Structure-function aspects of the Porphyromonas gingivalis tyrosine kinase Ptk1

Abstract

The development of synergistically pathogenic communities of Porphyromonas gingivalis and Streptococcus gordonii is controlled by a tyrosine-phosphorylation-dependent signaling pathway in P. gingivalis. The Ptk1 bacterial tyrosine (BY) kinase of P. gingivalis is required for maximal community development and for the production of extracellular polysaccharide. We show that the consensus BY kinase Walker A and B domains, the RK cluster, and the YC domain of Ptk1 are necessary for autophosphorylation and for substrate phosphorylation. Mass spectrometry showed that six tyrosine residues in a 16-amino-acid C-terminal region were phosphorylated in recombinant (r) Ptk1. Complementation of a ptk1 mutant with the wild-type ptk1 allele in trans restored community development between P. gingivalis and S. gordonii, and extracellular polysaccharide production by P. gingivalis. In contrast, complementation of Δptk1 with ptk1 containing a mutation in the Walker A domain failed to restore community development or extracellular polysaccharide production. rPtk1 was capable of phosphorylating the tyrosine phosphatase Ltp1 and the transcriptional regulator CdhR, both of which are involved in the development of P. gingivalis communities with S. gordonii.

Keywords: biofilms; extracellular polysaccharide; periodontal disease.

Figure 1

Domain architecture of the Ptk1 tyrosine kinase in P. gingivalis. Amino acid residues that were mutated are indicated in bold. TD: transmembrane domain, RK: arginine rich domain, YC: tyrosine rich C-terminal cluster.

Figure 2

Auto (A) and substrate (B) phosphorylation by Ptk1 and its phosphotransfer domain mutants. For autophosphorylation, recombinant FPtk1 (Ptk1:541–821) or FPtk1 with mutations in the Walker A (KS/MC), Walker B (D/N), RK (ERR/A) and YC (YA) domains were dephosphorylated with alkaline phosphatase and incubated with or without ATP in the presence of a phosphatase inhibitor. For substrate phosphorylation, Fptk1 and mutant derivatives were incubated with recombinant EpsD (PGN_0224) in the presence of ATP and a phosphatase inhibitor. The phosphorylation level of proteins was determined by Western blotting with phosphotyrosine antibodies.

Figure 3

Mass spectroscopy sequence coverage for FPtk1 (Ptk1:541–821). A. High confidence MS/MS data for 37 unique peptides comprising 87 exclusive unique and 394 total spectra were used to assign sequence coverage for 246 (blue upper case font) of 281 amino acids achieving 87.5% coverage. Non-detected residues are in lower case. MS/MS spectra for peptide in box is shown in (B). Phosphorylated residues are shown in red upper case, and bolded font. B. Example of MS/MS spectra used to assign site specific phosphorylation to MS/MS, in this case to tyrosine-775 (Y775) in the carbamidomethylated (c) tryptic peptide FTNMcLVLNDVSYSGSR.

Figure 4

Phosphorylation of Ltp1:C¹⁰S and CdhR by FPtk1. Recombinant proteins were dephosphorylated with alkaline phosphatase and incubated individually with or without FPtk1 along with ATP and in the presence of a phosphatase inhibitor. Protein phosphorylation was determined by Western blotting with phosphotyrosine antibodies.

Figure 5

Phosphotransfer by Ptk1 is required for P. gingivalis community development with S. gordonii and for extracellular polysaccharide production. A. P. gingivalis 33277, Δptk1 (Dptk1), Δptk1 + pptk1 (CDptk1) and Δptk1 + pptk1 containing a K614M/S615C mutation in the Walker A domain (CMDptk1) were reacted with a substrate of S. gordonii for 18 h. Total biovolumes of P. gingivalis and S. gordonii in a 213 × 213 μm area were obtained with by confocal microscopy, analyzed by Volocity and data are expressed as ratio of P. gingivalis (Pg) to S. gordonii (Sg). B. P. gingivalis strains were stained for extracellular polysaccharide with FITC-labeled concanavalin A and wheat germ agglutinin. Bacterial cells were stained with Syto-17. Fluorescent images were collected by confocal microscopy and the ratio of lectin staining of EPS to whole cell staining determined using Volocity. Error bars are SD, n = 3. ***P < 0.001; ****P < 0.0001, ns P > 0.05.