Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis.
The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.
Sato
Keiko
K
Divisions of Microbiology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Sakai
Eiko
E
Veith
Paul D
PD
Shoji
Mikio
M
Kikuchi
Yuichiro
Y
Yukitake
Hideharu
H
Ohara
Naoya
N
Naito
Mariko
M
Okamoto
Kuniaki
K
Reynolds
Eric C
EC
Nakayama
Koji
K
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002874:Chromosome Mapping; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D000080867:Gingipain Cysteine Endopeptidases; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016966:Porphyromonas gingivalis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D018829:Adhesins, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D003546:Cysteine Endopeptidases