RESUMO
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.
Escherichia coli uropatogênica (UPEC) é a principal causa de infecção do trato urinário (ITU). Entre os fatores de virulência produzidos pela UPEC, destacam- se a alfa hemolisina (HlyA) e o fator necrosante citotóxico 1 (CNF1). Tendo em vista que até 60% das cepas de UPEC produzem HlyA e cerca de 40% expressam CNF1, estas toxinas se tornam bons alvos para o diagnóstico e intervenções terapêuticas. A importância das toxinas HlyA e CNF1 nos casos de ITU causados por UPEC e os enormes desafios na produção de proteínas recombinantes, levou a este trabalho que se propôs a obter diferentes formas estruturais destas toxinas pela metodologia de clonagem e expressão heteróloga. Diferentes formas estruturais destas toxinas de alta massa molecular foram propostas, deste a produção integra a proteínas de baixo peso molecular. As toxinas recombinantes foram analisadas in silico, expressas e purificadas. Dentre as abordagens de clonagem e expressão destacamos para HlyA a estratégia de obtenção do imunógeno intermediário dessa toxina. Já para CNF1, tanto a construção da proteína integra quanto a proteína recombinante utilizando apenas sua porção catalítica. Além disso, analisou-se uma coleção bacteriana de UPEC para definir dados epidemiológicos locais da prevalência dos genes das toxinas e capacidade de causar hemólise e multinucleação. A genes hlyA e cnf1 estavam presentes nos isolados em 36% e 23%, respectivamente e o fenótipo de hemólise ocorreu em 33% e a multinucleação em 23% dos isolados bacterianos.
RESUMO
The role of uropathogenic Escherichia coli (UPEC) in colonization and infection of female patients with anatomical and functional abnormalities of the urinary system is elusive. In this study, the phenotype, genotype and the phylogeny of UPEC strains isolated from the urine of pediatric female patients with cystitis of normal and abnormal urinary tract were determined. Multiplex PCR results demonstrated that 86% of the strains isolated from female patients with normal urinary tract (NUT), belonged to the phylo-groups B2 and D. Their prevalence decreased to 23% in strains isolated from patients with abnormal urinary tract (AUT). More of the isolates from AUT patients produced a biofilm on polystyrene and polyvinyl chloride (PVC), adhered to epithelial cells, and encoded pap and sfa genes than strains isolated from female patients with NUT. In contrast, a higher number of hemolysin-producing strains with serogroups associated with UPEC were isolated from patients with NUT. In summary, the results suggest that cystitis in female patients with NUT is associated with ExPEC, whereas cystitis in female patients with AUT is associated with pathogenic intestinal E. coli strains that have acquired the ability to colonize the bladder.
RESUMO
The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542-723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555-565, aa600-610, and aa674-717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.