LAUSR

Development of alternative antibiotic approaches is urgently required with the emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa) strains. Pyocins are proteic antibiotics produced by Pa for intraspecies competition for resources. Due to its potency and specificity, these molecules may become ideal candidates as antibiotics for the treatment of Pa infections. Previous reports revealed that H2O2 generated by neutrophils during bacterial infection induces the expression of pyocin genes, indicating a central role of these molecules in host-pathogen interactions. To better understand how pyocins can be used as a potential antimicrobial agent, we characterized a newly identified pyocin S8. Recombinant pyocin S8 displayed potent killing activity against at least 16 MDR isolates, including carbapenemase (SPM-1, GIM-1, VIM-1, IMP-1 and GES-5)-producing Pa strains. We are investigating the role of H2O2 in inducing pyocin gene expression by using a chromosomal promoter S8 fused to a -lux reporter. However, preliminary data indicated that exposure the MDR Pa strains to 1 mM H2O2 for a period of 20 min before the pyocin addition did not improve its killing activity. Next, we determined the crystal structure of C-terminal cytotoxic domain of a mutant E736A pyocin S8 in complex with its immunity protein (S8 DNase E736A-ImS8) at 1.4 A resolution. The Glu736 localizes in DNase active site composed for 32 amino acid H-N-H (His-Asn-His) motif, which coordinates a single divalent metal ion responsible for catalysis. In our structure, we were not able to identify the electronic density of a metal ion, probably due the disorder of metal ion ligands composed by four histidine residues. These results indicate an important function of H-N-H motif for pyocin catalysis, consistent with the loss of killing activity of mutant E736A pyocin S8. Understanding and exploiting the mechanisms by which the pyocin S8 works might represent a promising strategy for future therapeutic applications.