Abstract
Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.
Highlights
Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia
With the aim of better understanding the membrane protein organization of E. coli and of identifying proteins to be included in vaccine formulations capable of preventing Extraintestinal pathogenic Escherichia coli (ExPEC) infections, in this study we describe the inactivation of the tolR gene in the pathogenic E. coli strain IHE3034 and the detailed characterization of the protein content of the purified outer membrane vesicles (OMVs)
In Gram-negative bacteria, several protocols based on detergents or carbonate extraction have been developed for the analysis of outer membrane proteins, but none of them are fully satisfactory, and in general, poor enrichment of membrane proteins and frequent contamination by cytosolic proteins occur [62, 63]
Summary
Construction of ⌬tolR IHE3034 Deletion Mutant—The ⌬tolR mutant was produced by replacing tolR coding sequence with a kanamycin resistance (kmr) cassette [37] To this aim, we used a three-step PCR protocol to fuse the tolR upstream and downstream regions to the kmr gene. The linear fragment (1 g), in which the kmr gene was flanked by the tolR upstream and downstream regions, was used to transform the recombination-prone IHE3034 E. coli strain (50 l at 1010 cells/ml, made electrocompetent by three washing steps in ice-cold 10% glycerol), and ⌬tolR mutants were selected by plating transformed bacteria on Luria-Bertani (LB) plates containing 25 g/ml kanamycin. The pellet was resuspended in 30 l of 200 mM Tris-HCl, pH 8.8, whereas the proteins from the ultracentrifuged supernatant and the filtered culture supernatant were precipitated by adding TCA and deoxycholate at a final concentration of 10 and 0.04%, respectively. Colorimetric staining was performed, after washing the membranes, with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce.) as described by the manufacturer
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