Two DHH subfamily 1 proteins in Streptococcus pneumoniae possess cyclic di-AMP phosphodiesterase activity and affect bacterial growth and virulence

Abstract

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Fig 1

Domain architecture and activity of S. pneumoniae DacA. (A) Domain architecture of DacA. The numbers indicate the positions of amino acids in the full-length protein. TM, transmembrane domain. (B) Enzymatic activity of DacA. ATP was incubated with purified S. pneumoniae DacA, and the reaction mixture was separated using HPLC. ATP and c-di-AMP standards, as well as the reaction with purified B. subtilis DisA, were used as controls.

Fig 2

Cleavage of BNPP by S. pneumoniae Pde1 and Pde2. (A and B) Domain architectures of Pde1 (A) and Pde2 (B). Two different constructs of Pde1, Pde1_51-657 and Pde1_109-657 as indicated by the amino acid positions, were expressed and purified. (C and D) Cleavage of BNPP by Pde1_109-657 (C) and Pde2 (D) in the presence of different metal cations (no cation was present in the control reaction). (E and F) Cleavage of BNPP by Pde1_109-657 (E) and Pde2 (F) under different pH conditions. The data shown in panels C to F are the means of three independent experiments. The error bars denote the standard error of the mean (SEM).

Fig 3

Phosphodiesterase activities of S. pneumoniae Pde1 and Pde2. (A) Pde1_51-657 or Pde2 protein was incubated with c-di-AMP or pApA, as indicated. Each sample was separated by HPLC and monitored at 254 nm. c-di-AMP, pApA, and AMP were used as standards. (B) Verification that AMP is the sole degradation product from c-di-AMP catalyzed by Pde2 using LC–MS-MS. AMP and c-di-AMP were eluted at 3.11 min and 5.72 min, respectively, which were identical to their standards (not shown). The m/z of each nucleotide is indicated. (C) Summary of the catalytic activities of pneumococcal DacA, Pde1, and Pde2 in homeostasis of c-di-AMP.

Fig 4

Cleavage of c-di-AMP and pApA by S. pneumoniae Pde1 and Pde2. (A) c-di-AMP at indicated concentrations was cleaved by Pde1 or Pde2 for 1 h. The enzymatic activity was determined as nmol c-di-AMP cleaved per mg protein per min. (B) pApA was cleaved by Pde2 for 10 min. Enzymatic activity was determined as μmol pApA cleaved by per mg protein per min. Note that different units are displayed for cleaved c-di-AMP (A) and pApA (B). The error bars indicate the SEM.

Fig 5

Construction of in-frame deletion mutants of pde1 and pde2 in S. pneumoniae. (A and B) The in-frame deletion mutants of S. pneumoniae pde1 (A) and pde2 (B) were generated using homologous recombination. The size (in bp) of each ORF is indicated above the gene. The length (in bp) of each intergenic region is also marked between adjacent genes. (C) Western blot analysis to verify the deletion of pde1 and pde2 using antibodies against Pde1 and Pde2, respectively. Subsequent blotting with a mouse anti-PdxS antibody (62) served as a loading control. (D) Detection of bacterial c-di-AMP levels. Bacteria were grown in THY broth to an OD₆₂₀ of 0.3 or 0.8. Each sample was then harvested, resuspended in 50 mM Tris-HCl (pH 8.0), and sonicated. The bacterial debris was removed, and the supernatant was taken to detect c-di-AMP levels using ELISA. The concentration of each sample was normalized with the actual OD₆₂₀. The data shown are the means of three independent experiments. The error bars denote the SEM. *, P < 0.05; **, P < 0.01 in a two-tailed t test using Prism 5.0a (GraphPad Software).

Fig 6

Growth phenotypes of the S. pneumoniae Δpde1 and Δpde2 mutants. (A) Morphology of the WT, Δpde1, Δpde2, and Δpde1 Δpde2 strains after Gram staining. The images were taken at a magnification of ×400. The insets are enlarged an additional 4-fold. The results shown are representative of three independent experiments. (B) Growth curves of the WT, Δpde1, Δpde2, and Δpde1 Δpde2 strains in THY broth. The same CFU from the bacterial stocks were inoculated, and bacterial growth was monitored hourly. The data shown are the means of three independent experiments. The error bars denote the SEM. (C) Response of WT, Δpde1, Δpde2, and Δpde1 Δpde2 strains to UV treatment. Bacterial serial dilutions were spotted onto TSA plates either with or without treatment with UV radiation. The plates were then incubated overnight, and the CFU were enumerated to determine the survival percentage of each bacterial strain. The data shown are the means of three independent experiments. The error bars denote the SEM. *, P < 0.05 in a two-tailed t test using Prism 5.0a (GraphPad Software).

Fig 7

Infection of mice with the pneumococcal phosphodiesterase mutants. Approximately 5 × 10⁶ CFU of the WT, Δpde1, Δpde2, or Δpde1 Δpde2 strain was inoculated intranasally in 50 μl PBS, and the mice were subsequently monitored for 9 days. The indicated P values are the statistical results for each mutant compared to the WT and analyzed by the log-rank (Mantel-Cox) test using Prism 5.0a (GraphPad Software).