Identification of proteins in Sporothrixschenckiisensu stricto in response to oxidative stress induced by hydrogen peroxide / Identificación de proteínas en Sporothrix schenckii sensu stricto en respuesta al estrés oxidativo inducido por peróxido de hidrógeno

Background: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. In order to colonize the host, the pathogen must neutralize the reactive oxygen species produced by the phagocytic cells during the respiratory burst. Little is known about these mechanisms in S. schenckii. Aims: To identify the proteins differentially expressed after the exposure of S. schenckiisensu stricto to different concentrations of H2O2. Methods: Yeast cells of S. schenckiisensu stricto were exposed to increasing concentrations of H2O2. Proteins differentially expressed in response to oxidative stress were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-MS/MS. RT-PCR assays were performed to evaluate the transcription of genes of the identified proteins. Results: Concentrations of H2O2 as high as 800 mM allowed cell growth, and 200 mM and 400mM were selected for comparative analysis by 2D-PAGE. This analysis revealed at least five differentially expressed proteins, which were identified as heat shock 70 kDa protein (Hsp70), chaperonin GroEL, elongation factor 1-β (EF1-β), a hypothetical protein, and mitochondrial peroxiredoxin (Prx1). RT-PCR revealed that the transcription of the genes coding for some of these proteins are differentially regulated. Conclusions: Based on these results, it is proposed that these proteins may be involved in the resistance of S. schenckii to oxidative stress, and play an important role in the fungus survival in the host

Antecedentes: La esporotricosis es una infección fúngica causada por el complejo Sporothrix schenckii. Para colonizar al huésped, los patógenos deben neutralizar las especies reactivas de oxígeno producidas por las células fagocíticas durante el estallido respiratorio. Poco se conoce sobre este mecanismo en S. schenckii. Objetivos: Identificar proteínas diferencialmente expresadas durante la exposición de S. schenckiisensu stricto a diferentes concentraciones de H2O2. Métodos: Levaduras de S. schenckiisensu stricto fueron expuestas a concentraciones crecientes de H2O2. Las proteínas diferencialmente expresadas en respuesta el estrés oxidativo fueron analizadas mediante electroforesis en geles de poliacrilamida en doble dimensión (2D-PAGE) e identificadas por MALDI-MS/MS. Se realizaron ensayos de RT-PCR para evaluar la transcripción de genes de las proteínas identificadas. Resultados: Concentraciones altas de H2O2 (800 mM) permitieron el crecimiento celular, y se seleccionaron las concentraciones de 200 y 400 mM para el análisis comparativo mediante 2D-PAGE. Este análisis reveló al menos cinco proteínas diferencialmente expresadas, identificadas como proteína de choque térmico de 70 kDa (Hsp70), chaperonina GroEL, factor de alargamiento 1-β (EF1-β), una proteína hipotética y peroxirredoxina mitocondrial (Prx1). La RT-PCR reveló que la transcripción de los genes que codifican para algunas de estas proteínas se regula diferencialmente. Conclusiones: Con estos resultados pensamos que estas proteínas podrían estar involucradas en la resistencia de S. schenckiisensu stricto al estrés oxidativo y jugar un papel importante en la supervivencia del hongo en el huésped

Identification of proteins in Sporothrixschenckiisensu stricto in response to oxidative stress induced by hydrogen peroxide.

BACKGROUND: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. In order to colonize the host, the pathogen must neutralize the reactive oxygen species produced by the phagocytic cells during the respiratory burst. Little is known about these mechanisms in S. schenckii. AIMS: To identify the proteins differentially expressed after the exposure of S. schenckiisensu stricto to different concentrations of H2O2. METHODS: Yeast cells of S. schenckiisensu stricto were exposed to increasing concentrations of H2O2. Proteins differentially expressed in response to oxidative stress were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-MS/MS. RT-PCR assays were performed to evaluate the transcription of genes of the identified proteins. RESULTS: Concentrations of H2O2 as high as 800mM allowed cell growth, and 200mM and 400mM were selected for comparative analysis by 2D-PAGE. This analysis revealed at least five differentially expressed proteins, which were identified as heat shock 70kDa protein (Hsp70), chaperonin GroEL, elongation factor 1-ß (EF1-ß), a hypothetical protein, and mitochondrial peroxiredoxin (Prx1). RT-PCR revealed that the transcription of the genes coding for some of these proteins are differentially regulated. CONCLUSIONS: Based on these results, it is proposed that these proteins may be involved in the resistance of S. schenckii to oxidative stress, and play an important role in the fungus survival in the host.

Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride.

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.

The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence.

Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.