Estratégias de clonagem de proteínas de alta massa molecular: α-hemolisina e CNF1 produzidas por Escherichia coli uropatogênica como modelos

Abstract

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infection (UTI). Among the virulence factors produced by UPEC, alpha hemolysin (HlyA) and cytotoxic necrotizing factor 1 (CNF1) stand out. Keeping in mind that up to 60% of UPEC strains produce HlyA and about 40% express CNF1, these toxins become good targets for diagnosis and therapeutic interventions. The importance of HlyA and CNF1 toxins in the cases of UTI caused by UPEC and the enormous challenges in the production of recombinant proteins, led to this work proposal to obtain different structural forms of these toxins through the methodology of cloning and heterologous expression, of integral toxins, low molecular mass and intermediate immunogenic toxins. Different structural forms of these high molecular weight toxins have been proposed, from which the production integrates to low molecular weight proteins. Recombinant toxins were analyzed in silico, expressed and purified. Among the cloning and expression approaches, we highlight for HlyA the strategy to obtain the intermediate immunogen of this toxin. For CNF1, both the construction of the full protein and the recombinant protein using only its catalytic portion. In addition, a bacterial collection of UPEC was analyzed to define local epidemiological data on the prevalence of toxin genes and the ability to cause hemolysis and multinucleation. The hlyA and cnf1 genes were present in the isolates in 36% and 23%, respectively, and the hemolysis phenotype occurred in 33% and the multinucleation in 23% of the bacterial isolates.