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Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

ABSTRACT: Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein. Immunochemical analysis of P. gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface. Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P. gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P. gingivalis chromosome. These data indicate that the ArgI protease of P. gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties.

SUBMITTER: Aduse-Opoku J

PROVIDER: S-EPMC173680 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

Aduse-Opoku J J Muir J J Slaney J M JM Rangarajan M M Curtis M A MA

Infection and immunity 19951201 12

Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer ...[more]

PMID: 7591131

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Project description:The gram-negative pathogen Porphyromonas gingivalis requires hemin for growth. Hemoglobin bound to haptoglobin and hemin complexed to hemopexin can be used as heme sources, indicating that P. gingivalis must have a means to remove the hemin from these host iron-binding proteins. However, the specific mechanisms utilized by P. gingivalis for the extraction of heme from heme-binding proteins and for iron transport are poorly understood. In this study we have determined that a newly identified TonB-dependent hemoglobin-hemin receptor (HmuR) is involved in hemoglobin binding and utilization in P. gingivalis A7436. HmuR shares amino acid homology with TonB-dependent outer membrane receptors of gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin, including HemR of Yersinia enterocolitica, ShuA of Shigella dysenteriae, HpuB of Neisseria gonorrhoeae and N. meningitidis, HmbR of N. meningitidis, HgbA of Haemophilus ducreyi, and HgpB of H. influenzae. Southern blot analysis confirmed the presence of the hmuR gene and revealed genetic variability in the carboxy terminus of hmuR in P. gingivalis strains 33277, 381, W50, and 53977. We also identified directly upstream of the hmuR gene a gene which we designated hmuY. Upstream of the hmuY start codon, a region with homology to the Fur binding consensus sequence was identified. Reverse transcription-PCR analysis revealed that hmuR and hmuY were cotranscribed and that transcription was negatively regulated by iron. Inactivation of hmuR resulted in a decreased ability of P. gingivalis to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sources. Escherichia coli cells expressing recombinant HmuR were shown to bind hemoglobin and hemin. Furthermore, purified recombinant HmuR was demonstrated to bind hemoglobin. Taken together, these results indicate that HmuR serves as the major TonB-dependent outer membrane receptor involved in the utilization of both hemin and hemoglobin in P. gingivalis.

Project description:Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0-2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.

Dataset Information

Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

Publications

Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

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