Glutamate is a key nutrient forPorphyromonas gingivalisgrowth and survival during intracellular autophagic life under nutritionally limited conditions

Abstract

AbstractPorphyromonas gingivalissurvives in special autophagic vacuoles that serve as major replicative habitats in human primary gingival epithelial cells (GECs). As an asaccharolytic strict anaerobe,P. gingivalisis dependent on amino acids and peptides for nutrient sources. However, it is largely unknown as toP. gingivalis’metabolic processing under the nutritionally limited intracellular environments such the vacuoles, especially the preferred amino acids and associated-metabolic machineries. Here we elucidate that a Glutamate (Glu) catabolic enzyme, glutamate dehydrogenase (GdhA) is highly enriched in the isolatedP. gingivalis-containing vacuoles. Interestingly, we found thatP. gingivalisinduces conversion of intracellular glutamine pool to Glu determined by analyses of theP. gingivalis-containing vacuoles and the whole infected-GECs. Critically, exogenous Glu-Glu dipeptide, a simple precursor of Glu, significantly increases the size of isolated intactP. gingivaliscontaining-vacuoles and live wild-typeP. gingivalisnumbers in GECs. In contrast, the isogenic GdhA-deficient-strain, ΔgdhAdisplayed a significant growth defect with collapsed-vacuoles in GECs. Next, we confirmed thatP. gingivalisuptakes14C-Glu and it preferentially utilizes Glu-Glu-dipeptide using a nutritionally reduced Tryptic-Soy-Broth (TSB) media supplemented with Glu-Glu. Contrary, ΔgdhA-strain showed no detectable growth especially in nutritionally reduced TSB media with Glu-Glu. Using Atomic-Force-Microscopy, we observed that, wild-typeP. gingivalisbut not ΔgdhAstrain notably increased the cell volume upon Glu-Glu supplementation, an indicator of higher metabolism and growth. Utilization of a human gingiva-mimicking organoid-system further validated the importance of Glu as an essential nutrient for the intramucosal colonization ofP. gingivalisvia the protected replicative vacuoles in GECs.ImportanceThis study reveals thatP. gingivalisheavily depends on preferential utilization of Glutamate (Glu) for autophagic vacuolar growth and survival in human GECs. Several novel observations are made to support this: (i) GdhA ofP. gingivalisis highly enriched in these vacuoles, (ii)P. gingivalisinduces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu inP. gingivaliswild-type strain infection which is opposite in a ΔgdhAstrain, (iv)P. gingivalisuptakes14C-Glu and preferentially utilizes Glu-Glu dipeptide, (v) similarly, wild-type strain shows growth increase in a nutritionally reduced bacterial culture media, and (vi) finally, Glu-Glu supplementation increases bacterial cell-volume ofP. gingivaliswild-type but not ΔgdhAstrain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu forP. gingivalisgrowth-rate, biomass induction and survival in nutritionally limited host subcellular environments.