RESUMEN
Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.
Asunto(s)
Aspergillus fumigatus/patogenicidad , Proteínas Fúngicas , Micosis/microbiología , Factores de Virulencia/genética , Animales , Antígenos Fúngicos/genética , Antígenos Fúngicos/metabolismo , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Encéfalo/microbiología , Encéfalo/patología , Línea Celular , Recuento de Colonia Microbiana , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Silenciamiento del Gen , Riñón/microbiología , Riñón/patología , Macrófagos/microbiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Micosis/mortalidad , Micosis/patología , Bazo/microbiología , Bazo/patología , Tasa de SupervivenciaRESUMEN
Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity of P. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles of P. marneffei PM1 grown at 25 and 37°C. Among â¼11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted in P. marneffei through sequence comparison, did not tend to be overexpressed at 37°C. These results suggest that P. marneffei may take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable to P. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions of P. marneffei genes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes of P. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation in P. marneffei.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Respuesta al Choque Térmico/genética , Penicillium/metabolismo , Transcripción Genética , Genes Fúngicos , Proteínas de Choque Térmico/genética , Calor , Penicillium/genética , Secuencias Repetidas en Tándem , TranscriptomaRESUMEN
Penicillium marneffei is the most important thermal dimorphic, pathogenic fungus endemic in China and Southeast Asia and is particularly important in HIV-positive patients. We report the 28,887,485-bp draft genome sequence of P. marneffei, which contains its complete mitochondrial genome, sexual cycle genes, a high diversity of Mp1p homologues, and polyketide synthase genes.
Asunto(s)
Genoma Fúngico , Penicillium/genética , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.
Asunto(s)
Genoma Fúngico , Penicillium/genética , Penicillium/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Factor de Apareamiento , Datos de Secuencia Molecular , Penicillium/clasificación , Péptidos/química , Péptidos/genética , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Using serum from guinea-pigs immunized with a DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus to screen a cDNA library of A. fumigatus, we cloned a novel immunogenic 57-kDa protein in A. fumigatus. We also cloned its 55-kDa homologue in Penicillium marneffei, which was possibly related to amino acid biosynthesis and metabolism, with homologues present only in the subphylum Pezizomycotina of Ascomycota. The recombinant 55-kDa protein of P. marneffei reacted strongly with guinea-pig serum immunized with P. marneffei and with the sera of patients with P. marneffei infection. A similar approach could be applied to immunogenic protein screening in other microorganisms for serological diagnosis, epidemiological studies and the study of vaccines.
Asunto(s)
Aspergillus fumigatus/genética , Aspergillus fumigatus/inmunología , Proteínas Fúngicas/inmunología , Técnicas Inmunológicas , Micosis/microbiología , Penicillium/inmunología , Penicillium/aislamiento & purificación , Animales , Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/genética , Secuencia de Bases , Southern Blotting , Western Blotting , Trasplante de Médula Ósea , Clonación Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Eliminación de Gen , Genes Fúngicos , Cobayas , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Penicillium/clasificación , Filogenia , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
BACKGROUND: Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. METHODOLOGY/PRINCIPAL FINDINGS: We examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P<0.0001). Seventy percent of mice died 60 days after challenge with MP1 knockdown mutant (P<0.0001). All mice died after challenge with MPLP1 to MPLP13 knockdown mutants, suggesting that only Mp1p plays a significant role in virulence. The mean fungal loads of PM1 and MP1 complemented mutant in the liver, lung, kidney and spleen were significantly higher than those of the MP1 knockout mutant. Similarly, the mean load of PM1 in the liver, lung and spleen were significantly higher than that of the MP1 knockdown mutant. Histopathological studies showed an abundance of yeast in the kidney, spleen, liver and lung with more marked hepatic and splenic necrosis in mice challenged with PM1 compared to MP1 knockout and MP1 knockdown mutants. Likewise, a higher abundance of yeast was observed in the liver and spleen of mice challenged with MP1 complemented mutant compared to MP1 knockout mutant. PM1 and MP1 complemented mutant survived significantly better than MP1 knockout mutant in macrophages at 48 hours (P<0.01) post-infection. The mean fungal counts of Pichia pastoris GS115-MP1 in the liver (P<0.001) and spleen (P<0.05) of mice were significantly higher than those of GS115 at 24 hours post-challenge. CONCLUSIONS/SIGNIFICANCE: Mp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.
Asunto(s)
Macrófagos/microbiología , Glicoproteínas de Membrana/inmunología , Talaromyces/patogenicidad , Factores de Virulencia/inmunología , Factores de Virulencia/aislamiento & purificación , Animales , Antígenos Fúngicos/genética , Antígenos Fúngicos/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Riñón/microbiología , Hígado/microbiología , Hígado/patología , Pulmón/microbiología , Glicoproteínas de Membrana/genética , Ratones , Mutación , Micosis/inmunología , Pichia/crecimiento & desarrollo , Pichia/fisiología , Bazo/microbiología , Bazo/patología , Talaromyces/genética , Talaromyces/crecimiento & desarrollo , Factores de Virulencia/genéticaRESUMEN
Despite the unique phenotypic properties and clinical importance of Penicillium marneffei, the polyketide synthase genes in its genome have never been characterized. Twenty-three putative polyketide synthase genes and two putative polyketide synthase nonribosomal peptide-synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than found in other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one polyketide synthase gene) and Coccidioides immitis (10 polyketide synthase genes). These genes were evenly distributed on the phylogenetic tree with polyketide synthase genes of Aspergillus and other fungi, indicating that the high diversity was not a result of lineage-specific gene expansion through recent gene duplication. The melanin-biosynthesis gene cluster had gene order and orientations identical to those in the Talaromyces stipitatus (a teleomorph of Penicillium emmonsii) genome. Phylogenetically, all six genes of the melanin-biosynthesis gene cluster in P. marneffei were also most closely related to those in T. stipitatus, with high bootstrap supports. The polyketide synthase gene of the melanin-biosynthesis gene cluster (alb1) in P. marneffei was knocked down, which was accompanied by loss of melanin pigment production and reduced ornamentation in conidia. The survival of mice challenged with the alb1 knockdown mutant was significantly better than those challenged with wild-type P. marneffei (P < 0.005). The sterilizing doses of hydrogen peroxide, leading to a 50% reduction in survival of conidia, were 11 min for wild-type P. marneffei and 6 min for the alb1 knockdown mutant of P. marneffei, implying that the melanin-biosynthesis gene cluster contributed to virulence through decreased susceptibility to killing by hydrogen peroxide.
Asunto(s)
Melaninas/biosíntesis , Familia de Multigenes/genética , Penicillium/genética , Sintasas Poliquetidas/genética , Animales , Secuencia de Bases , Desinfectantes/farmacología , Proteínas Fúngicas/genética , Silenciador del Gen , Genes Fúngicos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Micosis/mortalidad , Penicillium/efectos de los fármacos , Penicillium/patogenicidad , Penicillium/efectos de la radiación , Filogenia , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , Esporas Fúngicas/efectos de la radiación , Esporas Fúngicas/ultraestructura , Análisis de Supervivencia , Rayos UltravioletaRESUMEN
A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of Penicillium marneffei, the most important thermal dimorphic fungus causing respiratory, skin, and systemic mycosis in Southeast Asia. The sequences of 11 housekeeping genes were identical among 10 strains of P. marneffei, but those of MP1 and its 13 homologues, a novel superfamily of mannoproteins in the subdivision Pezizomycotina of Ascomycetes, mostly species of Penicillium and Aspergillus, showed significant variations. Therefore, a multilocus sequence typing (MLST) system for P. marneffei was constructed using MP1 (549 bp) and the four of its homologues (MPLP4 [337 bp], MPLP7 [347 bp], MPLP10 [546 bp], and MPLP13 [422 bp]) that showed the greatest variations. Among the 2,201 bp of the five loci, 183 polymorphic sites were observed in 44 strains of P. marneffei. The median number of alleles at each locus was five (range, 5 [MPLP4, MPLP7, and MPLP13] to 15 [MPLP10]). Four of the five genes had nonsynonymous substitution/synonymous substitution (d(n)/d(s)) ratios of >1. A total of 35 different sequence types (STs) were assigned to the 44 P. marneffei isolates, with 28 of the 35 STs identified only once. The discriminatory power was 0.9884. MP1 and its homologues were better than housekeeping genes for MLST in P. marneffei. Due to their more rapid evolutionary rates, lineage-specific genes may be better candidates than housekeeping genes for sequence-based typing, especially in microbes that evolve slowly or have evolved recently.
Asunto(s)
Técnicas de Tipificación Micológica/métodos , Penicillium/clasificación , Adulto , Anciano , Secuencia de Bases , Preescolar , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Penicillium/genética , Análisis de Secuencia de ADNRESUMEN
We cloned the Aspergillus fumigatus mannoprotein 2 (AFMP2) gene, which encodes a novel immunogenic protein (Afmp2p) of the antigenic mannoprotein superfamily, in A. fumigatus. Sequence analysis revealed that Afmp2p has 510 amino acid residues, with a predicted molecular mass of 51.5 kDa. Afmp2p has a putative N-terminal signal peptide, a putative C-terminal glycosylphosphatidylinositol membrane attachment signal sequence, and an upstream GAA cleavage site commonly used for cytoplasmic membrane attachment and implicated in fungal cell wall assembly. Upstream of the GAA cleavage site, Afmp2p contains a 302-amino-acid serine- and threonine-rich region as a site for potential O-glycosylation. Within this serine- and threonine-rich region, 13 repeats of ETSTPCE(T)(n) were observed. Western blot analysis of Afmp2p in A. fumigatus fungal cell lysate and culture supernatant and immunogold staining and electron microscopy showed that Afmp2p is predominantly secreted into the culture supernatant, whereas only minimal amounts can be detected in the cell lysate and cell wall. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against recombinant Afmp2p. The abundance of Afmp2p in secreted form, its minimal cross-reactivity with Afmp1p, and the presence of an antibody response against Afmp2p in patients with A. fumigatus infections suggest that Afmp2p is a good candidate for complementing Afmp1p in serodiagnosis of A. fumigatus infections.
Asunto(s)
Antígenos Fúngicos/genética , Aspergillus fumigatus/genética , Aspergillus fumigatus/inmunología , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Secuencia de Aminoácidos , Antígenos Fúngicos/química , Antígenos Fúngicos/inmunología , Aspergilosis/diagnóstico , Aspergilosis/inmunología , Aspergilosis/microbiología , Aspergillus fumigatus/ultraestructura , Secuencia de Bases , ADN de Hongos/genética , Proteínas Fúngicas/química , Genes Fúngicos , Humanos , Glicoproteínas de Membrana/química , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Secuencias Repetitivas de Aminoácido , Pruebas SerológicasRESUMEN
Acupuncture has been gaining popularity as a form of alternative medicine. In the past, only blood-borne viruses and anecdotal reports of bacterial infections have been associated with acupuncture. We report on four patients with mycobacterial infections complicating acupuncture who were encountered in a 2-year period. All had clinical and/or radiological lesions at acupuncture point- and meridian-specific locations. There was no other history of trauma or other clinical foci of infections, and the chest radiographs were normal. Histological studies of biopsy specimens of all four patients showed changes compatible with chronic inflammation, with granulomatous inflammation present in three patients and acid-fast bacilli present in two. Conventional biochemical tests and whole-cell fatty acid analysis for identification were inconclusive for all four nonpigmented mycobacteria recovered from tissue biopsies. 16S rRNA gene sequencing showed that the strains from two patients were Mycobacterium chelonae and that those from the other two were Mycobacterium nonchromogenicum. Alcohol resistance assay using the quantitative suspension test revealed that all four strains showed prolonged survival in 75% alcohol compared to other skin flora. Mycobacterial infections transmitted by acupuncture are an emerging problem. A high index of suspicion is essential to recognize this clinical syndrome, and strict implementation of proper infection control guidelines for acupuncture is mandatory.
Asunto(s)
2-Propanol/farmacología , Terapia por Acupuntura/efectos adversos , Farmacorresistencia Bacteriana , Mycobacterium chelonae/efectos de los fármacos , Adulto , Anciano , ADN Ribosómico/análisis , Ácidos Grasos/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium/efectos de los fármacos , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Old tuberculosis and bronchiectasis are the two most important causes of chronic structural changes of lungs in our locality. In the absence of radiologically visible mycetoma, the cause of hemoptysis in these two groups of patients is largely unknown. A 17-month prospective study was carried out to compare the prevalence of Aspergillus fumigatus and Aspergillus flavus antibodies in hemoptysis patients with old tuberculosis or bronchiectasis but no radiologically visible mycetoma (cases, n = 38), hemoptysis patients with other diagnosis (control group 1, n = 29), and patients with old tuberculosis or bronchiectasis but no hemoptysis (control group 2, n = 47) by a recently developed sensitive and specific A. fumigatus and A. flavus antibody assay. There were a significantly larger number of patients with antibody against A. fumigatus or A. flavus among the cases than among the patients in control groups 1 and 2 (P < 0.05 in both comparisons). Molds were not recovered from any of the patients. Among the 10 cases with Aspergillus antibody, eight and two had antibody against A. flavus and A. fumigatus, respectively. We conclude that there was an association between the presence of Aspergillus antibodies and hemoptysis in patients with old tuberculosis or bronchiectasis, suggesting that these patients probably had occult infections caused by the corresponding fungi. Development of serological tests against other Aspergillus species as well as other causes of mycetoma will probably increase the detection of occult mold infections in patients with existing parenchymal lung diseases, and treatment of fungal microinvasion may help to alleviate hemoptysis in these patients with bronchiectasis or old tuberculosis who have Aspergillus antibodies.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Aspergilosis/diagnóstico , Aspergillus/inmunología , Hemoptisis/etiología , Anciano , Aspergilosis/complicaciones , Aspergillus/aislamiento & purificación , Aspergillus flavus/aislamiento & purificación , Aspergillus fumigatus/aislamiento & purificación , Bronquiectasia/complicaciones , Carcinoma Broncogénico/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tuberculosis/complicacionesRESUMEN
We have previously reported the cloning and characterization of the MP1 gene in Penicillium marneffei and the AFMP1 gene in Aspergillus fumigatus and their use for serodiagnosis of penicilliosis and aspergilloma and invasive aspergillosis, respectively. In this study, we describe the cloning of the AFLMP1 gene, which encodes the homologous antigenic cell wall protein in Aspergillus flavus, the most common Aspergillus species associated with human disease in our locality and in other Asian countries and the second most common Aspergillus species associated with human disease in Western countries. AFLMP1 codes for a protein, Aflmp1p, of 273 amino acid residues, with a few sequence features that are present in Mp1p and Afmp1p, the homologous antigenic cell wall proteins in P. marneffei and A. fumigatus, respectively, as well as several other cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Aflmp1p antibody was generated with recombinant Aflmp1p protein purified from Escherichia coli to allow further characterization of Aflmp1p. Indirect immunofluorescence analysis indicated that Aflmp1p is present on the surface of the hyphae of A. flavus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. flavus develop a specific antibody response against Aflmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.