Investigating the bactericidal mechanism of anti-LPS antibodies against Pseudomonas aeruginosa serotype O6
Our group previously demonstrated that the S20 antibody, and scFv and Fab fragments derived therefrom, exerts direct bactericidal activity towards Pseudomonas aeruginosa strain O6a, 6d. By binding to the LPS, and more specifically the O-specific antigen (OSA), the S20 antibody was observed to induce severe cell wall damage by a mechanism that remained elusive. It was initially proposed that the S20 was an abzyme that possessed a potent catalytic activity towards strain O6a, 6d. The main goal of this dissertation was to elucidate the unusual bactericidal mechanism of the S20 antibody. The first objective was to determine if this activity is unique and harboured within the S20 antibody sequence per se. We surveyed additional anti-LPS monoclonal antibodies (mAbs) that have been published and were made available to us from the laboratory of Dr. Joseph Lam (University of Guelph). Targeting the OSA of serotype O6 with mAb MF23-1 and its Fab fragment induced direct bactericidal activity against all wild-type O6 strains that have an OSA+ phenotype. Since the S20 and MF23-1 antibodies share little sequence identity in their respective variable domains, one would not anticipate that their bactericidal activity be residing in their sequences per se as would be expected for a catalytic antibody. Instead, we provide the evidence to show that these antibodies are exploiting a vulnerable target on the surface of P. aeruginosa, the OSA of serotype O6. The second objective of this thesis was to study the mechanism of the bactericidal antibodies. Based on our high-resolution AFM images, we propose that the mechanism of action of the two bactericidal antibodies involves the following steps: 1- LPS-binding step; 2- Micellization of the LPS-rich OM; 3- Appearance of membrane pits; 4- Loosening of the OM from the peptidoglycan layer; 5- Sloughing of the OM; 6- Membrane blebbing. Our work has established a novel mechanism by which antibodies can mediate direct antimicrobial immunity without the recruitment of phagocytes or the complement system.