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Ecto-5'-nucleotidase: a candidate virulence factor in Streptococcus sanguinis experimental endocarditis.

Fan J, Zhang Y, Chuang-Smith ON, Frank KL, Guenther BD, Kern M, Schlievert PM, Herzberg MC - PLoS ONE (2012)

Bottom Line: Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P=0.98).In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P=0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo.In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5'-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P=0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P=0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.

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Characterization of Nt5e activity on S. sanguinis 133-79 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. For (A), (B), and (C), the Michaelis-Menten curves were showed as enzyme velocity (represented as nmole/min/106 cells) vs. concentration of ATP, ADP and AMP substrates. (D) Effect of Nt5e inhibitor APCP on AMPase activity of S. sanguinis 133-79. The curve was fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit was shown. (E) Michaelis-Menten curves of AMPase activity vs. substrate concentration in the absence and presence of APCP. (F) pH dependence of AMPase activity of Nt5e. Statistical analysis was performed using non-linear regression. The results were represented as mean±SE, n = 3; *significantly decreased compared to no inhibitor (P<0.05).
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pone-0038059-g002: Characterization of Nt5e activity on S. sanguinis 133-79 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. For (A), (B), and (C), the Michaelis-Menten curves were showed as enzyme velocity (represented as nmole/min/106 cells) vs. concentration of ATP, ADP and AMP substrates. (D) Effect of Nt5e inhibitor APCP on AMPase activity of S. sanguinis 133-79. The curve was fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit was shown. (E) Michaelis-Menten curves of AMPase activity vs. substrate concentration in the absence and presence of APCP. (F) pH dependence of AMPase activity of Nt5e. Statistical analysis was performed using non-linear regression. The results were represented as mean±SE, n = 3; *significantly decreased compared to no inhibitor (P<0.05).

Mentions: Platelets from some donors do not aggregate in response to strain SK36, reflecting donor specificity [21]. To determine whether the enzymatic activities were expressed by other platelet-aggregating strains, we also studied S. sanguinis 133-79. Whole cells of S. sanguinis 133-79 wild-type (wt) hydrolyzed the adenine nucleotides ATP (Figure 2A), ADP (Figure 2B) and AMP (Figure 2C), following Michaelis-Menten kinetics. The Km and Vmax for the different strains tested are summarized in Table 1. Using the Δnt5e strain, the ATPase activity was significantly decreased (P<0.01), whereas activity in this mutant was restored to wt levels by complementation (nt5e+) (Figure 2A). In the absence of nt5e, the ADPase activity was reduced, whereas activity in this mutant was restored to wt levels by complementation (nt5e+) (Figure 2B). Similarly, AMPase activity in S. sanguinis 133-79 was fully abrogated by deletion of nt5e (Figure 2C). When complemented, nt5e+ regained AMPase activity. Indeed, Δnt5e generated inorganic phosphate only with ATP as substrate, which may reflect activity of an ecto-ATPase on the cell surface of S. sanguinis as we reported [9]. When compared to NT5E, ecto-ATPase activity did not play a major role in the hydrolysis of extracellular adenine nucleotides. Collectively, these results suggest strongly that Nt5e on S. sanguinis 133-79 hydrolyzes the adenine nucleotides ATP, ADP and AMP.


Ecto-5'-nucleotidase: a candidate virulence factor in Streptococcus sanguinis experimental endocarditis.

Fan J, Zhang Y, Chuang-Smith ON, Frank KL, Guenther BD, Kern M, Schlievert PM, Herzberg MC - PLoS ONE (2012)

Characterization of Nt5e activity on S. sanguinis 133-79 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. For (A), (B), and (C), the Michaelis-Menten curves were showed as enzyme velocity (represented as nmole/min/106 cells) vs. concentration of ATP, ADP and AMP substrates. (D) Effect of Nt5e inhibitor APCP on AMPase activity of S. sanguinis 133-79. The curve was fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit was shown. (E) Michaelis-Menten curves of AMPase activity vs. substrate concentration in the absence and presence of APCP. (F) pH dependence of AMPase activity of Nt5e. Statistical analysis was performed using non-linear regression. The results were represented as mean±SE, n = 3; *significantly decreased compared to no inhibitor (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369921&req=5

pone-0038059-g002: Characterization of Nt5e activity on S. sanguinis 133-79 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. For (A), (B), and (C), the Michaelis-Menten curves were showed as enzyme velocity (represented as nmole/min/106 cells) vs. concentration of ATP, ADP and AMP substrates. (D) Effect of Nt5e inhibitor APCP on AMPase activity of S. sanguinis 133-79. The curve was fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit was shown. (E) Michaelis-Menten curves of AMPase activity vs. substrate concentration in the absence and presence of APCP. (F) pH dependence of AMPase activity of Nt5e. Statistical analysis was performed using non-linear regression. The results were represented as mean±SE, n = 3; *significantly decreased compared to no inhibitor (P<0.05).
Mentions: Platelets from some donors do not aggregate in response to strain SK36, reflecting donor specificity [21]. To determine whether the enzymatic activities were expressed by other platelet-aggregating strains, we also studied S. sanguinis 133-79. Whole cells of S. sanguinis 133-79 wild-type (wt) hydrolyzed the adenine nucleotides ATP (Figure 2A), ADP (Figure 2B) and AMP (Figure 2C), following Michaelis-Menten kinetics. The Km and Vmax for the different strains tested are summarized in Table 1. Using the Δnt5e strain, the ATPase activity was significantly decreased (P<0.01), whereas activity in this mutant was restored to wt levels by complementation (nt5e+) (Figure 2A). In the absence of nt5e, the ADPase activity was reduced, whereas activity in this mutant was restored to wt levels by complementation (nt5e+) (Figure 2B). Similarly, AMPase activity in S. sanguinis 133-79 was fully abrogated by deletion of nt5e (Figure 2C). When complemented, nt5e+ regained AMPase activity. Indeed, Δnt5e generated inorganic phosphate only with ATP as substrate, which may reflect activity of an ecto-ATPase on the cell surface of S. sanguinis as we reported [9]. When compared to NT5E, ecto-ATPase activity did not play a major role in the hydrolysis of extracellular adenine nucleotides. Collectively, these results suggest strongly that Nt5e on S. sanguinis 133-79 hydrolyzes the adenine nucleotides ATP, ADP and AMP.

Bottom Line: Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P=0.98).In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P=0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo.In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5'-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P=0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P=0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.

Show MeSH
Related in: MedlinePlus