Identification and characterization of a UbK family kinase in Porphyromonas gingivalis that phosphorylates the RprY response regulator

Abstract

Phosphorylation of proteins is a key component of bacterial signaling systems that can control important functions such as community development and virulence. We report here the identification of a Ubiquitous bacterial Kinase (UbK) family member, designated UbK1, in the anaerobic periodontal pathogen, Porphyromonas gingivalis. UbK1 contains conserved SPT/S, Hanks‐type HxDxYR, EW, and Walker A motifs, and a mutation analysis established the Walker A domain and the Hanks‐type domain as required for both autophosphorylation and transphosphorylation. UbK1 autophosphorylates on the proximal serine in the SPT/S domain as well as the tyrosine residue within the HxDxYR domain and the tyrosine residue immediately proximal, indicating both serine/threonine and tyrosine specificity. The orphan two‐component system response regulator (RR) RprY was phosphorylated on Y41 in the receiver domain by UbK1. The ubk1 gene is essential in P. gingivalis; however, overexpression of UbK1 showed that UbK1‐mediated phosphorylation of RprY functions predominantly to augment its properties as a transcriptional enhancer. These results establish that P. gingivalis possesses an active UbK kinase in addition to a previously described Bacterial Tyrosine family kinase. The RR RprY is identified as the first transcriptional regulator controlled by a UbK enzyme.

Phosphorylation of major Porphyromonas gingivalis virulence factors is crucial for their processing and secretion

“…On the other hand, processing of proRgpA is dependent on VimA. Furthermore, activation of gingipains is probably additionally reliant on a response regulator RprY (PGN_1186) that was found to be phosphorylated (Y 41 ) in our phosphoproteomic analysis (Table S1) in accordance with recent findings (Perpich et al, 2021;Shen et al, 2020). Regulation was observed at the mRNA level and included other T9SS cargo proteins such as PPAD (Shen et al, 2020).…”

“…Although phosphorylation of histidine and aspartate is mostly reserved for two-component systems, the best characterized kinases target serine, threonine, and tyrosine residues (Whitmore & Lamont., 2012). Recently, a new class of bacterial kinases belonging to the ubiquitous bacterial kinase (UbK) family has been described (Nguyen et al, 2017;Perpich et al, 2021). UbKs have dual specificity and phosphorylate both Ser/Thr and Tyr residues.…”

Protein Tyrosine and Serine/Threonine Phosphorylation in Oral Bacterial Dysbiosis and Bacteria-Host Interaction

“…Structurally, UbK contains a conserved domain: the Walker A motif, HxDxYR, SPT/S and EW motifs. Ubk1 is a UbK family member in P. gingivalis that can autophosphorylate on the tyrosine and serine residues within the HxDxYR and SPT/S domains, respectively ( Perpich et al., 2021 ).…”

“…gingivalis can also exert its pathogenic function. Specifically, RprY, an orphan two-component system response regulator, can be phosphorylated by UbK1 on Y41 residue, affecting its transcriptional function ( Shen et al., 2020 ; Perpich et al., 2021 ). The UbK in S. mutans has been reported associated with cell morphology and biofilm development ( Bitoun et al., 2014 ).…”

Post-translational Modifications in Oral Bacteria and Their Functional Impact

“…Consistently, the RprY with a Y41F substitution impaired P. gingivalis virulence in a murine model of alveolar bone loss ( Shen et al, 2020 ). Furthermore, a recent report showed that the Tyr phosphorylation of RprY is mediated by the Ubiquitous bacterial Kinase 1 (UbK1) ( Perpich et al, 2021 ). In addition to Thr phosphorylation of SpxB, the Tyr phosphorylation at positions 409, 415, and 588 was also identified, with a possibly significant role in intraspecies and interspecies competition in S. sanguinis ( Mu et al, 2021 ).…”