Diversidade, clonagem e caracterização de nucleases extracelulares de Aeromonas spp.

Abstract

Aeromonas spp. are Gram-negative bacteria generally associated to aquatic ecosystems. These bacterial are considered opportunistic pathogens of various animals, including man. Some species of this genus can secrete a wide variety of extracellular enzymes, such as, β-lactamases, lipases, amylases, proteases, nucleases, among others. Nucleases can play multiple roles related to metabolism, protection against exogenous genetic material and, in some cases, virulence factor. In this context, the aim of this study was to evaluate the differences in extracellular DNases activities and electrophoretic profiles between species and isolates of Aeromonas spp., cloning and heterologous expression of nucleases, effect of physical/chemical factors on the enzyme activity, evaluation of nuc, dns and aha3441 expression, and bioinformatics analysis of DNases Dns and Aha3441. The results showed a wide variation in intra- and interspecific DNases extracellular activity, without significant difference between clinical and environmental isolates. DNase zymograms of Aeromonas spp. isolates, using DNA as a substrate, allowed the identification of different DNase activity profiles. A total of six bands of apparent molecular mass ranging between 22 and 119 KDa were identified. Bands of 22 and 85 KDa were the most frequent among Aeromonas spp. isolates analyzed. The Dnases secreted by Aeromonas hydrophila IBAer119 are affected by the action of proteases produced by the isolate itself. The evaluation of dns, aha3441 and nuc gene expression of A. hydrophila IBAer119 allowed observing high transcription rates for dns and aha3441 genes, which coincide with the peak of extracellular DNase activity. Extracellular DNases identified in isolated A. hydrophila IBAer119 do not play preferably a nutritional role, indicating that its function can be related to protection against exogenous DNA or virulence. The enzymes Dns and Aha3441 from A. hydrophila IBAer119 were cloned and heterologously expressed in Escherichia coli DH5α. Both DNases uses Mg as a cofactor and are inhibited by EDTA and SDS. Experimental data indicates that Dns and Aha3441 have maximal activity at pHs between 7.0 and 9.0, and temperatures between 37°C and 60°C, for Dns, and between 37°C and 50°C, for Aha3441. Both enzymes exhibited high thermal stability when kept at 40°C for 50 minutes. Dns and Aha3441 showed ability to degrade RNA and DNA characterizing them as sugar non-specific nucleases. The sequence analysis of the protein identified the presence of a signal peptide with 20aa, for Dns enzyme, and 23aa, for Aha3441 enzyme, indicating that these enzymes are secreted by secII system. Moreover, the bioinformatics evaluation of the Dns allowed identifying an Endonuclease I superfamily domain, and with high similarity with DNases of the others bacterial genera. DNase Aha3441 has an EEP superfamily domain and OB subfamily folds, similar to those observed in another Aeromonas DNase called Nuc. The threedimensional model of Dns indicates the presence a catalytic site with ββα-metal topology, typical of other endonucleases. The three-dimensional model of Aha3441 showed high conformational similarity with the D chain of the BP DNase I (bovine pancreatic DNase). In addition, the metal binding, the phosphate binding, and the catalytic sites of Aha3441 showed high degree of conservation when compared with homologs of other bacteria, and even BP DNase I, indicating that Aha3441 shares the same mechanism of action.