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Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

ABSTRACT: We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS1126(1), indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS1126(2), was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis: (i) the transposition of the endogenous insertion sequence element IS1126 and (ii) the modulation of gingipain transcription and translation as a result of IS1126 transposition.

SUBMITTER: Simpson W

PROVIDER: S-EPMC96847 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

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Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

Simpson W W Wang C Y CY Mikolajczyk-Pawlinska J J Potempa J J Travis J J Bond V C VC Genco C A CA

Infection and immunity 19991001 10

We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determine ...[more]

PMID: 10496872

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Project description:Porphyromonas gingivalis, the major etiologic agent in adult periodontitis, produces large amounts of proteases that are important for its survival and pathogenesis. The activation/maturation of gingipains, the major proteases, in P. gingivalis involves a complex network of processes which are not yet fully understood. VimA, a putative acetyltransferase and virulence-modulating protein in P. gingivalis, is known to be involved in gingipain biogenesis. P. gingivalis FLL92, a vimA-defective isogenic mutant (vimA::ermF-ermAM) showed late-onset gingipain activity at stationary phase, indicating the likelihood of a complementary functional VimA homolog in that growth phase. This study aimed to identify a functional homolog(s) that may activate the gingipains in the absence of VimA at stationary phase. A bioinformatics analysis showed five putative GCN5-related N-acetyltransferases (GNAT) encoded in the P. gingivalis genome that are structurally related to VimA. Allelic exchange mutagenesis was used to make deletion mutants for these acetyltransferases in the P. gingivalisvimA-defective mutant FLL102 (ΔvimA::ermF) genetic background. One of the mutants, designated P. gingivalis FLL126 (ΔvimA-ΔPG1842), did not show any late-onset gingipain activity at stationary phase compared to that of the parent strain P. gingivalis FLL102. A Western blot analysis of stationary-phase extracellular fractions with antigingipain antibodies showed immunoreactive bands that were similar in size to those for the progingipain species present only in the ΔvimA-ΔPG1842 isogenic mutant. Both recombinant VimA and PG1842 proteins acetylated Y230, K247, and K248 residues in the pro-RgpB substrate. Collectively, these findings indicate that PG1842 may play a significant role in the activation/maturation of gingipains in P. gingivalisIMPORTANCE Gingipain proteases are key virulence factors secreted by Porphyromonas gingivalis that cause periodontal tissue damage and the degradation of the host immune system proteins. Gingipains are translated as an inactive zymogen to restrict intracellular proteolytic activity before secretion. Posttranslational processing converts the inactive proenzyme to a catalytically active protease. Gingipain biogenesis, including its secretion and activation, is a complex process which is still not fully understood. One recent study identified acetylated lysine residues in the three gingipains RgpA, RgpB, and Kgp, thus indicating a role for acetylation in gingipain biogenesis. Here, we show that the acetyltransferases VimA and PG1842 can acetylate the pro-RgpB gingipain species. These findings further indicate that acetylation is a potential mechanism in the gingipain activation/maturation pathway in P. gingivalis.

Project description:BackgroundDysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment-modelling that in the subgingival pocket.MethodsGrowth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR.ResultsThe P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis.ConclusionsIn the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Project description:Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

Dataset Information

Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

Publications

Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

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