title: Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis.
abstract: 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.
authors.affiliation: Divisions of Microbiology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Sato
authors.forename: Eiko
authors.initials: E
authors.lastname: Sakai
authors.forename: Paul D
authors.initials: PD
authors.lastname: Veith
authors.forename: Mikio
authors.initials: M
authors.lastname: Shoji
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Kikuchi
authors.forename: Hideharu
authors.initials: H
authors.lastname: Yukitake
authors.forename: Naoya
authors.initials: N
authors.lastname: Ohara
authors.forename: Mariko
authors.initials: M
authors.lastname: Naito
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Okamoto
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
authors.forename: Koji
authors.initials: K
authors.lastname: Nakayama
mesh_terms: 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
chemical_list: D018829:Adhesins, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D003546:Cysteine Endopeptidases