing that Ga(NO3)3 could be a potential therapy not only against bacteria but also as an antifungal drug.
Assuntos
Antifúngicos/farmacologia , Gálio/farmacologia , Antifúngicos/química , Aspergillus fumigatus/efeitos dos fármacos , Azóis/química , Azóis/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica , Gálio/química , Cinética , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Fungal infections have been commonly diagnosed in individuals with advanced HIV disease. Cryptococcosis, pneumocystosis, and histoplasmosis are the most frequent systemic mycoses in people suffering from HIV/AIDS. CASE REPORT: We report a case of multiple fungal infections in an advanced AIDS-patient. A 33-year-old HIV-positive man from Brazil was hospitalized due to diarrhea, dyspnea, emaciation, hypoxemia, extensive oral thrush, and a CD4+ T lymphocyte count of 20cells/mm(3). Honeycombed-structures consistent with Pneumocystis jirovecii were observed by direct immunofluorescence in induced sputum. Cryptococcus neoformans was recovered from respiratory secretion and cerebrospinal fluid cultures. Histopathology of the bone marrow also revealed the presence of Histoplasma capsulatum. Molecular assays were performed in a sputum sample. Nested-PCR confirmed the presence of P. jirovecii and H. capsulatum; qPCR multiplex was positive for C. neoformans and H. capsulatum. With the treatment of antifungal drugs the patient progressed satisfactorily. CONCLUSIONS: The diagnosis of several systemic mycoses demonstrates the vulnerability of advanced AIDS-patients. Thus, the detection of AIDS cases in the early stages of infection is necessary for a prompt and adequate introduction of HAART therapy, and the use of prophylaxis to control opportunistic infections.