Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood

Abstract

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

Keywords: Adenosine; Bacterial Pathogenesis; Enzyme Catalysis; Enzymes; Host-Pathogen Interactions; Metalloenzymes; Nucleoside Nucleotide Metabolism; Streptococcus.

FIGURE 1.

NudP is a monomeric nucleotidase. A, schematic representation of the NudP protein. White boxes highlight the two typical domains of 5′-nucleotidases: the metallophosphatase domain (Metallophos; residues 32–293, pfam00149) and the substrate-binding domain (5′-Nucleotid_C; residues 367–531, pfam02872). Filled black boxes represent the two transmembrane domains (residues 5–27 and 664–683) necessary for secretion (SP, signal peptide; residues 1–28) and LPKTN cell wall anchoring (inverted black triangle; residues 657–661). The position of the conserved NHE motif essential for the stabilization of the transition state of 5′-nucleotidases is highlighted (white triangle; residues 126–128) as well as its corresponding mutation to AGA in the catalytically inactive (cat⁻) mutant NudP*. B, analytical ultracentrifugation analysis of rNudP. rNudP (residues 28–656) produced and purified from E. coli is a monomer with an elongated shape. Sedimentation coefficients are expressed in Svedberg units where 1 S = 10⁻¹³ S.

FIGURE 2.

NudP is a manganese-dependent (deoxy)-NMP/NDP 5′-nucleotidase. A, pH optimum for rNudP phosphatase activity. Release of P_i by the action of the rNudP protein is optimal at pH 7.5. Quantification of P_i was performed with Biomol Green reagents after incubation of 1.5 nm rNudP rNudP with 200 μm AMP in the presence of 5 mm Mn²⁺ for 30 min. Mean and S.D. (error bars) are calculated from three experiments. B, effect of cations on rNudP phosphatase activity. The maximal rNudP phosphatase activity is manganese-dependent. Quantification of P_i was performed as in A except that the pH was fixed to 7.5, and 5 mm concentrations of the different cations were used. rNudP activity is reported as a relative activity (=100 × activity with cation X/maximal observed activity). Mean and S.D. (error bars) are calculated from three experiments. C, kinetic analysis of the rNudP phosphatase activity with AMP, ADP, and ATP as substrates. Experiments were performed at pH 7.5 in the presence of 1.5 nm rNudP, 5 mm Mn²⁺, and 200 μm substrates. Substrate degradation and product formation were followed by rapid resolution HPLC (filled triangles, adenosine; empty squares, AMP; empty circles, ADP; empty diamonds, ATP). Inactive rNudP* with the AGA mutation is shown only with AMP. Mean and S.D. (error bars) are calculated from at least three independent experiments.

FIGURE 3.

NudP is a cell wall-associated protein acting on extracellular nucleotides. A, immunodetection of NudP by Western analysis of GBS cell wall and secreted proteins. Specific antibodies directed against purified rNudP were used to detect NudP in the cell wall (CW) and in the concentrated culture supernatants (SN) of the NEM316 WT strain, the mutant expressing the inactivated AGA substitution form (NudP*), the corresponding isogenic complemented strain (WTbk), the inactive SrtA* mutant, and the nudP deletion mutant (ΔnudP). Similar amounts of total cell wall and secreted proteins, corresponding to the extraction from 10⁸ bacterial cells in midexponential phase in TH broth at 37 °C, were loaded. Shown is a representative experiment of at least three independent experiments. Note that NudP is specifically revealed by two bands migrating closely. As both were absent in the ΔnudP extracts, we assume that the smaller band is the result of proteolysis. B, kinetic analysis of the cell wall-associated phosphatase activity in the NudP* mutant, the WTbk complemented strain, and the SrtA* mutant with AMP and ADP as substrates. Experiments were performed at pH 7.5 in the presence of 0.1 μg of cell wall proteins, 5 mm Mn²⁺, and 200 μm substrates. Substrate degradation and product formation were followed by rapid resolution HPLC (filled triangles, adenosine; empty squares, AMP; empty circles, ADP).

FIGURE 4.

The NudP enzymatic activity contributes to GBS survival in blood and colonization of internal organs. A, NudP contributes to GBS survival in blood. 5 × 10⁷ bacterial cells of the nudP* mutant (white bars) or the isogenic WTbk complemented strain (black bars) were incubated at 37 °C with fresh blood of BALB/c mice supplemented with 150 μm adenosine (+ Ado) or guanosine (+ Gua). Aliquots were taken at the indicated time points (30 and 60 min), and bacterial survival was calculated after 24-h incubation on an agar plate as the total number of cfu at a given time versus the number of cfu at time 0. Mean and S.D. (error bars) are calculated from two independent experiments in duplicate, and statistical significance is indicated by asterisks (unpaired t test; ***, p < 0.001; **, p < 0.01). B, NudP contributes to GBS virulence in neonate animals. 5 × 10⁶ bacterial cells of the nudP* mutant (empty circles) or the isogenic WTbk complemented strain (filled squares) were inoculated intraperitoneally into 2-day-old neonatal Sprague-Dawley rats. Animal survival was followed for 5 days, and the mortality curve is the result of two independent experiments with 2 × 10 animals inoculated with bacterial strains. C, NudP contributes to colonization of internal organs. 5 × 10⁷ bacterial cells of the nudP* mutant (empty circles) or the isogenic WTbk complemented strain (filled squares) were injected intravenously into 5-week-old BALB/c mice. At 24 and 48 h postinfection, groups of eight mice were sacrificed, and organ colonization was quantified by cfu counting. One representative experiment is shown with statistical significance indicated by asterisks (unpaired t test; ***, p < 0.001; **, p < 0.01).