Effects of various culture environments on expression of major outer membrane proteins from<i>Porphyromonas gingivalis</i>

Murakami, Yukitaka; Imai, Masashi; Mukai, Yuichi; Ichihara, Shigeyuki; Nakamura, Hiroshi; Yoshimura, Fuminobu

doi:10.1016/s0378-1097(03)00896-6

Cited by 21 publications

(34 citation statements)

References 47 publications

(58 reference statements)

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“…In the absence of a specific antibody to Rag4, it was not possible to confirm the identity of this protein. However, Murakami et al recently determined by N-terminal sequencing that both RagA and a variant of RagB that can now be confirmed to be RagB4 are expressed in ATCC 33277 T (rag-4), where they represent two of the seven major outer membrane proteins (22,23). Sequence comparisons of the four rag alleles.…”

Section: Resultsmentioning

confidence: 99%

Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis

et al. 2005

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The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G؉C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.

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Section: Resultsmentioning

confidence: 99%

Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis

et al. 2005

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“…It was noteworthy that only trace amounts of RagA, RagB, and OmpA-like proteins existed in the supernatant fraction. The above-mentioned major protein identification, based on mobility in SDS-PAGE, was carried out previously (41)(42)(43).…”

Section: Resultsmentioning

confidence: 99%

“…P. gingivalis expresses two types of fimbriae, long fimbriae composed of FimA (fimbrilin) (67), and short fimbriae composed of a 75-kDa protein (21, 51, 68). Many studies have shown that FimA has various functions, which include attachment to and invasion of gingival epithelial cells (28, 64), coaggregation with other species of bacteria (2), attachment to salivary components (4), activation of fibroblasts and macrophages (24), induction of inflammatory cytokines, and bone resorption (23).Several proteins are often found in the outer membrane fraction, particularly RagA, RagB, Arg-gingipains (Rgps), Lysgingipain (Kgp), and the OmpA-like proteins (41,42). RagA is a TonB-dependent outer membrane receptor.…”

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Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors inPorphyromonas gingivalis

Masuda

Murakami

Noguchi³

et al. 2006

Appl Environ Microbiol

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Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptidelimiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.It has been reported that more than 80% of U.S. adults have overt gingivitis and about half of them have periodontitis (1). In periodontal diseases, teeth may be lost after inflammation spreads to the supporting periodontal tissues. Not only local events such as inflammation and trauma but also systemic and environmental factors are involved in establishment of periodontal diseases (9). The formation and maturation of plaque, especially subgingival plaque in periodontal pockets, are also involved (61). Although there are more than 600 species of bacteria in the mouth (30), specific species of gram-negative anaerobic bacteria, spirochetes, and motile rods are known to participate in formation of subgingival plaque (20,60). Porphyromonas gingivalis, a gram-negative anaerobe, is considered to contribute to periodontitis, since it is detected frequently in patients with these diseases (56, 60).Asaccharolytic P. gingivalis mainly obtains nutrients by degradation of proteins. This bacterium is known to have many potential virulence factors, including fimbriae, proteases, hemagglutinins, lipopolysaccharide, capsule polysaccharides, and major outer membrane proteins (26, 31). P. gingivalis expresses two types of fimbriae, long fimbriae composed of FimA (fimbrilin) (67), and short fimbriae composed of a 75-kDa protein (21, 51, 68). Many studies have shown that FimA has various functions, which include attachment to and invasion of gingival epithelial cells (28, 64), coaggregation with other species of bacteria (2), attachment to salivary components (4), activation of fibroblasts and macrophages (24), induction of inflammatory cytokines, and bone resorption (23)....

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“…The Pgm6/7 protein heterotrimers were partially digested with Staphylococcus aureus V8 protease to analyze the internal sequences as described previously (28). Heterotrimers and homotrimers were also analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MS).…”

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Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA inPorphyromonas gingivalis

Nagano¹,

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Murakami³

et al. 2005

J Bacteriol

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The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded ␤-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.

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Effects of various culture environments on expression of major outer membrane proteins fromPorphyromonas gingivalis

Cited by 21 publications

References 47 publications

Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis

Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors inPorphyromonas gingivalis

Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA inPorphyromonas gingivalis

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