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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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nt5e confers Nt5e activity on S. sanguinis SK36 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. (A), (B), and (C) were showed as enzyme velocity vs. concentration of ATP, ADP and AMP substrates, where the results were represented as mean±SE, n = 3. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test for multiple comparisons. *significantly decreased compared to wild-type strain SK36 (P<0.05). Δnt5e: 5′-nucleotidase deletion mutant; ΔnucH: extracellular nuclease deletion mutant; Δcnp: cyclo-nucleotide phosphodiesterase deletion mutant; and Δrad3: DNA repair ATPase deletion mutant.
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pone-0038059-g001: nt5e confers Nt5e activity on S. sanguinis SK36 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. (A), (B), and (C) were showed as enzyme velocity vs. concentration of ATP, ADP and AMP substrates, where the results were represented as mean±SE, n = 3. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test for multiple comparisons. *significantly decreased compared to wild-type strain SK36 (P<0.05). Δnt5e: 5′-nucleotidase deletion mutant; ΔnucH: extracellular nuclease deletion mutant; Δcnp: cyclo-nucleotide phosphodiesterase deletion mutant; and Δrad3: DNA repair ATPase deletion mutant.

Mentions: By mining the SK36 genome, 16 putative surface proteins with LPxTG motifs were identified. Of these proteins, we identified four putative cell-surface nucleotidases based upon the presence of an LPxTG motif and substrate specificities that suggest the ability to hydrolyze a nucleotide and water to a nucleoside and orthophosphate (Table S1). Putative Nt5e showed a substrate specificity that would most likely hydrolyze released platelet adenine nucleotides and yield adenosine. To test this possibility in strain SK36, allelic exchange mutants of nt5e and the other putative nucleotidases were each constructed using primers listed in Tables S3 (SK36) and compared for loss of hydrolysis of extracellular adenine nucleotides (Figure 1). Each strain was incubated with 10, 50, or 100 µM of ATP, ADP or AMP. Hydrolysis is reported as S. sanguinis Nt5e activity per cell. When strain SK36 was compared to deletion mutants of genes for the extracellular nuclease (nucH), cyclo-nucleotide phosphodiesterase (cnp) or DNA repair ATPase (rad3), only Δnt5e cells lost significant ability of whole cells to hydrolyze ATP (Figure 1A, P<0.05), ADP (Figure 1B, P<0.05) and AMP (Figure 1C, P<0.05) at each concentration examined. The ATPase, ADPase and AMPase activities of Δnt5e cells were also associated with a reduction in the lag time to platelet aggregation (Figure S1).


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)

nt5e confers Nt5e activity on S. sanguinis SK36 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. (A), (B), and (C) were showed as enzyme velocity vs. concentration of ATP, ADP and AMP substrates, where the results were represented as mean±SE, n = 3. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test for multiple comparisons. *significantly decreased compared to wild-type strain SK36 (P<0.05). Δnt5e: 5′-nucleotidase deletion mutant; ΔnucH: extracellular nuclease deletion mutant; Δcnp: cyclo-nucleotide phosphodiesterase deletion mutant; and Δrad3: DNA repair ATPase deletion mutant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369921&req=5

pone-0038059-g001: nt5e confers Nt5e activity on S. sanguinis SK36 whole cells.Nt5e activity was measured by the release of inorganic phosphate (Pi) from adenine nucleotides. (A), (B), and (C) were showed as enzyme velocity vs. concentration of ATP, ADP and AMP substrates, where the results were represented as mean±SE, n = 3. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test for multiple comparisons. *significantly decreased compared to wild-type strain SK36 (P<0.05). Δnt5e: 5′-nucleotidase deletion mutant; ΔnucH: extracellular nuclease deletion mutant; Δcnp: cyclo-nucleotide phosphodiesterase deletion mutant; and Δrad3: DNA repair ATPase deletion mutant.
Mentions: By mining the SK36 genome, 16 putative surface proteins with LPxTG motifs were identified. Of these proteins, we identified four putative cell-surface nucleotidases based upon the presence of an LPxTG motif and substrate specificities that suggest the ability to hydrolyze a nucleotide and water to a nucleoside and orthophosphate (Table S1). Putative Nt5e showed a substrate specificity that would most likely hydrolyze released platelet adenine nucleotides and yield adenosine. To test this possibility in strain SK36, allelic exchange mutants of nt5e and the other putative nucleotidases were each constructed using primers listed in Tables S3 (SK36) and compared for loss of hydrolysis of extracellular adenine nucleotides (Figure 1). Each strain was incubated with 10, 50, or 100 µM of ATP, ADP or AMP. Hydrolysis is reported as S. sanguinis Nt5e activity per cell. When strain SK36 was compared to deletion mutants of genes for the extracellular nuclease (nucH), cyclo-nucleotide phosphodiesterase (cnp) or DNA repair ATPase (rad3), only Δnt5e cells lost significant ability of whole cells to hydrolyze ATP (Figure 1A, P<0.05), ADP (Figure 1B, P<0.05) and AMP (Figure 1C, P<0.05) at each concentration examined. The ATPase, ADPase and AMPase activities of Δnt5e cells were also associated with a reduction in the lag time to platelet aggregation (Figure S1).

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