Common protein phosphorylation involves kinase-catalyzed addition of phosphate to amino acid side chains of histidine (His), aspartate (Asp), serine (Ser), threonine (Thr), and tyrosine (Tyr) in oral bacteria. Protein phosphorylation can be readily reversed by phosphatase-mediated hydrolysis (dephosphorylation) to regenerate unmodified hydroxyls, i.e., changing the residue from hydrophobic to hydrophilic (Mijakovic et al., 2016). Therefore, reversible protein phosphorylation can regulate the characteristics of proteins rapidly and react quickly to external stimuli. There are many donor sources of phosphate groups, the most utilized of which are active phosphate groups and adenosine triphosphate (ATP). Most phosphorylated side chains or kinases are residue-specific, and different phosphorylated amino acid side chains have different chemical stability and distinct impacts on hemostasis and quality control. Protein phosphorylation is widely involved in metabolic processes, including cell survival, virulence factor, and environmental adaptation in oral bacteria. Different phosphorylation modifications will be reviewed using the families of kinases and the residues they phosphorylate in this part (Figure 1).

Protein acetylation is another major regulatory PTM found ubiquitously in oral bacteria. The protein acetylation modifications are finely regulated by two distinct mechanisms: enzymatic and non-enzymatic acetylation (chemical acetylation; Figure 2). Enzymatic acetylation relies on protein acetyltransferase to transfer the acetyl group from Ac-CoA to lysine residue (Starai and Escalante-Semerena, 2004). Non-enzymatic acetylation requires acetyl phosphate (AcP) or Ac-CoA to serve as the donor of the acetyl group. Lysine acetylation is carried out covalently by adding an acetyl group to a lysine residue, changing the biochemical characteristics of target protein, such as their charge, stability, and/or conformation, and subsequently, altering the protein biological activity (Carabetta and Cristea, 2017; Sang et al., 2017).

Lysine acetylation has been shown to play an essential role in bacterial survival and virulence. Butler et al. (2015) identified 130 lysine-acetylated peptides from 92 P. gingivalis proteins, including the three gingipains RgpA, RgpB, and Kgp in a P. gingivalis W40 acetylome study. In a previous report, the inactivation of vimA, a putative acetyltransferase gene, resulted in the late-onset gingipain activity, compared with the wild-type W83, which is further explained in the following report. PG1842 (a functional VimA homolog) and VimA can acetylate pro-RgpB at residues K247 and K248, contributing to the activation/maturation of gingipains in P. gingivalis (Mishra et al., 2018).

In the acetylome of S. mutans, 973 acetylation sites were identified in 445 proteins, accounting for 22.7% of overall proteins, among which 617 acetylation sites were quantified in 302 proteins. Notably, the acetylation levels of glucosyltransferase B (GtfB), glucosyltransferase C (GtfC), and glucosyltransferase D (GtfD) in the biofilms decreased by 29, 22, and 37%, respectively, compared to planktonic conditions (Lei et al., 2020). Gtfs are the critical virulence factors by which S. mutans can utilize sucrose to synthesize EPS, promoting biofilm formation, the initiating factor for caries. The results from the acetylome study indicate that the acetylation of Gtfs is an important mechanism of regulating their catalytic activity and subsequently the biofilm formation.

Lysine acetylation is reversed by deacetylases. Lysine deacetylases (NAD⁺-dependent sirtuins; for example, CobB) can enzymatically remove the acetyl group from lysine side chains. Reports on the characterization of acetyltransferases and deacetylases emerge continuously, such as the acetyltransferase Pat and the deacetylase CobB from P. gingivalis. Li et al. (2018) reported that RprY could be modified by Pat with Ac-CoA serving as the acetyl group donor, and the acetylated RprY could be reversely deacetylated by CobB. Subsequently, the acetylation of RprY diminished its ability to bind the promoter DNA of nqrA (PGN_0115: Na⁺-translocating NADH-quinone reductase subunit A), affecting bacterial survival in oxidative stress.

Protein glycosylation is considered one of the most abundant forms of PTMs and identified as a critical regulator in bacterial physiology, such as communication, adhesion, and virulence (Nothaft and Szymanski, 2010). It entails the covalent attachment of a glycan group to the amino acid residue to form glycoproteins (Figure 3). The glycan donors are phosphate-activated sugars linked to nucleotides (UDP-, GDP-, and ADP-) or dolichol-related lipid groups. Protein glycosylation can be categorized into two major types, N-linkages and O-linkages, based on the glycosidic linkage; however, Cys-S-linked and Trp-C-linked glycoproteins are also known (Stepper et al., 2011; Yoshimoto et al., 2021). N-glycosylation binds glycans to the asparagine (Asn) residues, while O-glycosylation occurs at Ser or Thr residues. Protein glycosylation results from the modification of glycosidases and glycosyltransferases, whose activities depend on various bacterial processes and environmental stresses (Schaffer and Messner, 2017).

Citrullination is an essential PTMs in which peptidylarginine is catalyzed to peptidyl-citrulline by PADs. The reaction is referred to as delamination due to the exchange of an imine for a carbonyl group (Tilvawala and Thompson, 2019). The PAD-catalyzed reaction involves the hydrolysis of the guanidinium group of an arginine residue, leading to the loss of positive charge, generation of two hydrogen bond donors, and urea (Figure 4). Eventually, citrullination alters the charges of the substrate protein, possibly leading to significant changes in its structure and function (Wegner et al., 2010a). Although the existence of “decitrullinase” has been hypothesized, no such enzyme has been identified. The dysregulated PAD activity is observed in numerous inflammatory diseases including rheumatoid arthritis (RA), Alzheimer’s disease, atherosclerosis, and periodontal diseases (PDs) (Vossenaar et al., 2003; Ishigami et al., 2005; Kinloch et al., 2008; Jones et al., 2009).

Interestingly, P. gingivalis secrets an analogous enzyme of PAD, known as PPAD. At present, P. gingivalis is the only known human pathogen producing cysteine-dependent PPAD. PPAD contains N-terminal and C-terminal domains with different functions. N-terminal domains are involved in substrate binding, protein-protein, and protein-nucleic acid interactions. C-terminal domains function as the catalytic domain, which can convert arginine residue to citrullinate. P. gingivalis secretes PPAD in a secreted soluble state or outer membrane vesicle (OMW)-bound state. Thus, PPAD causes aberrant citrullination that contributes to inflammatory responses in PD and can be an etiological agent of RA.

Gingipains, primarily with arginine residues (RgpA and RgpB), are associated with PPAD because fewer citrullinated proteins are detected in the Rgp deletion mutant than the parent strains (Wegner et al., 2010b). In addition, there is increasing evidence that PPAD-catalyzed protein citrullination depends on the function of Rgp proteases. Rgp proteases can cleave polypeptides at arginine residues, freeing a C-terminal arginine residue for PPAD to citrullinate. It is worth noting that Rgps with arginine residues can be citrullinated by PPAD (Stobernack et al., 2016). In addition, PPAD can be autocitrullinated on arginine residue 352, reducing its activity and aid bacterial survival in periodontal pockets (Konig et al., 2015).

In addition to citrullinating bacterial proteins, PPAD has been shown to citrullinate specific human host proteins (Gabarrini et al., 2020). For example, PPAD can citrullinate the epidermal growth factor (EGF), blocking the recognition between the epithelium and the EGF signaling pathway molecules and impairing its ability to stimulate epidermal cell proliferation and migration (Pyrc et al., 2013). In addition, the pore-forming leukotoxin (LtxA) secreted by A. actinomycetemcomitans is involved in host protein citrullination by activating endogenous PAD2 and PAD4 in neutrophils (Engstrom et al., 2018). The mechanisms and pathways responsible for human protein citrullination might indicate associations between oral bacteria and several systematic diseases (atherosclerosis, rheumatic arthritis, and Alzheimer’s disease) (Olsen et al., 2018).

A recent analysis of P. gingivalis genomes of laboratory strains and clinical isolates unveiled a PPAD variant (PPAD-T2), with three amino-acid substitutions directly preceding the catalytic residue H²³⁶(G²³¹N/E²³²T/N²³⁵D) compared with PPAD from the reference strain (PPAD-T1). Functionally, PPAD-T2 exhibits weaker substrate binding capability but higher catalytic rates than PPAD-T1. In addition, like PPAD-1, PPAD-T2 citrullinates arginine at the C-terminal domain of the targeted protein (Bereta et al., 2019).

Additional PTMs have been identified and characterized in oral bacteria, with a vital role in bacterial physiological function and pathogenicity with the development of mass spectrometry. For example, a relatively recently discovered PTM is succinylation. Lysine succinylation has been defined as the transfer of a succinyl group to a lysine residue of the target protein, mediated either enzymatically or non-enzymatically with succinyl-CoA as the donor. Succinylation at lysine residues changes lysine’s charge from +1 to −1 and introduces a relatively larger structure moiety, leading to a mass increase of 100 Da, expected to contribute to significant changes in protein structure and function. Over the past few years, lysine succinylation has been identified in a few oral bacterial species. An example is P. gingivalis. Several important virulence factors, gingipains, fimbriae, RagB, and PorR, occur in lysine succinylation (Wu et al., 2019). Furthermore, protein succinylation and acetylation were extensively overlapped in P. gingivalis, including PGN_0377, PGN_0457, PGN_0497, PGN_0723, PGN_0724, PGN_0725, PGN_1176, PGN_1178, PGN_1341, and PGN_1367, which play a crucial role in the ribosome and metabolic processes (Zeng et al., 2020).

S-glutathionylation is the specific PTM of protein cysteine thiols by incorporating tripeptide glutathione (GSH), in which cysteine thiol and glutathione are classified as low-molecular-mass free (non-protein) thiols (Dalle-Donne et al., 2009). Thiols, organic sulfur derivatives (mercaptans), play a central role in coordinating the antioxidant signaling pathways. As a result, protein S-glutathionylation usually occurs in response to oxidative stress and prevents irreversible oxidation of cysteine residue, which is vital for maintaining intracellular redox homeostasis. A recent report showed that a thioredoxin-like protein (Tlp) is S-glutathionylated at Cys 41 residue, protecting S. mutans from reactive oxygen species (ROS) derived from hostile bacteria, e.g., S. sanguinis and S. gordonii (Li et al., 2020).