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Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.

ABSTRACT: 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 beta-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.

SUBMITTER: Nagano K

PROVIDER: S-EPMC545718 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.

Nagano Keiji K Read Erik K EK Murakami Yukitaka Y Masuda Takashi T Noguchi Toshihide T Yoshimura Fuminobu F

Journal of bacteriology 20050201 3

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 beta-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular mass ...[more]

PMID: 15659668

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Project description:Many bacteria switch between a sessile and a motile mode in response to environmental and host-related signals. Porphyromonas gingivalis, an oral anaerobe implicated in the etiology of chronic periodontal disease, has long been described as a nonmotile bacterium. And yet, recent studies have shown that under certain conditions, P. gingivalis is capable of surface translocation. Considering these findings, this work aimed to increase our understanding of how P. gingivalis transitions between sessile growth and surface migration. Here, we show that the peptidylarginine deiminase secreted by P. gingivalis (PPAD), an enzyme previously shown to be upregulated during surface translocation and to constrain biofilm formation, promotes surface translocation. In the absence of PPAD, the production of outer membrane vesicles (OMVs) was drastically reduced. In turn, there was a reduction in gingipain-mediated proteolysis and a reduced zone of hydration around the site of inoculation. Transcriptome sequencing (RNA-Seq) and metabolomics analyses also showed that these changes corresponded to a shift in arginine metabolism. Overall, this report provides new evidence for the functional relevance of PPAD and proteases, as well as the importance of PPAD activity in OMV biogenesis and release. Our findings support the model that citrullination is a critical mechanism during lifestyle transition between surface-attached growth and surface translocation by modulating OMV-mediated proteolysis and arginine metabolism.IMPORTANCE Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production, and many of the proteins packaged in these vesicles are proteolytic or protein-modifying enzymes. This includes key virulence determinants, such as the gingipains and PPAD (a unique peptidylarginine deiminase). Here, we show that PPAD activity (citrullination) is involved in OMV biogenesis. The study revealed an unusual mechanism that allows this bacterium to transform its surroundings. Since OMVs are detected in circulation and in systemic tissues, our study results also support the notion that PPAD activity may be a key factor in the correlation between periodontitis and systemic diseases, further supporting the idea of PPAD as an important therapeutic target.

Dataset Information

Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.

Publications

Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.

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