The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection

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.

The constructs generated in this study are available upon request. Source data are provided with this paper. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 134 partner repository with dataset identifiers PXD017971, PXD021888, PDX018769. SAXS data have been deposited at the SASBDB (SASBDB IDs: SASDK42 (20°C), SASDJQ5 (24°C), SASDJR5 (37°C). The CbpD structure prediction was based on templates provided by publicly available protein structures with PDB identifiers 6IF7, 2XWX, 2I1S. 4TX8, 1ED7, 2D49, 5IN1 and 4XZJ. For analysis of CbpD structural similarity the following publicly available structures were used: PDB identifiers 2XWX (for comparison with GbpA) and 4A5W (for comparison with the C5bC6-complex). All data required to evaluate the paper's conclusions are present in the paper, the Supplementary information, data sets and source data. Any additional data are available from the corresponding authors upon reasonable request.
No statistical pre-determination of sample size was performed. Sample sizes selected were based on results from pilot experiments or based on other published work that gave reliable statistical results.
No data were excluded from the analyses with one exception: To identify the organ-specific proteome responses to PA14 and CbpD infection, one out of the eight independent biological replicates in CbpD-infected mice showed only 10% of the total identified protein IDs compared to other ( n=7) independent biological replicates. Our additional analysis revealed a low Pearson correlation coefficient value for this sample compared to the rest of the replicates under CbpD-infection treatment. Thus this sample was considered as an outlier and omitted from the analysis.
All attempts at replication were successful. The number of experimental replicates are indicated in the respective figure legends.
For the in vivo infection model studies, mice were randomly distributed/grouped from housing cages to experimental cages that comprised control and treatment (different infection conditions) cages. Randomization was not relevant for the other experiments performed in this Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. In general, blinding was considered unnecessary for the experiments associated with this study as prior knowledge was required to carry out experiments and analysis. The only exception was the histopathological analysis, where slide evaluation and scoring was performed blindly.
A custom made affinity-purified CbpD antibody was generated by Davids Biotechnologie by immunizing rabbits with peptides KDGYNPEKPLAWSDLEPA and DAQGRDAQRHSLTLAQGANGA.
The commercially used antibodies that were described in the "Methods" are listed as follow: CbpD antibody recognizes secreted CbpD by Pseudomonas aeruginosa ( e.g. Supplementary Fig. 11a). Species reactivity: rat.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy Verified by the supplier (ATCC) at the time of distribution via a series of morphology, karyotyping, and PCR based approaches ( e.g., growth curves, cytochrome C oxidase I gene (COI) analysis and short tandem repeat (STR) profiling) Cell lines were negative to mycoplasma (tested and verified by ATCC) No commonly misidentified cell lines were used in this study.
Mus Musculus (strain CD-1 mice), eight-week-old female, kept in filter-top cages with access to food pellet and water under controlled ambient temperature (20-22°C) and relative humidity (30-70%), 12h light/12h dark cycle No wild animal were used in this study No field-collected samples were used in the study.
As described in the "Ethics Declarations" section, animal experiments/housing were conducted under the UC San Diego approved IRB protocol S00227M, and in accordance with the rules and regulations of the Institutional Animal Care and Use Committee.
The participant were healthy individuals, comprised of both male and female, age range 20-50 and from different ethnic groups Any healthy male and female were eligible to voluntarily participation and were recruited under the protocols that has been approved by UMC Medical Ethics Committee and by REK-Norway (2018/1586). The blood donors provided written informed consent and there were no known biases that contributed towards their study inclusion other than that they were healthy and working, studying or visiting the same institution where the experiment was conducted.
As described in the "Ethics Declarations" section: "Blood was drawn from several healthy volunteers (male and female) in accordance with ethical principles of the Helsinki Declaration and under the protocols that has been approved by UMC Medical Ethics Committee and REK-Norway (2018/1586). All blood donors provided written informed consent." Flow cytomteric analysis was performed on bacteria monoculture or immune cells (with focus on PMNs) obtained from blood . Sample preparation is described in detail in the "Methods" under sections: "Complement deposition on bacteria surface", "Whole-blood phagocytosis", and "Competition for surface-expressed receptor binding".

CellStream (Luminex) MACSQuant (Miltenyi biotech) FACSVerse (Becton Dickinson)
FlowJo and CellStream software 10000 events per condition (from gated cells) Gating of cells was carried out on the basis of forward and side scatter. The fluorescence intensity (FL) of 10,000 gated neutrophils/bacteria was measured for each sample. Phagocytosis/binding of antibody to the surface expressed receptors/ deposition of complement components on the bacterial surface ( C3b, Bb, C5b9) was identified when neutrophils/bacteria