D. Grenier et al., Role of gingipains in growth of Porphyromonas gingivalis in the presence of human serum albumin, INFEC IMMUN, 69(8), 2001, pp. 5166-5172
Porphyromonas gingivalis, a bacterium associated with active chronic period
ontitis lesions, produces several proteolytic enzymes that are thought to b
e involved in host colonization, perturbation of the immune system, and tis
sue destruction. The aim of the present study was to investigate the contri
bution of Arg- and Lys-gingipains produced by P. gingivalis to its growth.
Although all of the proteins studied were degraded by P. gingivalis, only h
uman serum albumin and transferrin supported growth during serial transfers
in a chemically defined medium (CDM). Growth studies with site-directed gi
ngipain-deficient mutants of P. gingivalis revealed that inactivation of bo
th gingipains prevents growth, whereas inactivation of either Arg- or Lys-g
ingipain activity extended the doubling times to 33 or 13 h, respectively,
compared to 9 h for the parent strain. Growth of the mutants and the parent
strain was similar when the CDM was supplemented with a protein hydrolysat
e instead of human serum albumin. Incubation of resting P. gingivalis ATCC
33277 cells with fluorophore-labeled albumin indicated that the proteolytic
fragments generated by the gingipains were internalized by the bacterial c
ells. Internalization of fluorophore-labeled albumin fragments was reduced
or completely inhibited in the proteinase-deficient mutants. Interestingly,
gingival crevicular fluid samples from diseased periodontal sites containe
d low-molecular-mass albumin fragments, whereas samples from healthy sites
did not. The critical role of proteinases in the growth of P. gingivalis wa
s further investigated using specific Arg- and Lys-gingipain inhibitors. Ad
ding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-
gingipain A and B inhibitor) was more efficient at inhibiting growth than c
athepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that
Arg-gingipains and, to a lesser extent, Lys-gingipain play an important rol
e in the growth of P. gingivalis in a defined medium containing a human pro
tein as the sole carbon and nitrogen source.