RESUMEN

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. Due to its zoonotic transmission in Brazil, S. brasiliensis represents a significant health threat to humans and domestic animals. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii. Fluorescent miltefosine localizes to the Sporothrix cell membrane and mitochondria and causes cell death through increased permeabilization. Milteforan decreases S. brasiliensis fungal burden in A549 pulmonary cells and bone marrow-derived macrophages and also has an immunomodulatory effect by decreasing TNF-α, IL-6, and IL-10 production. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis. IMPORTANCE: Sporotrichosis is an endemic disease in Latin America caused by different species of Sporothrix. This fungus can infect domestic animals, mainly cats and eventually dogs, as well as humans. Few drugs are available to treat this disease, such as itraconazole, terbinafine, and amphotericin B, but resistance to these agents has risen in the last few years. Alternative new therapeutic options to treat sporotrichosis are essential. Here, we propose milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, as a possible therapeutic alternative for treating sporotrichosis. Milteforan decreases S. brasiliensis fungal burden in human and mouse cells and has an immunomodulatory effect by decreasing several cytokine production.

Asunto(s)

Antifúngicos , Enfermedades de los Gatos , Sporothrix , Esporotricosis , Animales , Esporotricosis/tratamiento farmacológico , Esporotricosis/microbiología , Esporotricosis/veterinaria , Gatos , Sporothrix/efectos de los fármacos , Antifúngicos/farmacología , Enfermedades de los Gatos/tratamiento farmacológico , Enfermedades de los Gatos/microbiología , Humanos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Brasil , Pruebas de Sensibilidad Microbiana , Perros , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones

RESUMEN

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Asunto(s)

Aspergilosis , Aspergillus fumigatus , Proteínas Fúngicas , Evasión Inmune , Proteoma , Esporas Fúngicas , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/genética , Animales , Esporas Fúngicas/inmunología , Ratones , Proteoma/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/inmunología , Aspergilosis/inmunología , Aspergilosis/microbiología , Humanos , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Citocinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino

RESUMEN

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. BRI + IBX can inhibit the growth of A. fumigatus voriconazole- and caspofungin-resistant clinical isolates. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against viruses, bacteria, and fungi. In vitro, combination of BRI + IBX plays a fungicidal role, increases the fungal cell permeability, decreases the fungal survival in the presence of A549 epithelial cells, and appears as a promising antifungal therapeutic alternative against A. fumigatus. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis.

Asunto(s)

Antifúngicos , Aspergillus fumigatus , Aspergillus fumigatus/efectos de los fármacos , Humanos , Antifúngicos/farmacología , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Células A549 , Péptidos Antimicrobianos/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos

RESUMEN

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Asunto(s)

Antifúngicos , Criptococosis , Cryptococcus neoformans , Saccharomyces cerevisiae , Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Animales , Ratones , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Modelos Animales de Enfermedad , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Pruebas de Sensibilidad Microbiana , Caspofungina/farmacología , Femenino , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Anfotericina B/farmacología

RESUMEN

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic, brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. CAS-resistant strains with mutations in fks1 that encodes the 1,3-ß-D-glucan synthase are not IBX-resistant and BRI+IBX can inhibit their growth. The combination of BRI+IBX plays a fungicidal role, increases the fungal cell permeability and decreases the fungal survival in the presence of A549 epithelial cells.

RESUMEN

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. In Brazil, S. brasiliensis represents a vital health threat to humans and domestic animals due to its zoonotic transmission. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii, and by its antifungal activity when used to treat infected epithelial cells and macrophages. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis.

RESUMEN

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.

RESUMEN

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Asunto(s)

Aspergilosis , Micosis , Humanos , Ratones , Animales , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Caspofungina/farmacología , Caspofungina/uso terapéutico , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Modelos Animales de Enfermedad , Aspergilosis/microbiología , Micosis/tratamiento farmacológico , Aspergillus fumigatus , Candida albicans , Farmacorresistencia Fúngica

RESUMEN

In cystic fibrosis (CF), mucus plaques are formed in the patient's lungs, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can cocolonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and antibiofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms under normoxia and hypoxia conditions. We detected nine SM produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion levels were increased by the fungal presence. The roles of the two operons responsible for phenazine production (phzA1 and phzA2) were also investigated, and mutants lacking one of those operons were able to produce partial sets of phenazines. We detected a total of 20 SM secreted by A. fumigatus either in monoculture or in coculture with P. aeruginosa. All these compounds were secreted during biofilm formation in either normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, triacetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa under normoxia and hypoxia conditions. Overall, we showed how diverse SM secretion is during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation in normoxia and hypoxia. IMPORTANCE The interaction between Pseudomonas aeruginosa and Aspergillus fumigatus has been well characterized in vitro. In this scenario, the bacterium exerts a strong inhibitory effect against the fungus. However, little is known about the metabolites produced by the fungus to counterattack the bacteria. Our work aimed to annotate secondary metabolites (SM) secreted during coculture between P. aeruginosa and A. fumigatus during biofilm formation in both normoxia and hypoxia. The bacterium produces several different types of phenazines and pyoverdins in response to presence of the fungus. In contrast, we were able to annotate 29 metabolites produced during A. fumigatus biofilm formation, but only 8 compounds were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa upon either normoxia or hypoxia. In conclusion, we detected many SM secreted during A. fumigatus and P. aeruginosa biofilm formation. This analysis provides several opportunities to understand the interactions between these two species.

Asunto(s)

Fibrosis Quística , Gliotoxina , Aspergillus fumigatus , Biopelículas , Humanos , Hipoxia , Fenazinas/metabolismo , Fenazinas/farmacología , Pseudomonas aeruginosa/metabolismo

RESUMEN

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.

Asunto(s)

Aspergillus fumigatus , Águilas , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Caspofungina/farmacología , Águilas/metabolismo , Equinocandinas/metabolismo , Equinocandinas/farmacología , Proteínas Fúngicas/metabolismo , Pruebas de Sensibilidad Microbiana , Esporas Fúngicas/metabolismo

RESUMEN

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Asunto(s)

Aspergillus/crecimiento & desarrollo , Gliotoxina/farmacología , Metiltransferasas/genética , Factores de Transcripción/genética , Aspergillus/efectos de los fármacos , Aspergillus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus oryzae/efectos de los fármacos , Aspergillus oryzae/genética , Aspergillus oryzae/crecimiento & desarrollo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Gliotoxina/biosíntesis , RNA-Seq

RESUMEN

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.

Asunto(s)

Aspergillus fumigatus/clasificación , Cromosomas Fúngicos/genética , Heterogeneidad Genética , Histonas/metabolismo , Aspergilosis Pulmonar/microbiología , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Cromatina , Elementos Transponibles de ADN , Proteínas Fúngicas/metabolismo , Regulación de la Expresión Génica de las Plantas , Aptitud Genética , Código de Histonas , Humanos , Regiones Promotoras Genéticas , Metabolismo Secundario , Virulencia

RESUMEN

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.

RESUMEN

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.

Asunto(s)

Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidad , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Receptores Acoplados a Proteínas G/genética , Metabolismo Secundario , Animales , Aspergillus fumigatus/química , Regulación Fúngica de la Expresión Génica , Larva/microbiología , Macrófagos/microbiología , Masculino , Melaninas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mariposas Nocturnas/microbiología , Fagocitosis , Fosforilación , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

[This corrects the article DOI: 10.1016/j.tcsw.2018.03.002.].

RESUMEN

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.

Asunto(s)

Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Caspofungina/farmacología , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Antifúngicos/farmacología , Aspergilosis/microbiología , Femenino , Regulación Fúngica de la Expresión Génica , Biblioteca de Genes , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Transducción de Señal

RESUMEN

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.

Asunto(s)

Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/enzimología , Caspofungina/farmacología , Pared Celular/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Aspergillus fumigatus/genética , Pared Celular/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Glicerol/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Concentración Osmolar , Presión Osmótica , Fosforilación , Proteoma , Transducción de Señal

RESUMEN

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Asunto(s)

Aspergillus fumigatus/patogenicidad , Calcio/efectos adversos , Farmacorresistencia Fúngica , Aspergilosis Pulmonar/microbiología , Factores de Transcripción/genética , Animales , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Caspofungina , Pared Celular/metabolismo , Modelos Animales de Enfermedad , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Ratones , Mutación , Aspergilosis Pulmonar/inmunología , Estrés Fisiológico , Virulencia

RESUMEN

The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatusMSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatusmshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition.IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2 Although the mutation frequency of mshA (the A. fumigatusMSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.

Asunto(s)

Antifúngicos/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidad , Azoles/farmacología , Farmacorresistencia Fúngica , Proteína 2 Homóloga a MutS/genética , Animales , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Reparación de la Incompatibilidad de ADN , Femenino , Proteínas Fúngicas/genética , Larva/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Neutropenia , Homología de Secuencia , Virulencia

RESUMEN

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner.