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 terms: Oxidoreductases, Pathogens
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   The Pseudomonas aeruginosa lytic polysaccharide monooxygenase CbpD,
   prevalent in clinical isolates, has been proposed to act as a virulence
   factor. Here, the authors combine structural work, in silico
   simulations, enzymatic activity and in vitro and in vivo experiments to
   further delineate the role of CbpD and show that its deletion renders
   P. aeruginosa unable to establish a lethal systemic infection, leading
   to enhanced bacterial clearance in a mouse model of infection.

Introduction

   Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent
   enzymes that cleave glycosidic linkages by oxidation^[62]1–[63]4. Most
   characterized LPMOs act on recalcitrant polysaccharides such as
   cellulose (β-1,4(Glc)[n]) and chitin (β-1,4(GlcNAc)[n])^[64]5. Hence,
   current LPMO research focuses mainly on biomass degradation from
   fundamental and applied perspectives. All LPMOs possess a conserved
   active site with two histidine residues coordinating a copper ion in a
   so-called “histidine brace”^[65]3. Catalysis entails the reduction of
   the copper followed by activation of an oxygen-containing co-substrate
   to oxidize the C1 or C4 carbon of the polysaccharide substrate
   (reviewed in ref. ^[66]5). The co-substrate may be either dioxygen or
   hydrogen peroxide, the latter yielding catalytic rates several orders
   of magnitude higher than the former^[67]6–[68]8. LPMOs target multiple
   soluble and insoluble substrates, demonstrating their catalytic
   versatility (reviewed in ref. ^[69]9). Despite being primarily
   associated with biomass degradation machineries^[70]9,[71]10,
   genes encoding LPMOs are found in many pathogenic organisms and have
   been speculated to contribute to bacterial physiological processes or
   pathogenicity phenotypes^[72]11–[73]19. However, scant data exist to
   substantiate LPMO function during bacterial infection.

   One important pathogen that expresses a putative LPMO is Pseudomonas
   aeruginosa (PA), a frequently multidrug-resistant microorganism
   associated with nosocomial infections of surgical sites, urinary tract,
   lung, and blood^[74]20. PA accounts for ~20% of hospital-associated
   Gram-negative bacteremia cases, with high mortality (>30%),
   particularly in patients receiving inappropriate initial antimicrobial
   treatment^[75]21,[76]22. PA is known to explicitly disarm several
   aspects of the innate host defense and erodes efficacy of first-line
   antibiotics through an array of immune evasion factors and
   antibiotic-resistant determinants (reviewed in refs. ^[77]23,[78]24).

   The putative LPMO expressed by PA is called “chitin-binding protein D”
   (CbpD). Many publications refer to CbpD as a virulence factor (e.g.,
   ref. ^[79]25 and references within), yet no direct functional evidence