|Pratt, Ashley - Scripps Institute|
|Didonato, Michael - Scripps Institute|
|Shin, David - Scripps Institute|
|Cabelli, Diane - Brookhaven National Laboratory|
|Bruns, Cami - Scripps Institute|
|Gorringe, Andrew - Public Health England (PHE)|
|Langford, Paul - Imperial College|
|Kroll, Simon - Imperial College|
|Tainer, John - Brookhaven National Laboratory|
|Getzoff, Elizabeth - Scripps Institute|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2015
Publication Date: 12/1/2015
Publication URL: http://handle.nal.usda.gov/10113/5642484
Citation: Pratt, A.J., Didonato, M., Shin, D.S., Cabelli, D.E., Bruns, C.K., Belzer, C.A., Gorringe, A.R., Langford, P.R., Tabatabai, L.B., Kroll, S.J., Tainer, J.A., Getzoff, E.D. 2015. Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus. Journal of Bacteriology. 197(24):3834-3847. doi: 10.1128/JB.00343-15.
Interpretive Summary: Both Neisseria meningitides (Nm) and Brucella abortus (Ba) are pathogenic bacteria that contain a periplasmic enzyme, Cu,Zn superoxide dismutase (Cu,ZnSOD), that allows the pathogen to evade the immune surveillance system of the host that the bacterium has infected. To understand the basic molecular mechanism that allows the organism to escape the immune surveillance system, the genes encoding the periplasmic Cu,ZnSODs were cloned. The recombinant enzymes were purified to homogeneity and their three dimensional X-ray crystallographic structures were determined. The NmSOD exists as a dimer whereas the BaSOD is a monomer. Their structures have been compared by superimposition and similarities and differences were described. Furthermore, the X-ray structure provided the basis for the previously described synthetic SOD peptide that protected mice against virulent B. abortus challenge. This peptide is located at the substrate cleft of the enzyme, thereby inhibiting SOD enzyme action due to the binding of specific anti-peptide antibodies. This resulted in lowering the splenic bacterial load of virulent B. abortus by 4 logs compared to controls. Based on the three dimensional crystal structure, improved peptide vaccines can now be designed to control both pathogens.
Technical Abstract: Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens.