Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg‐ and Lys‐gingipain activities

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Abstract
Porphyromonas gingivalis, a key causative agent of adult periodontitis, is known to produce a variety of virulence factors including proteases. The aim of this study was to evaluate the participation of Arg‐ and Lys‐gingipain activities of P. gingivalis in the acquisition of iron from human transferrin and its subsequent utilization in growth. Iron‐saturated transferrin was found to support the long‐term growth of P. gingivalis. Our results indicated that P. gingivalis does not produce siderophore and does not possess ferric reductase and transferrin‐binding activities. Incubating transferrin with P. gingivalis resulted in degradation of the protein, a step that may be critical for the acquisition of iron from transferrin. Spontaneous and site‐directed mutants of P. gingivalis deficient in one or several proteases were used to demonstrate the key role of specific enzymes in degradation of transferrin and subsequent utilization for growth. The lack of both Arg‐ and Lys‐gingipain activities (mutants M1 and KDP128) was associated with an absence of degradation of transferrin and the incapacity of bacteria to grow in the presence of transferrin as the sole source of iron. It was also found that the Lys‐gingipain activity is more critical than the Arg‐gingipain activity since the mutant KDP112 (deficient in Arg‐gingipain A and B) could grow whereas the mutant KDP129 (deficient in Lys‐gingipain) could not. The fact that growth of mutant KDP112 was associated with a lower final optical density and a generation time much longer compared with the parent strain suggests that the Arg‐gingipain activity also participates in the acquisition of iron from transferrin. Selected inhibitors of cysteine proteases (TLCK, leupeptin and cathepsin B inhibitor II) were tested for their capacity to reduce or inhibit the growth of P. gingivalis under different iron conditions. All three inhibitors were found to completely inhibit growth of strain ATCC 33277 in a medium supplemented with transferrin as the source of iron. The inhibitors had no effects when the bacteria were grown in a medium containing hemin instead of transferrin. The ability of P. gingivalis to cleave transferrin may be an important mechanism for the acquisition of iron from this protein during periodontitis.