Characterization of a Novel Protein Kinase in Porphyromonas gingivalis

Objectives: Multispecies bacterial communities within the gingival compartment initiate and maintain an inflammatory-based state that leads to periodontal diseases. Interbacterial signaling based on small-molecules and coadhesion is crucial in defining the overall pathogenic properties of the community. Porphyromonas gingivalis utilizes signaling based on protein phosphorylation/dephosphorylation to sense its environment and regulate pathogenic potential. Here we expand our understanding of the protein phosphorylation signaling network in P. gingivalis, and characterize PGN_1020, a novel component of this system.
Methods: The amino acid sequence of PGN_1020 was aligned with YdiB, a member of a novel family of Ser/Thr/Tyr protein kinases, and modeled to the structure of YdiB using Phyre2. ³²P –based autophosphorylation assays were used to characterize the phosphorylation status and reaction conditions of PGN_1020. Putative substrates were identified by in vitro kinase assays. Amino acid substitutions were generated by site directed mutagenesis.
Results: Amino acid sequence alignment of PGN_1020 with other YdiB kinase family members showed that it contains characteristic conserved motifs SPT/S, HxDxYR, EW, and a Walker A ATP-binding motif. Modeling the structure of PGN_1020 onto YdiB showed the two proteins share a similar 3D structure. PGN_1020 had autophosphorylation activity in vitro and required Mn²⁺ for maximal activity. A T40A mutation in the Walker A motif markedly diminished autophosphorylation activity, while K39G and H78A mutations had little to no effect. PGN_1020 phosphorylates the P. gingivalis serine phosphatase, SerB, which is involved in suppression host innate immunity and intracellular invasion of gingival epithelial cells.
Conclusions:
PGN_1020 is a member of the YdiB family of bacterial protein kinases containing characteristic conserved domains including a Walker A motif essential for autokinase activity. PGN_1020 phosphorylates SerB, which may have relevance for host inflammatory responses and bacterial intracellular invasion.