Characterization of a bacterial tyrosine kinase in Porphyromonas gingivalis involved in polymicrobial synergy

Abstract

Interspecies communication between Porphyromonas gingivalis and Streptococcus gordonii underlies the development of synergistic dual species communities. Contact with S. gordonii initiates signal transduction within P. gingivalis that is based on protein tyrosine (de)phosphorylation. In this study, we characterize a bacterial tyrosine (BY) kinase (designated Ptk1) of P. gingivalis and demonstrate its involvement in interspecies signaling. Ptk1 can utilize ATP for autophosphorylation and is dephosphorylated by the P. gingivalis tyrosine phosphatase, Ltp1. Community development with S. gordonii is severely abrogated in a ptk1 mutant of P. gingivalis, indicating that tyrosine kinase activity is required for maximal polymicrobial synergy. Ptk1 controls the levels of the transcriptional regulator CdhR and the fimbrial adhesin Mfa1 which mediates binding to S. gordonii. The ptk1 gene is in an operon with two genes involved in exopolysaccharide synthesis, and similar to other BY kinases, Ptk1 is necessary for exopolysaccharide production in P. gingivalis. Ptk1 can phosphorylate the capsule related proteins PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and PGN_0613, a UDP-glucose dehydrogenase, in P. gingivalis. Knockout of ptk1 in an encapsulated strain of P. gingivalis resulted in loss of capsule production. Collectively these results demonstrate that the P. gingivalis Ptk1 BY kinase regulates interspecies communication and controls heterotypic community development with S. gordonii through adjusting the levels of the Mfa1 adhesin and exopolysaccharide.

Keywords: Community; Porphyromonas gingivalis; periodontitis; polymicrobial synergy; tyrosine kinase.

Figure 1

Ptk1 demonstrates ATPase activity and autophosphorylation. (A) Tyrosine kinase assay (Beacon) with the 541-821 C-terminal amino acid fragment of Ptk1 (FPtk1, 5 μg). Positive control and negative kit control were supplied in the assay kit. Negative kinase control is buffer only. Error bars are SD, n = 3. ***P < 0.001 compared to kinase control. (B) Concentration-dependent kinase activity of FPtk1. Negative control is kinase buffer. *P < 0.05; ***P < 0.001 compared to negative control. (C) Autophosphorylation of FPtk1. FPtk1 was dephosphorylated with alkaline phosphatase (AP) followed by treatment with phosphatase inhibitor and ATP or left untreated as a control. Phosphorylated FPtk1 was visualized by Western blotting with phosphotyrosine antibodies.

Figure 2

Physical and transcriptional organization of the ptk1 region in Porphyromonas gingivalis 33277. (A) Genetic organization of the ptk1 region. Transcriptional direction is indicated with arrows. Primer binding sites are indicated for reverse transcriptase PCR (RT-PCR). (B) RT-PCR of genomic or cDNA with primers spanning the intergenic regions between the open reading frames. A negative control without reverse transcriptase was included (No RT). (C) Part of the PGN_1522 and PGN_1523 coding regions with amino acid sequence along with the untranscribed intergenic region. The transcriptional start site for the PGN_1523-1525 codon was mapped by 5′ RACE to an A (indicated with arrow) 52 bp upstream of the methionine ATG start codon (red font) of PGN_1523. The start codon of the upstream PGN_1522, transcribed in the opposite direction, is indicated (red font).

Figure 3

Dephosphorylation of FPtk1 by Ltp1. (A) Incubation of FPtk1 with Ltp1 results in dephosphorylation and reduced kinase activity in the tyrosine kinase assay. Substitution of Cys with Ser at amino acid 10 (Ltp1 C10S) in the catalytically active domain of Ltp1 restored kinase activity. ***P < 0.001 compared to negative control. (B) Western immunoblot of FPtk1 incubated with Ltp1 or Ltp1 C10S, and with ATP or phosphatase inhibitor as indicated. The phosphorylation level of FPtk1 was determined by Western blotting with phosphotyrosine antibodies. (C) Densiometric analysis of band intensity from the blot in (B) using ImageJ to quantitate band density. *P < 0.05; ***P < 0.001 compared to FPtk1 + Ltp1.

Figure 4

ptk1 is required for maximal Porphyromonas gingivalis–Streptococcus gordonii community development. (A) CSLM visualization of P. gingivalis 33277, Δptk1, and Δptk1 + pptk1 (green) reacted with a substrate of S. gordonii (red) for 18 h. A series of 0.2 μm-deep optical fluorescent x–y sections (213 × 213 μm) were collected using an Olympus FV500 confocal microscope, digitally reconstructed into a 3D image and quantitated using Volocity software. (B) Total P. gingivalis biovolume was obtained with the 3D Find Objects function of Volocity. (C) Numbers of microcolonies using an object size cut-off algorithm of greater than 30 μm for the total 3D volume for P. gingivalis fluorescence. Error bars are SD, n = 3. ***P < 0.001 compared to Δptk1.

Figure 5

Ptk1 regulates cdhR (A) and mfa1 (B) expression in P. gingivalis–S. gordonii communities. Messenger RNA extracted from P. gingivalis 33277 or Δptk1 cells alone or in a community with S. gordonii was analyzed by qRT-PCR. 16s rRNA was used for normalization. *P < 0.05; ***P < 0.001 compared to 33277.

Figure 6

Exopolysaccharide production by P. gingivalis is reduced in mutants lacking ptk1. (A) Exoploysaccharide produced by P. gingivalis 33277 strains was stained with FITC-labeled concanavalin A and wheat germ agglutinin. Bacterial cells were stained with Syto-17. Fluorescent images were collected by confocal microscopy. (B) Ratio of lectin binding (green) to whole cell staining (red) from images in (A) using Volocity software. Error bars are SD, n = 3. **P < 0.01; ***P < 0.001 compared to Δptk1 (C) Transmission electron microscopy analysis of P. gingivalis W83 wild type and ptk1 deficient mutant. Ruthenium red staining was employed to visualize capsule. Arrows indicate presence of surface capsule in W83 versus reduced capsule in Δptk1. Magnification, ×27,500. Scale bar = 0.5 μm.

Figure 7

The capsule associated UDP-acetyl-mannosamine dehydrogenase (PGN_0224), and UDP-glucose 6-dehydrogenase (PGN_0613) are phosphorylated by Ptk1. Recombinant protein substrates were incubated with FPtk1 (Ptk1:541-821) in the presence of ATP and a phosphatase inhibitor. PGN_0261 (a sigma-54-dependent transcriptional regulator) was used as a control protein. The phosphorylation level of protein substrates was determined by Western blotting with phosphotyrosine antibodies.