The comparative susceptibility of Porphyromonas gingivalis to the killing mechanisms of the neutrophil Public Deposited

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  • June 7, 2019
Creator
  • Parsons, Rebecca Keyser
    • Affiliation: School of Dentistry, Oral and Craniofacial Biomedicine PhD Program
Abstract
  • Neutrophils are critical in defense against periodontal pathogens such as Porphyromonas gingivalis, yet the mechanisms employed by neutrophils to kill this species are unclear. Using a dual-color fluorescence assay, we investigated phagocytosis and killing of P. gingivalis by neutrophils from normally healthy human donors vs. subjects with either chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficiency. P. gingivalis was not killed in the absence of phagocytosis, and neutrophil phagocytosis required both complement and high titered specific antibody. Intraphagosomal killing of P. gingivalis required a functional NADPH oxidase, which required the availability of oxygen, as there was no killing by CGD neutrophils or by normal neutrophils when NADPH oxidase was inhibited either with anaerobiosis or diphenyl iodonium. Furthermore, MPO was not required for neutrophil-mediated killing of this pathogen, as MPO-deficient neutrophils were fully competent in killing P. gingivalis, and MPO inhibition with 4-aminobenzoic acid hydrazide did not block neutrophil killing of P. gingivalis. The relative susceptibility of P. gingivalis to the reactive oxygen species associated with NADPH oxidase-dependent killing was also investigated. H2O2, but not superoxide, was effective in killing P. gingivalis, as killing by the xanthine-xanthine oxidase system was blocked by catalase, but not superoxide dismutase. When delivered as a bolus, H2O2 killed P. gingivalis in a dose-, time-, and temperature-dependent fashion, while -OCl killed at a critical concentration, independent of time or temperature. Compared to other species tested, P. gingivalis was remarkably sensitive to H2O2. Despite having very different susceptibilities to H2O2, all test bacteria showed similar susceptibilities to the MPO-H2O2 system and to its product, -OCl. When administered to logarithmically-varied bacterial densities, H2O2 killing was dependent on concentration, but not target density; while -OCl killing was lost with increasing target density. Together, these data support the hypothesis that -OCl is contact-lethal. In contrast, the time- and temperature-dependent properties associated with MPO-independent H2O2-mediated killing are consistent with the conversion of H2O2 to a more toxic product by the bacteria itself. The involvement of hydroxyl radical in this bactericidal activity was suggested by the protection afforded by dimethyl sulfoxide against H2O2 killing and azide protection against neutrophil-mediated killing of P. gingivalis.
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Oral Biology."
Advisor
  • Arnold, Roland
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  • Chapel Hill, NC
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  • Open access
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