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.

The functions of bacterial plasmid-encoded toxin–antitoxin (TA) systems are unambiguous in the sense of controlling cells that fail to inherit a plasmid copy. However, its role in chromosomal copies is contradictory, including stress-response-promoting fitness and antibiotic treatment survival. A hybrid pathogenic Escherichia coli strain may have the ability to colonize distinct host niches, facing contrasting stress environments. Herein, we determined the influence of multiple environmental stress factors on the bacterial growth dynamic and expression profile of previously described TA systems present in the chromosome of a hybrid atypical enteropathogenic and extraintestinal E. coli strain. Genomic analysis revealed 26 TA loci and the presence of five type II TA systems in the chromosome. Among the tested stress conditions, osmotic and acid stress significantly altered the growth dynamics of the hybrid strain, enhancing the necessary time to reach the stationary phase. Using qPCR analyses, 80% of the studied TA systems were differentially expressed in at least one of the tested conditions, either in the log or in the stationary phase. These data indicate that type II TA systems may contribute to the physiology of pathogenic hybrid strains, enabling their adaptation to different milieus.

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.