RESUMO
Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Farmacorresistência Fúngica Múltipla , Hidroximetilglutaril-CoA Redutases/metabolismo , Oxigenases de Função Mista/metabolismo , Esterol 14-Desmetilase/metabolismo , Animais , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Azóis/uso terapêutico , Cruzamentos Genéticos , Farmacorresistência Fúngica Múltipla/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/genética , Itraconazol/farmacologia , Itraconazol/uso terapêutico , Larva/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Oxigenases de Função Mista/genética , Mariposas/efeitos dos fármacos , Mutação , Esterol 14-Desmetilase/genética , Análise de Sobrevida , Triazóis/farmacologia , Triazóis/uso terapêutico , Virulência/efeitos dos fármacos , Voriconazol/farmacologia , Voriconazol/uso terapêuticoRESUMO
Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.
Assuntos
Aspergillus/crescimento & desenvolvimento , Aspergillus/genética , Cruzamentos Genéticos , Genes Fúngicos Tipo Acasalamento , Animais , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus/classificação , Aspergillus/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Animais de Doenças , Humanos , Lepidópteros , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência de DNA , VirulênciaAssuntos
Aspergillus fumigatus/imunologia , Asma/diagnóstico , Asma/imunologia , Proteínas de Bactérias/imunologia , Endopeptidases/imunologia , Alérgenos/imunologia , Aspergillus fumigatus/enzimologia , Humanos , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologia , Índice de Gravidade de DoençaAssuntos
Aspergilose/genética , Esofagite Eosinofílica/genética , Rinite Alérgica/genética , Fator de Transcrição STAT3/genética , Adulto , Antifúngicos/uso terapêutico , Aspergilose/diagnóstico por imagem , Aspergilose/terapia , Desbridamento , Esofagite Eosinofílica/diagnóstico por imagem , Esofagite Eosinofílica/terapia , Evolução Fatal , Haploinsuficiência , Humanos , Imunoterapia , Imageamento por Ressonância Magnética , Masculino , Rinite Alérgica/diagnóstico por imagem , Rinite Alérgica/terapiaRESUMO
The most common cause of invasive aspergillosis (IA) in patients with chronic granulomatous disease (CGD) is Aspergillus fumigatus followed by A. nidulans; other aspergilli rarely cause the disease. Here we review two clinical cases of fatal IA in CGD patients and describe a new etiologic agent of IA refractory to antifungal therapy. Unlike typical IA caused by A. fumigatus, the disease caused by the new species was chronic and spread from the lung to multiple adjacent organs. Mycological characteristics and the phylogenetic relationship with other aspergilli based on the sequence analysis of Mcm7, RPB2, and Tsr1 indicated that the new species, which we named as A. tanneri, belongs to Aspergillus section Circumdati. The species has a higher amphotericin B, voriconazole, and itraconazole MIC and causes more chronic infection in CGD mice than A. fumigatus. This is the first report documenting IA in CGD patients caused by a species belonging to the Aspergillus section Circumdati that is inherently resistant to azoles and amphotericin B. Unlike the results seen with many members of Aspergillus section Circumdati, ochratoxin was not detected in filtrates of cultures grown in various media. Our phenotypic and genetic characterization of the new species and the case reports will assist future diagnosis of infection caused by A. tanneri and lead to more appropriate patient management.
Assuntos
Antifúngicos/uso terapêutico , Aspergilose/microbiologia , Aspergillus/classificação , Aspergillus/genética , Farmacorresistência Fúngica , Adolescente , Anfotericina B/farmacologia , Animais , Antifúngicos/farmacologia , Aspergilose/diagnóstico , Aspergilose/tratamento farmacológico , Aspergilose/patologia , Aspergillus/efeitos dos fármacos , Aspergillus/isolamento & purificação , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/tratamento farmacológico , Doença Granulomatosa Crônica/microbiologia , Doença Granulomatosa Crônica/patologia , Humanos , Itraconazol/farmacologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Microscopia , Dados de Sequência Molecular , Filogenia , Pirimidinas/farmacologia , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Falha de Tratamento , Triazóis/farmacologia , Voriconazol , Adulto JovemRESUMO
Invasive aspergillosis is a major threat for patients suffering from chronic granulomatous disease (CGD). Although Aspergillus fumigatus is the most commonly encountered Aspergillus species, the presence of A. nidulans appears to be disproportionately high in CGD patients. The purpose of this study was to investigate the involvement of the NADPH oxidase and the resulting reactive oxygen species (ROS) in host defense against fungi and to clarify their relationship toward A. nidulans. Murine CGD alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from healthy controls and CGD patients were challenged with either A. fumigatus or A. nidulans. Analysis of the antifungal effects of ROS revealed that A. nidulans, in contrast to A. fumigatus, is not susceptible to ROS. In addition, infection with live A. nidulans did not result in any measurable ROS release. Remarkably, human CGD PMN and PBMC and murine CGD AM were at least equipotent at arresting conidial germination compared to healthy controls. Blocking of the NADPH oxidase resulted in significantly reduced damage of A. fumigatus but did not affect A. nidulans hyphae. Furthermore, the microbicidal activity of CGD PMN was maintained toward A. nidulans but not A. fumigatus. In summary, antifungal resistance to A. nidulans is not directly ROS related. The etiology of A. nidulans infections in CGD cannot be explained by the simple absence of the direct microbicidal effect of ROS. In vivo, the NADPH oxidase is a critical regulator of innate immunity whose unraveling will improve our understanding of fungal pathogenesis in CGD.
Assuntos
Aspergillus nidulans/imunologia , Leucócitos Mononucleares/fisiologia , Neutrófilos/fisiologia , Animais , Feminino , Humanos , Peróxido de Hidrogênio , Hifas/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de OxigênioRESUMO
We report here a case of disseminated skin infection caused by Mucor velutinosus, a recently described new species. We believe this to be the first published report of a clinical case of mucormycosis due to M. velutinosus, as well as a rare case of dissemination from a deep site to skin.
Assuntos
Dermatomicoses/diagnóstico , Fungemia/diagnóstico , Leucemia Mieloide Aguda/complicações , Mucor/isolamento & purificação , Mucormicose/diagnóstico , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Dermatomicoses/microbiologia , Feminino , Fungemia/microbiologia , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Técnicas Microbiológicas , Microscopia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mucor/classificação , Mucormicose/microbiologia , Filogenia , Análise de Sequência de DNAAssuntos
Aspergilose/microbiologia , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Ar , Aspergilose/epidemiologia , Meio Ambiente , Humanos , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/fisiologiaRESUMO
A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits.
RESUMO
BACKGROUND: Invasive aspergillosis (IA) is most commonly caused by the morphospecies Aspergillus fumigatus. However, genetic-based methods indicate that organisms phenotypically identified as A. fumigatus actually constitute a mold complex, designated Aspergillus section fumigati subgenus fumigati. METHODS: Multilocus sequencing and analysis was performed on fungi identified as A. fumigatus from the clinical culture collection maintained at the National Institutes of Health from 2000 through 2008, with a focus on the internal transcribed spacer 1 and 2 regions of ribosomal DNA (rDNA), beta-tubulin, and rodlet A genes. We reviewed the medical records, radiology, and histopathology of corresponding patients. To confirm identification of Neosartorya udagawae isolates, mating studies were performed with reference strains. Antifungal susceptibility testing was performed by broth microdilution and read at 48 hours. RESULTS: Thirty-six cases of infection attributed to A. fumigatus were identified; 4 were caused by N. udagawae (3 in patients with chronic granulomatous disease and 1 in a patient with myelodysplastic syndrome). Disease due to N. udagawae was chronic, with a median duration of 35 weeks, compared with a median duration of 5.5 weeks for patients with chronic granulomatous disease who had infection due to A. fumigatus sensu stricto (P < .05 , Mann-Whitney U test). Infection spread across anatomical planes in a contiguous manner and was refractory to standard therapy. Two of the 4 patients died. N. udagawae demonstrated relatively higher minimum inhibitory concentrations to various agents, compared with those demonstrated by contemporary A. fumigatus sensu stricto isolates. CONCLUSIONS: To our knowledge, this is the first report documenting infection due to N. udagawae. Clinical manifestations were distinct from those of typical IA. Fumigati-mimetics with inherent potential for antifungal resistance are agents of IA. Genetic identification of molds should be considered for unusual or refractory IA.
Assuntos
Aspergilose Pulmonar Invasiva/microbiologia , Micoses/microbiologia , Neosartorya/classificação , Neosartorya/isolamento & purificação , Adulto , Animais , Impressões Digitais de DNA/métodos , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Feminino , Proteínas Fúngicas/genética , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Neosartorya/genética , Radiografia Torácica , Análise de Sequência de DNA , Tubulina (Proteína)/genéticaRESUMO
Gliotoxin is a member of the epipolythiodioxopiperazine class of toxins and is both the major and the most potent toxin produced by Aspergillus fumigatus. Since the discovery of the putative gliotoxin biosynthetic 12-gene cluster in the genome of A. fumigatus, five different laboratories have attempted to determine the role of this toxin in the virulence of A. fumigatus. The genes in the cluster that have been disrupted to study the pathobiological importance of gliotoxin include gliZ that encodes a transcription factor and gliP that encodes a nonribosomal peptide synthase. Two of the five laboratories have reported gliotoxin to be an important virulence determinant of A. fumigatus, while the other three laboratories have shown it to be unimportant. Comparisons of the data generated among the five laboratories revealed that the immunosuppressive regimen used for mice was the key factor that contributed to the observed disparity. Regardless of either the mouse strains used or the route of infection, immunosuppression with a combination of cyclophosphamide and corticosteroids (neutropenic mice) showed gliotoxin to be unimportant. The mice immunosuppressed with corticosteroids alone, however, revealed that gliotoxin is an important virulence determinant of A. fumigatus. These studies indicate that the neutropenic mice model is inadequate to reveal the pathobiological importance of fungal secondary metabolites in invasive pulmonary aspergillosis.
Assuntos
Aspergillus fumigatus/patogenicidade , Gliotoxina/toxicidade , Fatores de Virulência/toxicidade , Animais , Aspergillus fumigatus/genética , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Genes Fúngicos , Gliotoxina/biossíntese , Hospedeiro Imunocomprometido , Camundongos , Família Multigênica , Fatores de Virulência/genéticaRESUMO
Invasive aspergillosis (IA) remains the primary cause of morbidity and mortality in chronic granulomatous disease (CGD) patients, often due to infection by Aspergillus species refractory to antifungals. This motivates the search for alternative treatments, including immunotherapy. We investigated the effect of exogenous type I interferon (IFN) activation on the outcome of IA caused by three Aspergillus species, A. fumigatus, A. nidulans, and A. tanneri, in CGD mice. The animals were treated with poly(I):poly(C) carboxymethyl cellulose poly-l-lysine (PICLC), a mimetic of double-stranded RNA, 24 h preinfection and postinfection. The survival rates and lung fungal burdens were markedly improved by PICLC immunotherapy in animals infected with any one of the three Aspergillus species. While protection from IA was remarkable, PICLC induction of type I IFN in the lungs surged 24 h posttreatment and returned to baseline levels by 48 h, suggesting that PICLC altered early events in protection against IA. Immunophenotyping of recruited leukocytes and histopathological examination of tissue sections showed that PICLC induced similar cellular infiltrates as those in untreated-infected mice, in both cases dominated by monocytic cells and neutrophils. However, the PICLC immunotherapy resulted in a marked earlier recruitment of the leukocytes. Unlike with conidia, infection with A. nidulans germlings reduced the protective effect of PICLC immunotherapy. Additionally, antibody depletion of neutrophils totally reversed the protection, suggesting that neutrophils are crucial for PICLC-mediated protection. Together, these data show that prophylactic PICLC immunotherapy prerecruits these cells, enabling them to attack the conidia and thus resulting in a profound protection from IA.IMPORTANCE Patients with chronic granulomatous disease (CGD) are highly susceptible to invasive aspergillosis (IA). While Aspergillus fumigatus is the most-studied Aspergillus species, CGD patients often suffer IA caused by A. nidulans, A. tanneri, and other rare species. These non-fumigatus Aspergillus species are more resistant to antifungal drugs and cause higher fatality rates than A. fumigatus Therefore, alternative therapies are needed to protect CGD patients. We report an effective immunotherapy of mice infected with three Aspergillus species via PICLC dosing. While protection from IA was long lasting, PICLC induction of type I IFN surged but quickly returned to baseline levels, suggesting that PICLC was altering early events in IA. Interestingly, we found responding immune cells to be similar between PICLC-treated and untreated-infected mice. However, PICLC immunotherapy resulted in an earlier recruitment of the leukocytes and suppressed fungal growth. This study highlights the value of type I IFN induction in CGD patients.
Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergillus/patogenicidade , Doença Granulomatosa Crônica/tratamento farmacológico , Doença Granulomatosa Crônica/microbiologia , Interferon Tipo I/uso terapêutico , Pulmão/metabolismo , Pulmão/microbiologia , Neutrófilos/citologia , Animais , Aspergilose/imunologia , Aspergilose/microbiologia , Citometria de Fluxo , Doença Granulomatosa Crônica/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/efeitos dos fármacosRESUMO
Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. DeltaCgChsI and DeltaCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of DeltaCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, DeltaCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, DeltaCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.
Assuntos
Quitina Sintase/fisiologia , Colletotrichum/enzimologia , Proteínas Fúngicas/fisiologia , Hifas/enzimologia , Zea mays/microbiologia , Sequência de Bases , Quitina Sintase/química , Quitina Sintase/genética , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/patogenicidade , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hifas/crescimento & desenvolvimento , Hifas/ultraestrutura , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Delayed diagnosis in invasive aspergillosis (IA) contributes to its high mortality. Gliotoxin (GT) and bis-methyl-gliotoxin (bmGT) are secondary metabolites produced by Aspergillus during invasive, hyphal growth and may prove diagnostically useful. Because IA pathophysiology and GT's role in virulence vary depending on the underlying host immune status, we hypothesized that GT and bmGT production in vivo may differ in three mouse models of IA that mimic human disease. We defined temporal kinetics of GT and bmGT in serum, bronchoalveolar lavage fluid (BALF) and lungs of A. fumigatus-infected chronic granulomatous disease (CGD), hydrocortisone-treated, and neutropenic mice. We harvested lungs for assessment of fungal burden, histology and GT/bmGT biosynthetic genes' mRNA induction. GT levels were higher in neutropenic versus CGD or steroid-treated lungs. bmGT was persistently detected only in CGD lungs. GT, but not bmGT, was detected in 71% of sera and 50% of BALF of neutropenic mice; neither was detected in serum/BALF of CGD or steroid-treated mice. Enrichment of GT in Aspergillus-infected neutropenic lung correlated with fungal burden and hyphal length but not induction of GT biosynthetic genes. In summary, GT is detectable in mouse lungs, serum and BALF during neutropenic IA, suggesting that GT may be useful to diagnose IA in neutropenic patients.
Assuntos
Aspergilose/etiologia , Aspergilose/metabolismo , Aspergillus/imunologia , Gliotoxina/biossíntese , Interações Hospedeiro-Patógeno/imunologia , Animais , Aspergilose/mortalidade , Aspergilose/patologia , Modelos Animais de Doenças , Doença Granulomatosa Crônica/complicações , Camundongos , Camundongos Knockout , Neutropenia/complicações , Aspergilose Pulmonar/etiologia , Aspergilose Pulmonar/metabolismo , Aspergilose Pulmonar/mortalidade , Aspergilose Pulmonar/patologia , Fatores de Risco , Esteroides/farmacologiaAssuntos
Aspergillus/imunologia , Imunoglobulina E/metabolismo , Aspergilose Pulmonar Invasiva/imunologia , Síndrome de Job/imunologia , Fator de Transcrição STAT3/genética , Idade de Início , Aspergillus/patogenicidade , Bronquiectasia , Células Cultivadas , Suscetibilidade a Doenças , Estudos de Associação Genética , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Aspergilose Pulmonar Invasiva/epidemiologia , Aspergilose Pulmonar Invasiva/genética , Aspergilose Pulmonar Invasiva/fisiopatologia , Síndrome de Job/epidemiologia , Síndrome de Job/genética , Síndrome de Job/fisiopatologia , Mutação/genética , Fator de Transcrição STAT3/imunologia , Análise de SobrevidaRESUMO
Invasive pulmonary aspergillosis is a life-threatening mycosis that only affects patients with immunosuppression, chemotherapy-induced neutropenia, transplantation, or congenital immunodeficiency. We studied the clinical, genetic, histological, and immunological features of 2 unrelated patients without known immunodeficiency who developed extrapulmonary invasive aspergillosis at the ages of 8 and 18. One patient died at age 12 with progressive intra-abdominal aspergillosis. The other patient had presented with intra-abdominal candidiasis at age 9, and developed central nervous system aspergillosis at age 18 and intra-abdominal aspergillosis at age 25. Neither patient developed Aspergillus infection of the lungs. One patient had homozygous M1I CARD9 (caspase recruitment domain family member 9) mutation, while the other had homozygous Q295X CARD9 mutation; both patients lacked CARD9 protein expression. The patients had normal monocyte and Th17 cell numbers in peripheral blood, but their mononuclear cells exhibited impaired production of proinflammatory cytokines upon fungus-specific stimulation. Neutrophil phagocytosis, killing, and oxidative burst against Aspergillus fumigatus were intact, but neither patient accumulated neutrophils in infected tissue despite normal neutrophil numbers in peripheral blood. The neutrophil tissue accumulation defect was not caused by defective neutrophil-intrinsic chemotaxis, indicating that production of neutrophil chemoattractants in extrapulmonary tissue is impaired in CARD9 deficiency. Taken together, our results show that CARD9 deficiency is the first known inherited or acquired condition that predisposes to extrapulmonary Aspergillus infection with sparing of the lungs, associated with impaired neutrophil recruitment to the site of infection.
Assuntos
Aspergilose/imunologia , Proteínas Adaptadoras de Sinalização CARD/deficiência , Infiltração de Neutrófilos , Adolescente , Adulto , Aspergilose/genética , Aspergillus fumigatus , Proteínas Adaptadoras de Sinalização CARD/genética , Criança , Homozigoto , Humanos , Pulmão , Masculino , Mutação , Neutrófilos/imunologiaRESUMO
Seed coats from Chorisia speciosa form a hydrogel on contact with water. When the hydrogel was solubilized and the solution centrifuged, subsequent ethanol precipitation gave a polysaccharide (F-I) composed of rhamnose, galactose and uronic acid in a molar ratio of 25:44:31. Analysis of F-I by HPSEC-MALLS showed a homogenous polymer with high molecular weight. It consisted of a main chain of (1-->4)-linked beta-galactopyranosyl units as indicated by NMR spectral and methylation data analysis, with rhamnose, galactose and glucuronic acid as non-reducing end units. This fraction interfered with adhesion of Colletotrichum graminicola, a causal agent of anthracnose, to polystyrene slides and to leaves of corn, thus delaying infection in the latter.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Plantas/química , Sementes/química , Adesão Celular/efeitos dos fármacos , Cromatografia em Gel , Colletotrichum/efeitos dos fármacos , Colletotrichum/fisiologia , Espectroscopia de Ressonância Magnética , Peso Molecular , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Zea mays/microbiologiaRESUMO
The genus Aspergillus contains etiologic agents of aspergillosis. The clinical manifestations of the disease range from allergic reaction to invasive pulmonary infection. Among the pathogenic aspergilli, Aspergillus fumigatus is most ubiquitous in the environment and is the major cause of the disease, followed by Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, and several species in the section Fumigati that morphologically resemble A. fumigatus. Patients that are at risk for acquiring aspergillosis are those with an altered immune system. Early diagnosis, species identification, and adequate antifungal therapy are key elements for treatment of the disease, especially in cases of pulmonary invasive aspergillosis that often advance very rapidly. Incorporating knowledge of the basic biology of Aspergillus species to that of the diseases that they cause is fundamental for further progress in the field.
Assuntos
Aspergillus fumigatus/classificação , Aspergillus fumigatus/patogenicidade , Aspergilose Pulmonar Invasiva/diagnóstico , Aspergilose Pulmonar Invasiva/epidemiologia , Aspergilose Pulmonar Invasiva/terapia , HumanosRESUMO
Chronic granulomatous disease (CGD) patients have recurrent life-threatening bacterial and fungal infections. Olfactomedin 4 (OLFM4) is a neutrophil granule protein that negatively regulates host defense against bacterial infection. The goal of this study was to evaluate the impact of Olfm4 deletion on host defense against Staphylococcus aureus and Aspergillus fumigatus in a murine X-linked gp91phox-deficiency CGD model. We found that intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox and Olfm4 double-deficient mice compared with CGD mice. The activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) and serum levels of IL-1ß, IL-6, IL-12p40, CXCL2, G-CSF, and GM-CSF in Olfm4-deficient as well as gp91phox and Olfm4 double-deficient mice were significantly higher than those in WT and CGD mice after challenge with S. aureus. We did not observe enhanced defense against A. fumigatus in Olfm4-deficient mice using a lung infection model. These results show that Olfm4 deletion can successfully enhance immune defense against S. aureus, but not A. fumigatus, in CGD mice. These data suggest that OLFM4 may be an important target in CGD patients for the augmentation of host defense against bacterial infection.
Assuntos
Glicoproteínas/genética , Doença Granulomatosa Crônica/complicações , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Aspergilose/sangue , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Catepsina C/metabolismo , Citocinas/sangue , Deleção de Genes , Glicoproteínas/metabolismo , Doença Granulomatosa Crônica/sangue , Doença Granulomatosa Crônica/enzimologia , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Neutrófilos/enzimologia , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologiaRESUMO
A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.