Porphyromonas gingivalis, an important periodontal pathogen, has been increasingly recognized for its potential role in the onset and progression of various systemic diseases, including rheumatoid arthritis, cardiovascular diseases, and neurodegenerative disorders [1]. A critical aspect of its pathogenicity is its interaction with the host immune system, particularly through its lipopolysaccharides (LPSs). Like other Gram-negative bacteria, P. gingivalis LPS generally consists of polysaccharides (O-antigen), core oligosaccharides, and lipid A (endotoxin). However, the unique characteristic of P. gingivalis LPS is that, depending on environmental factors such as haemin levels and phosphate availability, P. gingivalis LPS can exist in different isomeric forms, tetra-acylated (LPS_1,435/1,449) and penta-acylated (LPS_1,690), which selectively activate Toll-like receptor 2 (TLR2) and TLR4 in the host. These unique acylation patterns, which dynamically respond to environmental conditions, play a crucial role in evading host immune detection and ultimately increasing the survival of periodontal pathogens [2-4].

Meanwhile, it is notable that human TLRs may play a key role in neuropathic pain and migraine etiology by activating microglia. The pathogenesis of migraine may involve TLR-mediated crosstalk between neurons and immune cells [5]. Notably, a growing body of research has shown that TLR4 is a key receptor associated with persistent pain [6-9]. Given that different LPS isoforms of P. gingivalis engage with TLR2 and TLR4 in distinct ways, resulting in varied immune responses, this adds credibility to the hypothesis that these interactions may influence pain signaling pathways.

In addition to the immune evasion mechanisms based on the heterogeneity of LPS, P. gingivalis relies on various survival strategies, including the synthesis of inorganic polyphosphate (polyP). PolyP is a chain-like polymer made up of dozens to hundreds of orthophosphate (Pi) units connected by high-energy phosphoanhydride bonds [10]. In fact, polyP is not unique to P. gingivalis; this ubiquitous compound is found in bacteria, fungi, algae, plants, and animals [10]. In bacteria, intracellular polyP serves diverse roles, including acting as an adenosine triphosphate source and substitute, regulating intracellular ion levels, facilitating DNA entry, and participating in stress responses and pathogen virulence [10,11]. Polyphosphate kinase (PPK), which is responsible for polyP synthesis, is highly conserved across many bacterial species, including P. gingivalis, Escherichia coli, Helicobacter pylori, Salmonella enterica subsp. enterica Serovar Typhimurium, and Lactobacillus plantarum [10,12]. Mutants lacking the ppk gene, which encodes PPK, exhibit defects in motility, quorum sensing, biofilm formation, and virulence. For example, deletion of the ppk gene in E. coli results in an inability to express the sigma factor of RNA polymerase (RpoS) [10]. RpoS is a regulatory protein that controls more than 50 genes involved in stationary-phase adaptation, providing resistance to starvation, heat, oxidative stress, and ultraviolet irradiation [10]. Ultimately, ppk mutants die during the stationary phase after only a few days [10]. The ppk mutants of Pseudomonas aeruginosa exhibit apparently normal flagella and pili under conditions favorable for swimming and twitching but fail to develop an additional flagellum required for swarming [10].

However, it is crucial to distinguish between the roles of intracellular polyP and exogenous polyP. While intracellular polyP plays essential roles within the bacterial cell, exogenous polyP has garnered significant attention as an antimicrobial agent, inhibiting the growth of various Gram-positive bacteria, fungi, and some Gram-negative bacteria, including P. gingivalis and Prevotella intermedia [11,13]. However, the full scope of the functions of intracellular polyP in P. gingivalis, particularly under stress conditions, remains incompletely understood.

Understanding the pathogenic mechanisms of P. gingivalis is crucial due to its far-reaching effects beyond the oral cavity. This study aims to elucidate the role of intracellular polyP in the survival of P. gingivalis. To achieve this, this study centered on examining gene expression alterations in the ppk-deficient mutant P. gingivalis CW120 when exposed to exogenous polyP known for its antimicrobial properties, using the parent strain P. gingivalis 381 as a reference for comparison.

1. Bacterial Strains

The bacterial strains used in this study included the wild-type P. gingivalis 381 and its isogenic mutant P. gingivalis CW120. Both strains were provided by the Department of Microbiology, College of Dentistry, Kyung Hee University in 2005. The P. gingivalis CW120 strain is identical to the one created by Chen et al. [12], harboring an inactivated ppk gene due to the insertion of an erythromycin resistance gene, thereby preventing normal PPK synthesis.

2. Media and Chemicals

P. gingivalis strains were cultured anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C in half-strength brain heart infusion (BHI; Difco) supplemented with yeast extract (5 mg/mL), hemin (5 μg/mL), and vitamin K₁ (0.2 μg/mL). Sodium polyP with an average chain length of 75 (Sigma), was dissolved in distilled water to a 10% (wt/vol) solution, sterilized using a 0.22-μm filter, and stored at −20°C until needed. In a preliminary experiment, we observed that at concentrations of 0.04% and above, the growth of P. gingivalis was completely inhibited by exogenous polyP, and the extracted RNA was too degraded to perform microarray analysis. Therefore, 0.03% polyP was selected as the optimal concentration, which inhibited bacterial growth while allowing for the extraction of high-quality RNA.

3. Total RNA Extraction and cDNA Synthesis

Cultures of P. gingivalis strains 381 and CW120 were grown to an early exponential phase with an optical density at 600 nm (OD₆₀₀) of 0.3, and then split into two portions. One portion remained untreated, while the other was exposed to 0.03% polyP. Both bacterial cultures were incubated for 2 hours under anaerobic conditions, after which the cells were collected. Total RNA was extracted from the harvested cells using Trizol Reagent (Invitrogen). The quality of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies), and its quantity was determined by spectrophotometry, with an OD260/280 ratio ranging from 1.99 to 2.09. Subsequently, cDNA was synthesized from 20 μg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen).

4. Microarray and Validation

Four Cy3-labeled cDNA samples from each P. gingivalis strain (381 and CW120, exposed or not exposed to exogenous polyP) were hybridized onto DNA microarrays (Nimblegen Systems) containing the full genome of P. gingivalis. Each microarray chip included five genome replicates. The genomes were represented by an average of 19 different 60-mer probes per gene, with at least three mismatches compared to other 60-mers. Quality control was conducted using on-chip control oligonucleotides. Data extraction was performed using an Axon GenePix 4000B microarray scanner, and quantile normalization was applied across the replicate arrays. Genes showing more than a 2-fold change in expression were identified as differentially expressed genes (DEGs) based on t-tests (p<0.05). All statistical analyses were performed with R version 4.1.

To validate the microarray findings, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed on seven selected genes: PG0593 (htrA protein), PG1089 (transcription regulator), PG1019 (putative lipoprotein), PG1180 (hypothetical protein), PG1983 (clustered regularly interspaced short palindromic repeats-associated protein TM1791 family), PG0885 (chorismate mutase), and PG1181 (transcriptional regulator, tetR family). These genes were selected based on consistent expression patterns across P. gingivalis CW120, 381, and W83, representing a range of different cellular functions. The expression data for P. gingivalis W83 were obtained from a previously published study [14]. Specific primers for these genes, as well as for the P. gingivalis 16S rRNA gene used for normalization, were designed based on the sequences provided in the referenced literature [14]. The transcriptional ratios obtained from qRT-PCR were then converted to logarithmic values and compared with the average log₂ ratio values from the microarray analysis.

By applying a filtering criterion of a 2-fold or greater change in expression and a significance level of p<0.05, 583 genes in P. gingivalis CW120 were identified as DEGs following exogenous polyP treatment (Supplementary Fig. 1). Among these DEGs, 273 were upregulated, while 265 were downregulated. The functions of 117 upregulated and 164 downregulated genes (a total of 281 genes) were identified using the National Center for Biotechnology Information P. gingivalis genome database (Supplementary Table 1, Supplementary Fig. 2), while the remaining genes had unknown functions. To validate the microarray findings, qRT-PCR was conducted on randomly selected genes and normalized using the 16S rRNA gene. The expression ratios obtained showed a strong correlation with the microarray results (r=0.9307) (Fig. 1A). This strong correlation indicates that the microarray results are reliable and consistent with the more accurate real-time PCR measurements.

The gene expression changes in the P. gingivalis CW120 were generally similar to those in the wild-type 381 strain (Fig. 1B). However, the upregulation of protein synthesis-related genes and the downregulation of energy metabolism-related genes (p<0.05, ≥2-fold change in expression) were more pronounced in the mutant strain. Notably, both strains exhibited significant changes in the expression of genes related to the cell envelope (p<0.05, ≥2-fold change in expression).

Among 36 DEGs associated with protein synthesis, the majority (31 genes) were upregulated, while only 5 were downregulated. Most of the upregulated genes encoded ribosomal proteins (Table 1, Fig. 2A). A total of 46 DEGs were associated with energy metabolism, with 6 genes upregulated and 40 downregulated. Among the downregulated genes, those involved in electron transport were the most prevalent, followed by genes related to anaerobic/fermentation, and amino acids and amines (Table 2, Fig. 2B).

Regarding the cell envelope, there were 38 DEGs in total, with 14 genes upregulated and 24 downregulated (Table 3, Fig. 2C). Among the genes related to the biosynthesis and degradation of surface polysaccharides and LPS, the number of downregulated genes was more than double that of the upregulated ones. There were no upregulated genes involved in the biosynthesis and degradation of peptidoglycan (murein), while genes encoding N-acetylglucosaminyl transferase, Mur ligase domain protein, uridine diphosphate (UDP)-N-acetylmuramate—L-alanine ligase, and UDP-N-acetylmuramoylalanine—D-glutamate ligase were downregulated. Additionally, several genes related to cell division, such as filamenting temperature-sensitive (Fts)Z, FtsQ, and FtsA, as well as genes associated with DNA replication and the biosynthesis of purine and pyrimidine, showed significant downregulation upon exposure to exogenous polyP in the CW120 strain (Table 4).

In this study, we aimed to investigate the role of the ppk gene in P. gingivalis by analyzing changes in gene expression in both the CW120 mutant and the wild-type 381 strains under stress conditions induced by the addition of polyP75, known for its inhibitory effects on bacterial growth. Among the genes related to energy metabolism, the number of downregulated genes was over six times greater than that of upregulated genes. This widespread downregulation encompassed key areas of energy metabolism, including amino acids and amines, anaerobic/fermentation pathways, and electron transport processes. This trend was particularly pronounced in the mutant CW120 strain compared to the wild-type 381. These findings suggest that exposure to exogenous polyP leads to a significant reduction in overall energy metabolism in P. gingivalis, with the ppk gene likely playing a crucial role in the bacterial response to such stress conditions.

The expression changes observed in cell envelope-related genes, particularly those involved in LPS synthesis and degradation, were similar between the wild-type 381 and the CW120 mutant strains, indicating that the ppk gene might not be directly linked to these specific alterations. However, the exposure to exogenous polyP induced significant modifications in LPS expression, which could have profound implications for the virulence of P. gingivalis and its potential association with orofacial pain. LPS is a crucial factor in P. gingivalis’s interaction with the host immune system, particularly through TLRs. Changes in LPS expression or structure could alter how these receptors, especially TLR2 and TLR4, recognize and respond to the bacterium. Such modifications might enhance or diminish the inflammatory response, potentially leading to variations in immune system activation and the development of chronic pain conditions. These findings suggest that while the ppk gene itself may not be directly influencing LPS-related gene expression, the presence of exogenous polyP could still play a significant role in modulating host-pathogen interactions through changes in LPS, thereby affecting the bacterium's pathogenic potential.

In addition to P. gingivalis, several other bacteria have been shown to modulate TLR expression in response to stress conditions. For example, E. coli and S. enterica have been reported to influence TLR expression during oxidative and osmotic stress [15]. These interactions highlight the broader relevance of stress-induced bacterial modulation of TLR pathways in host immune responses. This suggests that the mechanisms we observed in P. gingivalis may extend to other pathogens, further emphasizing the importance of studying bacterial stress responses in relation to TLR signaling. Additional research is needed to fully elucidate how exogenous polyP influences LPS structure and function, and to determine the broader implications of these changes for P. gingivalis pathogenicity and its role in chronic inflammatory conditions and orofacial pain.

During bacterial cell division, DNA replication occurs first, followed by the attachment of each DNA strand to opposite cell membranes. This process leads to cytokinesis, where the cell membrane invaginates in the middle, eventually splitting into two daughter cells. A key protein in this process is FtsZ, a tubulin-like protein that polymerizes to form the division septum [16,17]. In P. gingivalis, genes coding for FtsZ and other division-related proteins, such as FtsW, FtsQ, and FtsA, are clustered together. The findings indicate that the expression of all these genes was significantly downregulated in the presence of polyP, suggesting that polyP exposure disrupts the normal cell division process, which is further supported by the observed decrease in genes involved in purine and pyrimidine biosynthesis and DNA replication. This implies that polyP inhibits not just nucleic acid synthesis and DNA replication, but also the entire bacterial proliferation process.

Interestingly, despite the overall inhibition of growth, division, and energy metabolism by polyP, there was a marked increase in the expression of various ribosomal proteins (r-proteins), particularly in the CW120 mutant compared to the wild-type 381. Ribosomes are the sites of translation, and an increase in r-proteins generally suggests heightened protein synthesis, which seems contradictory given the reduced bacterial growth and division. This paradoxical phenomenon might be explained by the autogenous feedback regulation observed in E. coli and Bacillus subtilis, where certain r-proteins, such as L1, S4, and S8, act as key regulators within the same operon to inhibit the expression of other r-proteins [18-20]. This regulation likely occurs at the translational level due to structural similarities between the rRNA binding sites and the mRNA of the r-proteins, as hypothesized by Nomura et al. [18]. Although the mechanisms of ribosomal protein synthesis in P. gingivalis have not been fully explored, it is plausible that similar structural analogies exist in this organism. If so, excessive expression of specific r-protein subunits could suppress the expression of others, potentially interfering with normal ribosome assembly and translation processes. The lack of significant changes in genes related to amino acid synthesis, despite the increased expression of r-proteins, further supports the notion that increased r-protein levels do not necessarily correlate with enhanced protein synthesis in the context of polyP exposure. In summary, this study provides significant insights into the role of the ppk gene in P. gingivalis, particularly under stress conditions induced by exogenous polyP. The findings demonstrate that polyP exposure leads to widespread transcriptional changes, including the downregulation of energy metabolism and cell division-related genes, while paradoxically upregulating ribosomal protein genes. These results highlight the complex regulatory networks within P. gingivalis that respond to environmental stressors. Importantly, while LPS-related gene expression changes were observed, these changes were not directly linked to the ppk gene, suggesting other regulatory mechanisms are at play. Nevertheless, the alteration of LPS expression and its potential impact on TLR-mediated immune responses underscores the possible link between polyP exposure and the modulation of bacterial virulence, which could contribute to the pathogenesis of orofacial pain.

The significance of this study lies in its contribution to understanding how P. gingivalis adapts to stress, providing a potential target for therapeutic interventions aimed at mitigating the bacterium’s role in chronic inflammatory conditions. However, the study’s limitations include the focus on transcriptional changes without assessing the corresponding protein levels or the functional outcomes of these changes in a host environment. Future studies should explore the proteomic and functional aspects of these findings, particularly in relation to how polyP-induced LPS modifications influence host-pathogen interactions and the development of pain. Such research could pave the way for new strategies to manage or prevent P. gingivalis-associated diseases, including those that manifest as chronic pain conditions.

No potential conflict of interest relevant to this article was reported.

The data sets used in this study are available from the corresponding author upon reasonable request.

None.

We gratefully acknowledge the Department of Microbiology, College of Dentistry, Kyung Hee University, for providing the bacterial strains used in this study.

Supplementary data is available at https://doi.org/10.14476/jomp.2024.49.4.91.