Supplementary Material for: Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis

<br>Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as <i>Porphyromonas gingivalis.</i> This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with <i>P. gingivalis</i> W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for <i>P. gingivalis</i> colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) <i>P. gingivalis</i> W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to <i>P. gingivalis-</i>related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for <i>P. gingivalis-</i>mediated aspiration pneumonia.