Identificação de genes associados a produção de ocratoxina A em Aspergillus westerdijkiae

AUTOR(ES)

Daniele Sartori

DATA DE PUBLICAÇÃO

2009

RESUMO

Mycotoxins, secondary metabolites of fungi, are toxic compounds found in great variety of foods, and when ingested can be very hazardous to animal and human health. Among the diverse mycotoxins, special interest has been given to the ochratoxin A (OA), produced by some species of Aspergillus and Penicillium. This mycotoxin is frequently found mainly in food such as grains and grapes, and when ingested and accumulated they can cause nephotoxicity, hepatotoxicity, immunossupressive and carcinogenic effects. Ochratoxins are formed from cyclic pentaketides, derived from dihydroisocoumarin linked to L-phenylalanine, but information on their biosynthesis and regulation are still limited. The objective of this study was to identify genes and proteins with differential expression between OA producing and non-producing strains, using the techniques Representational Differential Analysis, Proteomic Analysis and Real Time PCR. Initially, differentially expressed genic fragments (400 bp approximately) were obtained between OA producing (UEL91) and non-producing strains (ITAL163) grown in the permissive medium (YES). Approximately 260 transcripts were obtained and three oxidases denoted P450-AL, P450-PH and OXI-1 were of special interest. The oxidases P450-AL and OXI-1 showed a higher differential expression (32 and 20-fold respectively), and this result was confirmed through Real Time PCR. Due to this fact, both oxidases genes were analyzed for the association between the amount of OA produced and transcripts expressed in permissive (YES), semi- permissive (CY) and restrictive (EM) media for the production of OA. Association was found between the level of both gene transcripts and the amount of OA, although in different magnitudes. In a second study, it was used a mutant strain of Aspergillus westerdijkiae (ITAL142-T10) with reduced OA production. This mutant has an interrupted gene by a T-DNA integration, similar to the locus An09g05800 of Aspergillus niger which codes for a putative transcription factor PHD (Rum1), involved in chromatin-mediated regulatory events. The mutant strain (ITAL142-T10) was used for differential proteomic analysis with the wild type strain (ITAL142) grown in permissive medium for OA production, with the objective of identifying possible proteins under regulation of the transcription factor PHD. Among the 23 proteins with higher levels of expression in the wild type strain, proteins of stress response (36.4%) and the regulatory subunit 26S of the proteasome (9.1%) were the ones with highest frequency. Being usual the sharing of regulatory genes involved in mycotoxin biosynthesis and with the process of fungi sporulation, an analysis of conidia (production and morphological characteristics) was carried in ITAL142 and ITAL142-T10 in MEA and YES media. No alteration was found regarding conidiation in MEA, but a greater number of conidia were observed in YES medium. Then, the proteins, 14-3-3 and the regulatory subunit 26S of the proteasome were transcriptionally analyzed by Real Time PCR. Both transcripts showed higher relative expression in ITAL142 compared to ITAL142-T10, confirming the data of differential expression observed by the proteomic analysis. Thus, both studies brought contributions concerning the elucidation of genes associated with OA biosynthesis.

ASSUNTO(S)

microbiologia aspergilos ocratoxinas microbiology aspergillus

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