RESUMO
The toxin-antitoxin (TA) systems are genetic modules associated with some bacterial processes, such as cell formation persistence, biofilm formation, protection against bacteriophages and responses to stressful environments. In these systems, the toxin is related to the inhibition of physiological processes and the antitoxin provides protection to the cell against the toxin. Five TA type II systems present in the genome of the hybrid strain BA1250 (atypical enteropathogenic E. coli and extraintestinal E. coli strain) were analyzed. The hybrid strain BA1250 can colonize different host niches and can face different stress environments, therefore, through transcription analysis under stress conditions such as nutritional, oxidative, osmotic and acid, the CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM and PasI-PasT were evaluated both in the log and stationary phases of BA1250 strain. We observed significant differences in the expression levels of the CcdB-CcdA, YhaV-PrlF, YoeB-YefM and PasT-PasI systems in the presence of nutritional stress in the stationary phase. In acid stress, an increase in the gene expression of YhaV, YefM and PasT toxins in stationary phase was observed. In contrast, the analysis of the MazF-MazE system did not show any change in expression levels under the stress conditions evaluated. These data indicate that these four TA systems seem to be involved in the stress response of the BA1250 strain, therefore involved in the physiological processes of BA1250.
Os sistemas toxina-antitoxina (TA) são módulos genéticos que estão associados a alguns processos das bactérias, como formação de células persistentes, formação de biofilme, proteção contra bacteriófagos e respostas a ambientes estressantes. Nesses sistemas, a toxina está relacionada à inibição de processos fisiológicos e a antitoxina fornece proteção à célula contra a toxina. Cinco sistemas TA tipo II presentes no genoma da cepa híbrida BA1250 (E. coli enteropatogênica atípica e cepa de E. coli extraintestinal) foram analisados. A cepa híbrida BA1250 pode colonizar diferentes nichos do hospedeiro e pode enfrentar diferentes ambientes de estresse, por tanto, por meio de análises de transcrição sob condições de estresse como nutricional, oxidativo, osmótico e ácido, os sistemas CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM e PasI-PasT foram avaliados tanto na fase log quanto estacionária do cultivo da cepa BA1250. Observamos diferenças significativas nos níveis de expressão dos sistemas CcdB-CcdA, YhaV-PrlF, YoeB-YefM e PasT-PasI na presença de estresse nutricional na fase estacionária. No estresse ácido foi observado um aumento de expressão gênica das toxinas YhaV, YefM e PasT em fase estacionária. Em contraste, a análise do sistema MazF-MazE não apresentou nenhuma alteração nos níveis de expressão nas condições de estresse avaliadas. Esses dados indicam que esses quatro sistemas TA parecem estar envolvidos na resposta ao estresse da cepa BA1250, portanto envolvidos em processos fisiológicos da BA1250.
RESUMO
Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.
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.
