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Updated: Jul 23, 2025
Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens
Published on: April 5, 2019
Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa
Fatemeh Askarian1, Chih-Ming Tsai1, Gabriele Cordara2
1Division of Host-Microbe Systems & Therapeutics, Department of Pediatrics, University of California San Diego, La Jolla, CA 92093.
Abstract:
Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.
Insights
Pseudomonas aeruginosa CbpD impairs host defenses and causes pneumonia. Antibodies targeting non-catalytic CbpD domains protect against infection, offering potential vaccine strategies.
Area of Science:
- Microbiology
- Immunology
- Structural Biology
Background:
- Pseudomonas aeruginosa (PA) CbpD is a lytic polysaccharide monooxygenase (LPMO) involved in chitin degradation.
- LPMOs are crucial in various biological processes, and their role in bacterial virulence is an area of active research.
Purpose of the Study:
- To investigate the role of CbpD in PA virulence and host immune response.
- To determine the structural basis of CbpD function and identify potential vaccine targets.
Main Methods:
- Intratracheal challenge model in mice using wild-type and ΔCbpD PA strains.
- X-ray crystallography and small-angle X-ray scattering for structural analysis.
- AlphaFold2 for domain modeling and structure-based epitope mapping.
- Immunization studies with recombinant CbpD and antibody characterization.
Main Results:
- PA ΔCbpD mutant showed reduced virulence, mortality, and enhanced clearance compared to wild-type PA.
- High-resolution crystal structure of CbpD LPMO domain solved; other domains modeled.
- Immunization with full-length CbpD or its noncatalytic domains induced protective opsonic antibodies.
- Antibodies against the catalytic AA10 domain were not protective and were prevalent in cystic fibrosis patients.
Conclusions:
- CbpD contributes to PA virulence by hindering host complement function and opsonophagocytic clearance.
- Non-catalytic domains of CbpD represent promising targets for developing effective anti-PA vaccines.
- LPMO family proteins in other pathogens warrant investigation for novel vaccine development.
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