A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion

Abstract

Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epithelial cells. In contrast, mutation of a second HAD family serine phosphatase of P. gingivalis (SerB1170), or of a serine transporter, did not affect invasion. A pull-down assay identified GAPDH and heat-shock protein 90 as potential substrates for SerB653. Furthermore, exogenous phosphatase regulated microtubule dynamics in host cells. These data indicate that P. gingivalis has adapted a formerly metabolic enzyme to facilitate entry into host cells by modulating host cytoskeletal architecture. Our findings define a virulence-related role of a HAD family phosphatase and reveal an invasin of an important periodontal pathogen.

Fig. 1.

Amino acid sequence, hydrophobicity, and structural motifs for SerB proteins. (A and B) PG0653 and (C) PG1170. ACT domains in SerB653 are indicated by gray bars. Phosphoserine phosphatase regions are designated by black bars in 653 and 1170. HAD superfamily motifs are shown in bold italics, with catalytic residues indicated by a star. Goldman–Engelman–Steitz analysis (14) of SerB653 is shown in B. Predicted transmembrane domains are boxed in A. Amino acid sequence of strain 33277 PG1170 is shown above substituted residues.

Fig. 2.

SerB enzymes are active on phosphorylated peptide substrates and use aspartate as the nucleophile. (A) Phosphatase activity on peptides: serine phosphopeptide RRApSVA (Upstate, Charlottesville, VA), threonine phosphopeptide RRApTVA (Promega, Madison, WI), and tyrosine phosphopeptide RRLIEDAEpYAARG (Upstate). (B) Phosphatase activity of the SerB653 D198N mutant. Purified His-tagged enzymes were tested against the amino acid substrate phosphoserine.

Fig. 3.

Allelic exchange mutants and gingival epithelial cell invasion. For all graphs, invasion levels are represented as total colony-forming units per 2 × 10⁵ cells. Statistical significance between invasion levels was measured by using an unpaired t test. ∗, P < 0.001; ∗∗, P < 0.05. (A) Antibiotic protection assays in HIGK cells using P. gingivalis strain 33277 and the isogenic mutants described in Materials and Methods. (B) Invasion of HIGK cells with P. gingivalis W83 and its isogenic SerB653 mutant. (C) Invasion of primary GEC with 33277 and W83 parental and SerB653 mutant strains. (D) Invasion of HIGK cells with the complemented SerB653 mutant. Statistical significance is between mutant and complemented strains.

Fig. 4.

The SerB653 mutant adheres normally, but internalization is delayed. (A) Comparison of adherence efficiency between wild-type strains 33277, W83, and selected mutants, by using formalin-fixed HIGK cells. An unpaired t test comparing 33277 and the serB653::ermF mutant showed no significant difference (P = 0.36). A P. gingivalis 33277 fimbrial- (fimA) deficient mutant (19) was used as a control for reduced adherence (P < 0.001 compared to 33277, indicated by ∗). Results are from two independent experiments in triplicate. (B) Quantitation of invasion over 3 hr. Fluorescent microscopy images (×20) of P. gingivalis 33277 invasion of HIGK cells were quantitated by conversion of fluorescent intensity to grain counts by using Morphometrics image analysis software.

Fig. 5.

SerB653 interaction with gingival epithelial cell extracts. (A) Pull-down assays were performed as described in Materials and Methods. Proteins were separated by SDS/PAGE, and bands unique to the SerB653–HIGK interaction lane are indicated by arrows. (B and C) Peptide identification of the 85- and 37-kDa proteins. Peptides identified by MALDI-TOF analysis for each protein band are shown.

Fig. 6.

Addition of exogenous His-SerB653 protein to HIGK cells induces microtubule rearrangements. (A) Cells stained for α-tubulin (red) and with DAPI and images taken at ×60 magnification. Arrows indicate the presence of enriched tubulin at the cell surface. Controls contained enzyme buffer only. Additional controls of SerB653 (heat-inactivated) and SerB1170 did not induce tubulin rearrangements (not shown). (B) Magnification (×100) of cells at the 30-min time point.