Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Degree Program

Oral Biology, MS

Committee Chair

Lamont, Richard

Committee Co-Chair (if applicable)

Uriarte, Silvia

Committee Member

Uriarte, Silvia

Committee Member

Scott, David

Author's Keywords

Periodontitis; Peptoanaerobacter; neutrophil; immune response; oral pathogen

Abstract

Peptoanaerobacter stomatis is a newly appreciated taxon, present in high numbers in oral biofilms from periodontal patients. However, little is known about this organism’s potential virulence and its interaction with the host immune response. Therefore, to better understand the role of P. stomatis in periodontitis, we studied its interactions with neutrophils. Human neutrophils were challenged with P. stomatis strain CM2 or S. aureus AF488 (positive control) for 30 min. Bacterial internalization was examined by ImageStreamX imaging flow cytometry. Bacterial viability was assessed by colony-forming unit assays and Baclight assays. Phagocytosis-stimulated respiratory burst response was measured by flow cytometry. The fusion of specific and azurophil granules with bacteria-containing phagosomes was tested by immunostaining using confocal microscopy. Neutrophil granule exocytosis was measured by ELISA and flow cytometry. After 30 min of incubation, 20% of neutrophils were infected with P. stomatis. Approximately, 43 % of the bacterial inoculum was killed within a 30 min interaction with neutrophils. P. stomatis induced a higher respiratory burst response compared to S. aureus. When neutrophils were challenged with S. aureus, 64% of the bacteria-containing phagosomes were enriched for the specific granule marker, lactoferrin; similarly, 52% of P. stomatis-containing phagosomes were enriched with the granule marker. Likewise, 82% of P. stomatis-containing phagosomes were enriched with the azurophil granule marker, elastase; while 89% of the phagosomes containing S. aureus were enriched for this granule marker. P. stomatis challenge stimulated the degranulation of the four neutrophil granule subtypes. These results suggest that although neutrophils had a low phagocytic efficiency for P. stomatis; this periodontal pathogen was able to trigger the neutrophil killing mechanisms contributing to promote the chronic inflammation and tissue destruction that characterize periodontal diseases. These data demonstrate previously unexplored aspects of this newly appreciated taxon and how these organisms modulate neutrophil function.

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