Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence

Abstract

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3',5')-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pde_pg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pde_pg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe-microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Fig. 1. Comprehensive analysis of cyclic mono- and di-nucleotide second messengers in P. gingivalis 381.

A Cellular concentration of ubiquitous bacterial second messengers in the cells of P. gingivalis in biofilm or planktonic modes of growth. Mononucleotide second messengers are in the low-nanogram range (inside box). B c-di-AMP in P. gingivalis ∆pde_pg. C c-di-AMP in P. gingivalis ∆cdaR. Graphs represent the mean ± SE (three biological replicates) of nucleotide second messengers which were analyzed with a student’s t test (**P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant). Scatter plots in Supplementary Fig. 9 display the data distribution. Standard curves are presented in Supplementary Fig. 10. ND not detected, WT wild type.

Fig. 2. Both pde_pg and cdaR genes are required for the synthesis of c-di-AMP and biofilm formation while they regulate differentially the cellular level of c-di-AMP in P. gingivalis in response to biologically relevant stimuli.

A, B Cellular levels of ATP and c-di-AMP in biofilm cells in response to pyruvate availability. C Monospecies biofilm formation with WT, ∆pde_pg, and ∆cdaR; in the presence or absence of pyruvate. D, E Differential regulation of c-di-AMP levels in the WT, ∆pde_pg, and ∆cdaR mutants in response to 10% human serum (D) or 10% saliva (E). Graphs represent the mean ± SE (three biological replicates) of c-di-AMP concentrations which were analyzed with a Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant). Scatter plots in Supplementary Fig. 9 display the data distribution. Standard curves are presented in Supplementary Fig. 10. WT wild-type.

Fig. 3. Comparison of planktonic growth rate and biofilm formation.

A Graph shows that ∆pde_pg displays a lower rate of planktonic growth than the wild-type and ∆cdaR displays a significant growth defect. Cells were cultivated in the CDM-HSA-HK medium. B Biofilm assay using 96-well plates shows that both mutants produce less biofilms than WT in the CDM-HSA-HK medium. However, complementation of the mutants with relevant genes restored biofilm formation to the level of the wild type (C). Graphs represent mean biomass of biofilms ± SE (three biological replicates) at 48 h, as determined by safranin staining, which were analyzed using ANOVA test (Shapiro–Wilk test: P > 0.05). Asterisks indicate pairs of significantly different values (post hoc Tukey’s HSD test: ****P < 0.0001; ns not significant).

Fig. 4. Effects of defective regulation of c-di-AMP levels on biofilm formation of P. gingivalis WT, ∆pde_pg, and ∆cdaR and its colocalization with S. gordonii.

CLSM images of mono- and dual-species biofilms were analyzed by IMARIS image analysis software. The resultant biofilm parameters are summarized in the table. The cell community dimensions are provided as µm³/µm⁻² and the values of three biological replicates were calculated per unit that represent mean biovolume/unit ± SE, compactness/unit ± SE, dead/live ratio, and S. gordonii/P. gingivalis ratio, as determined by appropriate staining methods. Sg S. gordonii, Pg P. gingivalis, WT wild-type.

Fig. 5. Effects of defective regulation of the cellular c-di-AMP level on P. gingivalis cell envelope, the immunoreactivity of LPS, and gingipain activities.

A Cells were stained with osmium tetroxide and uranyl acetate and imaged by transmission electron microscopy. Cells of ∆cdaR mutant displayed a significant shape and cell envelope heterogeneity represented by rod- and round-shaped cells with fully or partially intact cell envelopes that are overloaded with OMVs varying in shape and size (1); cells with an intact monolayer of membrane encompassing agglomerated cytoplasmic materials (2) or entirely void round-shaped structures with an intact monolayer of the membrane (3), cells displaying bare peptidoglycan layers (4). B ELISA assays of cell lysates using anti-P. gingivalis LPS monoclonal antibody. The graph represents the mean ± SE of the immunoreactivity of cell lysates (three biological replicates). The standard curve is presented in Supplementary Fig. 10. C Gingipain-dependent proteolytic activities of P. gingivalis strains cells. Graphs represent the mean ± SE (three biological replicates) of the activity of arginine (BAPNA) and lysine (ALPNA) gingipains which were analyzed with a Student’s t test (**P < 0.01; ***P < 0.001; ****P < 0.0001). Scatter plots in Supplementary Fig. 9 display the data distribution. Sup cell-free supernatant, BAPNA N-α-benzoyl-l-arginine-p-nitroanilide, ALPNA N-α-acetyl-l-lysine-p-nitroanilide.

Fig. 6. Survival of G. mellonella larvae injected with ∼10⁸ CFU/ml of P. gingivalis WT, ∆pde_pg, and ∆cdaR mutants.

The control group was injected with sterile PBS. Survival data were plotted using the Log-rank (Mantel–Cox) test. Graph shows the average of two biological replicates and the standard error (n = 15 larvae per group).