Immunization with Pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of severe nosocomial and community acquired infections, these infections are major health problems for cystic fibrosis patients and immune-compromised individuals. The emergence of multidrug-resistant isolates highlights the need to develop alternative strategies for treatment of P. aeruginosa infections. Outer membrane vesicles (OMVs) are spherical nanometer-sized proteolipids that are secreted from numerous of pathogenic Gram-negative bacteria, and a number of studies have confirmed the protective efficacy for use of OMVs as candidate vaccines. In this study, OMVs from P. aeruginosa (PA_OMVs) were isolated, formulated with aluminum phosphate adjuvant and used as a vaccine in a mouse model of acute lung infection. The results confirmed that active immunization with PA_OMVs was able to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa. Cytokines assay validated that immunization with PA_OMVs was efficient to induce a mixed cellular immune response in mice. Further, high level of specific antibodies was detected in mice immunized with PA_OMVs, and results from opsonophagocytic killing assay and passive immunization suggested that humoral immune response may be critical for PA_OMVs mediated protection. These findings demonstrated that PA_OMVs may be served as a novel candidate vaccine for the prevention of P. aeruginosa infection.

Identification of BamC on the Surface of E. coli.

In order to relate the structural architecture of the BAM complex to its function in outer membrane protein assembly, the arrangement of each component within the complex is vital. This chapter explores the structure and topology of BamC, using a range of biochemical techniques to probe the topology and surface exposure.

Crystallographic analysis of the C-terminal domain of the Escherichia coli lipoprotein BamC.

In Gram-negative bacteria, the BAM complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one ß-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD and BamE. Here, the crystal structure of the C-terminal domain of E. coli BamC (BamC(C): Ala224-Ser343) refined to 1.5 Å resolution in space group H3 is reported. BamC(C) consists of a six-stranded antiparallel ß-sheet, three α-helices and one 3(10)-helix. Sequence and surface analysis reveals that most of the conserved residues within BamC(C) are localized to form a continuous negatively charged groove that is involved in a major crystalline lattice contact in which a helix from a neighbouring BamC(C) binds against this surface. This interaction is topologically and architecturally similar to those seen in the substrate-binding grooves of other proteins with BamC-like folds. Taken together, these results suggest that an identified surface on the C-terminal domain of BamC may serve as an important protein-binding surface for interaction with other BAM-complex components or substrates.

Identification and characterization of oxylipid-protein and peptide conjugates by mass spectrometry.

The modification of proteins by reactive products of lipid peroxidation is associated with a large number of diseases and biological aging; thus, methods that enable the characterization of oxylipid-protein and/or peptide conjugates are highly in demand. This unit outlines a chemical labeling approach to identifying and characterizing proteins modified by lipid peroxidation products. It also outlines two approaches for mass spectrometry-based identification and detailed characterization of oxylipid conjugates. The first combines chemical labeling of oxylipid-protein conjugates using an aldehyde-specific biotinylation reagent, electrophoretic separation, and mass spectrometry-based identification of the biotinylated proteins. In the second approach, protein extracts are treated with the aldehyde-specific reagent, proteolyzed using trypsin, and the biotinylated peptides are enriched using immobilized monomeric avidin. The enriched peptide fractions are submitted to tandem mass spectrometry for determining the peptide sequence information, site of the modification, and chemical nature of the oxylipid.