Extracellular metalloproteases of pseudomonas aeruginosa and burkholderia cenocepacia as potential immunotherapeutic targets

Abstract

Pseudomonas aeruginosa and Burkholderia cenocepacia are the causative agents of chronic airway infections and a major cause of mortality in cystic fibrosis patients. Despite advances in anti-microbial therapy, the treatment of infections caused by P. aeruginosa and B. cenocepacia remains a challenge. The study of virulence factors produced by the bacteria is being pursued to develop new therapeutic leads. P. aeruginosa produces LasB, an extracellular metalloprotease that is required for maximal virulence. In this study, we cloned and sequenced the zmpA gene encoding for PSCP, an extracellular metalloprotease of B. cenocepacia. The deduced amino acid sequence of PSCP indicates that it has a pre-prometalloenzyme structure, similar to the thermolysinlike metalloproteases. By creating isogenic PSCP mutants, we determined that PSCP is required for the maximal virulence of B. cenocepacia K56-2 in an experimental model of chronic lung infection.

Monoclonal antibodies (MAbs) toward peptide 15, (34 IHGFTEQNSG349) a neutralizing epitope of P. aeruginosa LasB, were produced that reacted with and neutralized the enzymatic activity of LasB and PSCP. The anti-peptide 15 MAb 3123 could be detected in lung homogenates 48 hours after administration to rats infected with P. aeruginosa. Anti-peptide 15 MAb 5201 or MAb 3123 were passively administered to the lungs of rats chronically infected with P. aeruginosa. Quantitative histopathology analysis revealed that animals passively treated with MAb 5201 or 3123 had 51 -78 % less histopathological changes in the lung than control animals. The decrease in histopathological changes could be maintained for at least one week without continued administration of the MAb. These studies suggest that anti-peptide 15 MAbs may be potential therapeutic agents for CF patients infected with P. aeruginosa.

Recombinant single chain variable fragment antibodies (scFvs) were produced using the neutralizing MAbs as templates. The scFvs retained the ability to neutralize LasB, indicating that recombinant Abs may be an option for anti-metalloprotease therapeutics. These studies provide the initial steps toward producing anti-peptide 15 immunotherapeutic agents for use in P. aeruginosa or B. cenocepacia respiratory infections.