Genome-Wide Study of Pseudomonas aeruginosa Outer Membrane Protein Immunogenicity Using Self-Assembling Protein Microarrays

Abstract

Pseudomonas aeruginosa is responsible for potentially life-threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. P. aeruginosa infection is notable for the appearance of a humoral response to some known antigens, such as flagellin C, elastase, alkaline protease, and others. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. Here, we report a comprehensive study of all 262 outer membrane and exported P. aeruginosa PAO1 proteins by a modified protein microarray methodology called the nucleic acid-programmable protein array. From this study, it was possible to identify 12 proteins that trigger an adaptive immune response in cystic fibrosis and acutely infected patients, providing valuable information about which bacterial proteins are actually recognized by the immune system in vivo during the natural course of infection. The differential detections of these proteins in patients and controls proved to be statistically significant (P < 0.01). The study provides a list of potential candidates for the improvement of serological diagnostics and the development of vaccines.

Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing

“…Increased fitness was not specific for Tn insertions into genes encoding for all outer membrane proteins (OMPs) although, as among the Tn insertions in the 262 genes predicted to encode for OMPs (17,18), only 68 were able to colonize the cecum (Fig. 2A).…”

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

“…In this study, we screened a P. aeruginosa protein library (15) and identified OprL, PopB, and FpvA as Th17-stimulating protein antigens. We further evaluated PopB, as it is a highly conserved, known virulence factor, and found that PopB possesses strong Th epitope(s) as well as one or more nonprotective B-cell epitopes.…”

“…Moreover, PopB has been reported to elicit a humoral immune response in patients with cystic fibrosis (CF) with P. aeruginosa lung infection (15,31), indicating its immunogenicity during infection. We did not assess the vaccine potential of FpvA due to the recently described high genetic variation of the fpvA gene, resulting in extensive sequence polymorphism of this protein when expressed by different P. aeruginosa strains (32,33).…”

“…The construction of the P. aeruginosa outer membrane and secreted protein library followed previously described methods (15).…”

“…We used a library of P. aeruginosa outer membrane and secreted proteins identified by bioinformatics, with their respective genes cloned into expression vectors (15). The proteins were produced with an in vitro transcription and translation system and used to stimulate splenocytes from mice immunized with a live-attenuated P. aeruginosa vaccine strain.…”