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.
Highlights
The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce
To gain further insight into the chitin-binding protein D” (CbpD) structure, homology modeling, molecular dynamics simulations, and small-angle X-ray scattering (SAXS) analysis were performed. These approaches revealed that CbpD is a monomeric, elongated protein, whose conformation may be affected by post-translational modifications (Fig. 1a–c and Supplementary Figs. 3−5, Supplementary Tables 1–3, and Supplementary Results)
Pseudomonas aeruginosa (PA) is an important cause of nosocomial bacteremia with high associated mortality[52], and as normal human serum (NHS) resulted in distinct proteome modulation in ΔCbpD compared to WT, we explored if the deletion of CbpD affected PA survival in freshly collected human blood
Summary
The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. We show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. We combine studies of CbpD structure and enzymatic activity with analysis of its immune evasion properties ex vivo and in vivo to unravel the function of this protein in PA bloodstream infection. We show that this highly conserved and prevalent tri-modular LPMO is a chitin-oxidizing virulence factor that enhances resistance of the bacterium to whole bloodmediated killing and virulence during ex vivo and in vivo infection by attenuation of the terminal complement cascade
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