bioRxiv2024
Porphyromonas gingivalis survives in special autophagic vacuoles that serve as major replicative habitats in human primary gingival epithelial cells (GECs). As an asaccharolytic strict anaerobe, P. gingivalis is dependent on amino acids and peptides for nutrient sources. However, it is largely unknown as to P. 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 isolated P. gingivalis-containing vacuoles. Interestingly, we found that P. gingivalis induces conversion of intracellular glutamine pool to Glu determined by analyses of the P. gingivalis-containing vacuoles and the whole infected-GECs. Critically, exogenous Glu-Glu dipeptide, a simple precursor of Glu, significantly increases the size of isolated intact P. gingivalis containing-vacuoles and live wild-type P. gingivalis numbers in GECs. In contrast, the isogenic GdhA-deficient-strain, {Delta}gdhA displayed a significant growth defect with collapsed-vacuoles in GECs. Next, we confirmed that P. gingivalis uptakes 14C-Glu and it preferentially utilizes Glu-Glu-dipeptide using a nutritionally reduced Tryptic-Soy-Broth (TSB) media supplemented with Glu-Glu. Contrary, {Delta}gdhA-strain showed no detectable growth especially in nutritionally reduced TSB media with Glu-Glu. Using Atomic-Force-Microscopy, we observed that, wild-type P. gingivalis but not {Delta}gdhA strain 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 of P. gingivalis via the protected replicative vacuoles in GECs. ImportanceThis study reveals that P. gingivalis heavily 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 of P. gingivalis is highly enriched in these vacuoles, (ii) P. gingivalis induces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu in P. gingivalis wild-type strain infection which is opposite in a {Delta}gdhA strain, (iv) P. gingivalis uptakes 14C-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 of P. gingivalis wild-type but not {Delta}gdhA strain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu for P. gingivalis growth-rate, biomass induction and survival in nutritionally limited host subcellular environments.