ABSTRACT
The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.
Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Mixed Function Oxygenases/metabolism , Polysaccharides/metabolism , Pseudomonas Infections/enzymology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/pathogenicity , Animals , Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Cell Death , Complement System Proteins/metabolism , Humans , Mice , Microbial Viability , Oxidation-Reduction , Protein Domains , Proteome/metabolism , Proteomics , Pseudomonas Infections/blood , Substrate Specificity , Transcription, Genetic , Virulence , Virulence Factors/metabolismABSTRACT
Does sodium dodecyl sulfate (SDS) denature proteins through electrostatic SDS-protein interactions? We show that a protein completely lacking charged side chains is unfolded by SDS in a manner similar to charged proteins, revealing that formal protein charges are not required for SDS-induced protein unfolding or binding.
