Dataset Information

Sequence-independent processing site of the C-terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis.

ABSTRACT: The Gram-negative periodontal pathogen Porphyromonas gingivalis produces a family of outer membrane-anchored proteases, the gingipains, shown to play an essential role in virulence of the organism. The C-terminal domain (CTD) of gingipains and other secreted proteins is known to be the targeting signal for maturation and translocation of the protein through the outer membrane. The CTD is subsequently cleaved during the secretion process. Multiple alignment of various CTDs failed to define a consensus sequence at the putative CTD processing site. Using mutagenesis, we were able to show that cleavage at the site is not dependent on a specific residue and that recognition of the site is independent of local sequence. Interestingly, length of the junction between the CTD and adjacent Ig-like subdomain has a critical influence on post-translational glycan modification of the protein, whereby insertion of additional residues immediately N-terminal to the cleavage site results in failure of glycan modification and release of soluble protease into the culture medium. Various hypotheses are presented to explain these phenomena. Knowledge of the role CTDs play in maturation of gingipains has broader application for understanding maturation of CTD homologues expressed by bacteria of the Bacteriodetes phylum.

SUBMITTER: Zhou XY

PROVIDER: S-EPMC3773331 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Sequence-independent processing site of the C-terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis.

Zhou Xiao-Yan XY Gao Jin-Long JL Hunter Neil N Potempa Jan J Nguyen Ky-Anh KA

Molecular microbiology 20130719 5

The Gram-negative periodontal pathogen Porphyromonas gingivalis produces a family of outer membrane-anchored proteases, the gingipains, shown to play an essential role in virulence of the organism. The C-terminal domain (CTD) of gingipains and other secreted proteins is known to be the targeting signal for maturation and translocation of the protein through the outer membrane. The CTD is subsequently cleaved during the secretion process. Multiple alignment of various CTDs failed to define a cons ...[more]

PMID: 23869473

Similar Datasets

Project description:The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P. gingivalis W50. RI is a heterodimer in which the catalytic alpha chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free alpha chain, and RIB is a highly posttranslationally modified form of the alpha chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2. In the present work, the prR2 was insertionally inactivated in P. gingivalis W50 in order to establish the contribution of this locus to the arginine-specific protease activity of this periodontal bacterium. Loss of prR2 function had several effects on prpR1-derived enzymes. First, the total Arg-X activity was reduced by approximately 50% relative to that of the parent strain. The reduction in total activity was a consequence of decreased concentrations of the monomeric enzymes derived from the prpR1, while the heterodimeric enzyme, RI, was unaffected by this mutation. Second, the chromatographic behavior of both the soluble and vesicle- or membrane-associated monomeric enzymes was radically different from the behavior of RIA and RIB from the parent strain. Finally, the vesicle- or membrane-associated enzyme in the prR2 mutant strain lacked the extensive posttranslational additions which are found on RIB in P. gingivalis W50. These data suggest that the product(s) of the prR2 plays a significant role in the maturation pathway of prpR1-derived enzymes, and this may contribute to the coconservation of these two genes in P. gingivalis.

Project description:Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively. The contribution to pathogenicity of each of the proteinase genes of P. gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain. Whole-cell Lys-x proteolytic activity of the RgpA(-) and RgpB(-) mutants was not significantly different from that of wild-type W50, whereas the Kgp(-) mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA(-) and RgpB(-) isogenic mutants did not die but developed lesions. Mice challenged with the Kgp(-) isogenic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp(-) mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 10(9) viable-cell dose. All the mice challenged with the RgpA(-) mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB(-) mutant at this dose. Wild-type phenotype was restored to the RgpB(-) mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene. There was no difference between the pNJR12::rgpB-complemented RgpB(-) mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model. These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P. gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp >> RgpB > or = RgpA.

Dataset Information

Sequence-independent processing site of the C-terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis.

Publications

Sequence-independent processing site of the C-terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets