Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants.
The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the DeltaragA mutant reduced RagB expression considerably and the DeltaragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the DeltaragA and DeltaragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the DeltaragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein-protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.
Nagano
Keiji
K
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
Murakami
Yukitaka
Y
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
Nishikawa
Kiyoshi
K
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
Sakakibara
Junpei
J
Oral and Maxillofacial Surgery II, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.|Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
Shimozato
Kazuo
K
Oral and Maxillofacial Surgery II, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
Yoshimura
Fuminobu
F
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
D018829:Adhesins, Bacterial / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D016866:Bacteroidaceae Infections; D002473:Cell Wall / Q000737:chemistry; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000096:biosynthesis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D000080867:Gingipain Cysteine Endopeptidases; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000235:genetics; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000382:microbiology; D014774:Virulence
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; C484343:RagA protein, Porphyromonas gingivalis; C484346:RagB protein, bacteria; D003546:Cysteine Endopeptidases