Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum

Abstract

Fungal infections have been increasing significantly throughout the world due to the incidence of serious infectious diseases that primarily weaken immunocompromised patients. Due to high resistance to antifungal drugs in use, new molecules are becoming the target of researches and in this class are so-called antifungal peptides. These studies are being carried out due to the need to develop new active molecules against pathogenic fungi, since these are becoming increasingly resistant to existing drugs and increasing the number of immunocompromised hosts. This study investigated the presence of the antifungal peptide in Penicillium crustosum using as reference the homologous sequence of the PgAFP gene of Penicillium chrysogenum. Applying molecular biology techniques, the homologous PgAFP gene of P. crustosum was obtained and sequenced, as well as the peptide was isolated from the fungus growth in stressful liquid culture medium for expression of the peptide. The gene was amplified by the conventional PCR technique, in which specific oligonucleotides (primers) were designed for the PgAFP gene. The complete PcAFP gene sequence of 405 bp genomic fragment was obtained with primers designed upstream and downstream of the PgAFP gene. The genomic sequence was compared to sequences deposited in the NCBI database with the Blastn tool and the gene showed 96.30% identity to the P. chrysogenum PgAFP gene. Comparison of the complete PcAFP gene sequence with the PgAFP gene revealed a 279 bp region encoding the antifungal peptide. This sequence has been translated into its amino acid sequence and was compared to the PgAFP sequence of P. chrysogenum peptide model demonstrating 98.21% homology with this peptide. The PcAFP modeling revealed the conserved signal peptide of the antifungal family of proteins, three-dimensional conserved structure with 3 disulfide bonds and -core conserved domain found in the PAFs. The peptide was analyzed with the pI / Mw prediction tool, in which it obtained 6.48 kDa and pI 8.83. The extract obtained from the AFPIM culture medium supernatant showed a 6.9 kDa peptide on Tricine SDS-PAGE 16% polyacrylamide gel, however, not yet conclusive for the P. crustosum peptide. The extracellular crude extract from the liquid culture was tested for antimicrobial activity against bacteria and yeast. In the broth microdilution assay the microorganisms were exposed to different dilutions of the extracellular extract containing the peptide, where activity against Staphylococcus aureus and Candida albicans was obtained. In conclusion, the isolation of this peptide in P. crustosum will contribute with the characterization of a new antifungal peptide with potential application, besides increasing the knowledge about the structure and function of these bioactive molecules. Key words: AFP gene, Antifungal Peptides, Penicillium crustosum