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1.
BMC Infect Dis ; 20(1): 800, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33115429

RESUMO

BACKGROUND: Talaromyces marneffei infection is an important opportunistic infection associated with acquired immune deficiency syndrome (AIDS). However, it is unusual in patients with non-AIDS and other non-immunosuppressed conditions. We report a case of delayed diagnosis of disseminated T. marneffei infection in non-AIDS, non-immunosuppressive and non-endemic conditions. CASE PRESENTATION: We describe a previously healthy 24-year-old man who complained of a 3-month history of intermittent diarrhea and a recent week of uncontrollable high fever. The HIV antibody test was negative. Enhanced abdominal computed tomography (CT) and integrated 18F-2-deoxy-2-fluoro-D-glucose position emission tomography/computed tomography (FDG PET/CT) both suspected malignant lymphoma. However, a large number of yeast-like cells were found in macrophages in cervical lymph node samples by hematoxylin and eosin stain and silver hexamine stain. Subsequent blood culture suggested T. marneffei infection. Metagenomic Next Generation Sequencing (mNGS) results suggested T. marneffei as the dominant pathogen. Unfortunately, the patient continued to develop acute liver failure and died due to adverse events associated with amphotericin B. CONCLUSIONS: Early diagnosis in HIV-negative patients who are otherwise not immunosuppressed and endemic poses a serious challenge. T. marneffei infection is an FDG-avid nonmalignant condition that may lead to false-positive FDG PET/CT scans. Nevertheless, integrated FDG PET/CT is necessary in patients with fever of unknown origin in the early period to perform earlier biopsy for histopathology and culture in highly avid sites and to avoid delays in diagnosis and treatment.


Assuntos
Linfoma/diagnóstico por imagem , Micoses/diagnóstico , Talaromyces/genética , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Anfotericina B/efeitos adversos , Anfotericina B/uso terapêutico , Antifúngicos/efeitos adversos , Antifúngicos/uso terapêutico , China , Diagnóstico Tardio , Diagnóstico Diferencial , Evolução Fatal , Febre , HIV/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/mortalidade , Masculino , Micoses/tratamento farmacológico , Micoses/microbiologia , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Talaromyces/isolamento & purificação , Tomografia Computadorizada por Raios X , Adulto Jovem
2.
Nat Commun ; 11(1): 4864, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978392

RESUMO

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal ß-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.


Assuntos
Tolerância a Medicamentos/fisiologia , Fungos/enzimologia , Fungos/metabolismo , Xilosidases/química , Xilosidases/metabolismo , Biocatálise , Domínio Catalítico , Fungos/efeitos dos fármacos , Glicoconjugados/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese , Especificidade por Substrato , Talaromyces/enzimologia , Talaromyces/genética , Xilosidases/genética
3.
Int J Food Microbiol ; 319: 108491, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31935649

RESUMO

Accurate identification of fungi occurring on agrofood products is the key aspect of any prevention and pest management program, offering valuable information in leading crop health and food safety. Fungal species misidentification can dramatically impact biodiversity assessment, ecological studies, management decisions, and, concerning toxigenic fungi, health risk assessment, since they can produce a wide range of toxic secondary metabolites, referred to as mycotoxins. Since each toxigenic fungal species can have its own mycotoxin profile, a correct species identification, hereby attempted with universal DNA barcoding approach, could have a key role in mycotoxins prevention strategies. Currently, identification of single marker for species resolution in fungi has not been achieved and the analysis of multiple genes is used, with the advantage of an accurate species identification and disadvantage of difficult setting up of PCR-based diagnostic assays. In the present paper, we describe our strategy to set up a DNA-based species identification of fungal species associated with maize ear rot, combining DNA barcoding approach and species-specific primers design for PCR based assays. We have (i) investigated the appropriate molecular marker for species identification, limited to mycobiota possibly occurring on maize, identifying calmodulin gene as single taxonomically informative entity; (ii) designed 17 sets of primers for rapid identification of 14 Fusarium, 10 Aspergillus, 2 Penicillium, and 2 Talaromyces species or species groups, and finally (iii) tested specificity of the 17 set of primers, in combination with 3 additional sets previously developed.


Assuntos
Calmodulina/genética , Fungos/classificação , Fungos/genética , Micotoxinas/análise , Zea mays/microbiologia , Aspergillus/classificação , Aspergillus/genética , Aspergillus/metabolismo , Biodiversidade , Contaminação de Alimentos/análise , Inocuidade dos Alimentos , Fusarium/classificação , Fusarium/genética , Fusarium/metabolismo , Penicillium/classificação , Penicillium/genética , Penicillium/metabolismo , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Talaromyces/classificação , Talaromyces/genética , Talaromyces/metabolismo
5.
Med Mycol ; 58(2): 181-186, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31131856

RESUMO

Talaromyces (Penicillium) marneffei is an emerging pathogen that causes significant morbidity and mortality in immunocompromised patients in endemic regions such as southeast Asia. The diagnosis of disseminated T. marneffei infection remains challenging in clinical practice. In the study, a well-validated real-time quantitative polymerase chain reaction (qPCR) target region of ITS1-5.8S-ITS2 and a Platelia galactomannan (GM) assay were compared for their diagnostic performance using serum samples from patients with or without human immunodeficiency virus (HIV). The results showed that this novel qPCR method is highly sensitive and specific for T. marneffei DNA detection in serum samples, and the limit of detection and species-specificity of qPCR were five copies of DNA and 100%, respectively. For detection in serum samples from 36 talaromycosis patients, the sensitivity of qPCR was 86.11% (31/36), including 20/20 (100%) patients with fungemia and 11/16 (68.75%) patients without fungemia. For the GM assay, the sensitivity was 80.56% (29/36) when the GM optical density cutoff index was ≥0.5, including 19/20 (95%) patients with fungemia and 10/16 (62.5%) patients without fungemia. These results indicate that the novel qPCR and GM assays can be used as a valuable tool in the diagnosis of T. marneffei infection. Serum samples are convenient hematological specimens for T. marneffei DNA quantification. Combining the GM assay and qPCR is more scientific and appropriate for diagnosing T. marneffei infection in endemic areas.


Assuntos
DNA Fúngico/sangue , Fungemia/diagnóstico , Mananas/sangue , Reação em Cadeia da Polimerase em Tempo Real , Talaromyces/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Pré-Escolar , China , DNA Intergênico/sangue , Feminino , Fungemia/microbiologia , Infecções por HIV/microbiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Talaromyces/patogenicidade , Adulto Jovem
6.
Folia Microbiol (Praha) ; 65(2): 323-328, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31250361

RESUMO

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 µg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.


Assuntos
Endófitos/crescimento & desenvolvimento , Tabebuia/microbiologia , Talaromyces/crescimento & desenvolvimento , Talaromyces/metabolismo , Tripanossomicidas/metabolismo , Técnicas de Cocultura , Endófitos/química , Endófitos/genética , Phanerochaete/química , Phanerochaete/genética , Phanerochaete/crescimento & desenvolvimento , Phanerochaete/metabolismo , Folhas de Planta/microbiologia , Talaromyces/química , Talaromyces/genética , Terpenos/análise , Terpenos/metabolismo , Terpenos/farmacologia , Tripanossomicidas/análise , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento
7.
Med Mycol ; 58(3): 351-361, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290549

RESUMO

Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.


Assuntos
Genes Fúngicos , Talaromyces/genética , Talaromyces/patogenicidade , Fatores de Virulência/genética , Animais , Meios de Cultura/química , Feminino , Deleção de Genes , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células RAW 264.7 , Organismos Livres de Patógenos Específicos , Talaromyces/crescimento & desenvolvimento , Virulência
8.
Fungal Biol ; 123(10): 745-762, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31542192

RESUMO

A sample of isolates from Talaromyces pinophilus (55 isolates) and closely related species (76 isolates) was sequenced at four loci, the data were analyzed using maximum likelihood analysis and the GCPSR. The isolates were subjected to growth studies on the recommended media for description of Talaromyces species. On the basis of the combined data, five new species were segregated out of T. pinophilus and placed in newly described species. The T. pinophilus species complex contains ten species. The three other new species, Talaromyces argentinensis, T. californicus and T. louisianensis were not a part of the T. pinophilus species complex but occurred in Talaromyces sect. Talaromyces. T. argentinensis produces a teleomorphic state and is phylogenetically and morphologically distinct from other Talaromyces species.


Assuntos
Talaromyces/classificação , Filogenia , Microbiologia do Solo , Talaromyces/genética , Talaromyces/crescimento & desenvolvimento , Talaromyces/isolamento & purificação
9.
Appl Environ Microbiol ; 85(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31492671

RESUMO

Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (X n U4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing X n U4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system.IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a ß-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.


Assuntos
Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Talaromyces/enzimologia , Xilanos/metabolismo , Domínio Catalítico , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Glucuronatos/metabolismo , Hidrólise , Cinética , Oligossacarídeos/metabolismo , Especificidade por Substrato , Talaromyces/genética , Madeira/microbiologia , Xilose/metabolismo
10.
Emerg Infect Dis ; 25(9): 1765-1768, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31441765

RESUMO

Talaromyces marneffei and other Talaromyces species can cause opportunistic invasive fungal infections. We characterized clinical Talaromyces isolates from patients in California, USA, a non-Talaromyces-endemic area, by a multiphasic approach, including multigene phylogeny, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and phenotypic methods. We identified 10 potentially pathogenic Talaromyces isolates, 2 T. marneffei.


Assuntos
Micoses/epidemiologia , Talaromyces/isolamento & purificação , Adulto , California/epidemiologia , Humanos , Masculino , Micoses/microbiologia , Filogenia , Talaromyces/genética
11.
World J Microbiol Biotechnol ; 35(6): 84, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134444

RESUMO

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.


Assuntos
Dosagem de Genes , Pectinas/metabolismo , Pichia/genética , Poligalacturonase/biossíntese , Poligalacturonase/genética , Talaromyces/enzimologia , Talaromyces/genética , Reatores Biológicos , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Hidrólise , Pichia/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/genética , Temperatura , Fatores de Tempo
12.
Fungal Genet Biol ; 130: 54-61, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075360

RESUMO

Talaromyces marneffei (T. marneffei) is a medically important opportunistic dimorphic fungus that infects both humans and bamboo rats. However, the mechanisms of transmission and pathogenicity of T. marneffei are poorly understood. In our study, we combined Illumina and PacBio sequencing technologies to sequence and assemble a complete genome of T. marneffei. To elucidate the transmission route and source, we sequenced three additional T. marneffei isolates using Illumina sequencing technology. Variations among isolates were used to develop a multilocus sequence typing (MLST) system comprising five housekeeping genes that can be used to discriminate between isolates derived from different sources. Our analysis revealed that human and bamboo rat share identical genotypes in these five loci. Thus, we hypothesized that T. marneffei is transmitted to humans through inhalation of spores in the surrounding environment into the lungs and that the bamboo rat can serve as an important natural reservoir for pathogens. Furthermore, we also identified temperature-dependent polyketide synthases, non-ribosomal peptide synthetases and secreted proteins as putative pathogenicity-related factors. In addition, we identified antifungal drug targets that can be investigated in future studies to elucidate the mechanisms underlying drug resistance. In summary, our study presents the basic features of the T. marneffei genome and provides insights into the transmission and pathogenicity of T. marneffei, which warrant fundamental experimental research.


Assuntos
Genoma Fúngico/genética , Genômica , Talaromyces/genética , Fatores de Virulência/genética , Animais , Antifúngicos , DNA Fúngico , Proteínas Fúngicas/genética , Genes Essenciais/genética , Genótipo , Humanos , Tipagem de Sequências Multilocus , Peptídeo Sintases/genética , Filogenia , Policetídeo Sintases/genética , Ratos , Metabolismo Secundário/genética , Talaromyces/efeitos dos fármacos , Talaromyces/isolamento & purificação , Virulência , Sequenciamento Completo do Genoma
13.
Appl Environ Microbiol ; 85(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31003983

RESUMO

In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. ß-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilose/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cinética , Especificidade por Substrato , Talaromyces/genética , Xilanos/metabolismo
14.
Prep Biochem Biotechnol ; 49(6): 606-615, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929565

RESUMO

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m-3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.


Assuntos
Dextranase/biossíntese , Pichia/genética , Proteínas Recombinantes/biossíntese , Oxirredutases do Álcool/genética , Biomassa , Reatores Biológicos , Dextranase/genética , Fermentação , Proteínas Fúngicas/análise , Glicerol/análise , Glicerol/metabolismo , Engenharia Metabólica/métodos , Metanol/análise , Metanol/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Talaromyces/enzimologia , Talaromyces/genética
15.
Virulence ; 10(1): 277-291, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30880596

RESUMO

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.


Assuntos
Ácido Aspártico Proteases/genética , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Talaromyces/crescimento & desenvolvimento , Talaromyces/genética , Animais , Evolução Molecular , Proteínas Fúngicas/genética , Humanos , Camundongos , Células THP-1 , Talaromyces/patogenicidade
16.
Protein Eng Des Sel ; 32(1): 33-40, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30715529

RESUMO

Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.


Assuntos
Celulose 1,4-beta-Celobiosidase/química , Proteínas Fúngicas/química , Temperatura Alta , Mutação , Talaromyces/enzimologia , Celulose 1,4-beta-Celobiosidase/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Estrutura Secundária de Proteína , Talaromyces/genética
17.
Mycoses ; 62(5): 450-457, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30597630

RESUMO

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong-Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV-negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T. marneffei by MALDI-TOF MS using Bruker database expanded with in-house generated T. marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five-locus sequence revealed that the seven strains were scattered amongst other T. marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI-TOF MS is useful for rapid identification. Highly diverse T. marneffei sequence types were observed.


Assuntos
Antifúngicos/farmacologia , Técnicas Microbiológicas/métodos , Micoses/epidemiologia , Micoses/patologia , Talaromyces/isolamento & purificação , Adulto , Idoso , Feminino , Genótipo , Hong Kong , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Técnicas de Tipagem Micológica , Micoses/diagnóstico , Micoses/microbiologia , Estudos Retrospectivos , Fatores de Risco , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise de Sobrevida , Talaromyces/classificação , Talaromyces/efeitos dos fármacos , Talaromyces/genética
18.
Microb Cell Fact ; 18(1): 14, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30691469

RESUMO

BACKGROUND: Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production. RESULTS: By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR+ versus ΔxlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts. CONCLUSIONS: The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.


Assuntos
Carbono/metabolismo , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Talaromyces/enzimologia , Transativadores/genética , Transcrição Genética , Biomassa , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Polissacarídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Talaromyces/genética , Fatores de Transcrição/genética
19.
Lett Appl Microbiol ; 68(5): 415-422, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30636057

RESUMO

Talaromyces species are typical fungi capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species-specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species-specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100 pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat-resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species-specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The heat-resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time-consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.


Assuntos
DNA Fúngico/genética , Proteínas Fúngicas/genética , Reação em Cadeia da Polimerase/métodos , Talaromyces/genética , Mirtilos Azuis (Planta)/microbiologia , Microbiologia de Alimentos , Especificidade da Espécie , Esporos Fúngicos/química , Talaromyces/classificação , Talaromyces/isolamento & purificação
20.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670555

RESUMO

Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.


Assuntos
Ácido Araquidônico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Micoses/microbiologia , Talaromyces/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Ácido Araquidônico/química , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas , Micoses/genética , Micoses/imunologia , Domínios Proteicos , Talaromyces/química , Talaromyces/genética , Fatores de Virulência/genética
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