Proteomics Characterization of Outer Membrane Vesicles from the Extraintestinal Pathogenic Escherichia coli ΔtolR IHE3034 Mutant

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 ⌬tolR 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. Molecular & Cellular Proteomics 7:473-485, 2008.

Production of Outer Membrane Vesicles and Outer Membrane Tubes by Francisella novicida

“…Approximately 16% of the vesicle-associated proteins were previously shown to be OM associated in Francisella spp. (see Table S4 in the supplemental material) (52,53), and 20% have homologs that are OMV-associated in other bacteria (see Table S5 in the supplemental material) (48,54,55). Consistent with the derivation of the vesicles from the OM, OM-associated proteins are prominent among the most abundant vesicle-associated proteins (comprising ϳ15% of NSAF values) and include the major Francisella antigens and T cell epitopes FopA, FopB, and LpnA (Table 1) (53,56).…”

Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

“…Importantly, OM vesicles are not a product of cell death since they contain newly synthesized proteins and are produced without concomitant bacterial lysis (96,99,144). Several OM vesicle proteomes have been evaluated recently, and all were determined to be enriched in envelope components, although some cytosolic and inner membrane proteins were also present in these preparations (10,40,84,86,87,124,141).…”

“…NarE is a putatively vesicle-associated protein of Neisseria meningitidis vesicles that exhibits ADP-ribosyltransferase and NAD-glycohydrolase activities typically associated with toxicity (93). Toxic vesicles produced by extraintestinal E. coli (ExPEC) include a hemolysin; an RTX toxin, which becomes surface bound; as well as cytolethal distending toxin, which has a lipid binding domain to bind the OM (6,10).…”

Exploiting Antigenic Diversity for Vaccine Design

“…"Reverse vaccinology" is a typical example of this, validated in different human pathogens (26,27). The fact that the majority of antigens eliciting antibody-mediated protective immunity are surface-exposed proteins and secreted toxins have also promoted a number of proteomic strategies that allow the precise characterization, both in qualitative and quantitative terms, of membrane-associated and secreted proteins (28,29). However, it remains intellectually challenging to understand which properties make these few proteins protec- tive.…”