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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 is a trypsin-cleavable surface protein of S. sanguinis and affects platelet aggregation lag time.(A) Gel filtration chromatography of 7-minute tryptic digest of S. sanguinis 133-79. 6.4mg was placed on a column of Sephadex G-100 and chromatographed as described under “Materials and methods”. (B) SDS-PAGE analysis of S. sanguinis tryptic digest fractions from gel filtration chromatography. All samples contained 15 µg of protein solubilized in 1% (w/v) SDS sample buffer. These samples were electrophoresed on a 10% gel, and stained with Coomassie Blue. Lane A, trypsin. Lane B, flow-through from void volume. Lane C, starting 7-minute crude tryptic digest. Lane D, Sephadex G-100 fraction 1. Lane E, Sephadex G-100 fraction 2. Lane F, Sephadex G-100 fraction 3. Lane G, Sephadex G-100 fraction 4. Lane H, Sephadex G-100 fraction 5. (C) PRP was stirred in an aggregometer. Wild type and Δnt5e strains were added at the S. sanguinis 133-79-labeled arrowhead and aggregation was measured as increasing light transmission. The start of aggregation of each strain was indicated by arrow. The aggregation tracing in response to the nt5e+ strain (not shown) was indistinguishable from the wild type. (D) Response leading to aggregation was recorded as the mean lag-time to onset of aggregation±SE, N = 4; Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test for multiple comparisons. * significantly decreased compared to wt (P<0.05).
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pone-0038059-g003: Nt5e is a trypsin-cleavable surface protein of S. sanguinis and affects platelet aggregation lag time.(A) Gel filtration chromatography of 7-minute tryptic digest of S. sanguinis 133-79. 6.4mg was placed on a column of Sephadex G-100 and chromatographed as described under “Materials and methods”. (B) SDS-PAGE analysis of S. sanguinis tryptic digest fractions from gel filtration chromatography. All samples contained 15 µg of protein solubilized in 1% (w/v) SDS sample buffer. These samples were electrophoresed on a 10% gel, and stained with Coomassie Blue. Lane A, trypsin. Lane B, flow-through from void volume. Lane C, starting 7-minute crude tryptic digest. Lane D, Sephadex G-100 fraction 1. Lane E, Sephadex G-100 fraction 2. Lane F, Sephadex G-100 fraction 3. Lane G, Sephadex G-100 fraction 4. Lane H, Sephadex G-100 fraction 5. (C) PRP was stirred in an aggregometer. Wild type and Δnt5e strains were added at the S. sanguinis 133-79-labeled arrowhead and aggregation was measured as increasing light transmission. The start of aggregation of each strain was indicated by arrow. The aggregation tracing in response to the nt5e+ strain (not shown) was indistinguishable from the wild type. (D) Response leading to aggregation was recorded as the mean lag-time to onset of aggregation±SE, N = 4; Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test for multiple comparisons. * significantly decreased compared to wt (P<0.05).

Mentions: To show that Nt5e is a cell wall-associated enzyme, we characterized a cell surface protein fraction with the requisite enzyme activities. Recovery of the cell surface proteins fragments from S. sanguinis 133-79 was maximal after 7 minutes of TPCK-trypsin digestion (data not shown). The 7-minute tryptic digests (crude digest) of S. sanguinis 133-79 were chromatographed on a column of Sephadex G-100, pooled (Figure 3A), fractions resolved using SDS-PAGE (Figure 3B), and analyzed for platelet interactions (Table 2). Fraction 3 (G100-3) had the greatest ability to inhibit S. sanguinis-induced platelet aggregation, but had no effect on platelet-S. sanguinis adhesion (data not shown). After separation of Fraction 3 by two-dimensional SDS gel electrophoresis and analysis by mass spectrometry, two putative 5′-nucleotidase superfamily proteins were identified (Table 3) [13]. The crude digest and fraction G100-3 showed 5′-nucleotidase activities (Table 2). Compared with the crude digest, G100-3 had higher ATPase, ADPase and AMPase activities, which correlated with the inhibition of platelet aggregation (Table 2). In the plasma, both ADP removal and adenosine generation can inhibit platelet aggregation [24]. Taken together, 5′-nucleotidase is a protein cleaved from the surface of S. sanguinis 133-79, which appeared to metabolize adenine nucleotides to modulate platelet aggregation.


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 is a trypsin-cleavable surface protein of S. sanguinis and affects platelet aggregation lag time.(A) Gel filtration chromatography of 7-minute tryptic digest of S. sanguinis 133-79. 6.4mg was placed on a column of Sephadex G-100 and chromatographed as described under “Materials and methods”. (B) SDS-PAGE analysis of S. sanguinis tryptic digest fractions from gel filtration chromatography. All samples contained 15 µg of protein solubilized in 1% (w/v) SDS sample buffer. These samples were electrophoresed on a 10% gel, and stained with Coomassie Blue. Lane A, trypsin. Lane B, flow-through from void volume. Lane C, starting 7-minute crude tryptic digest. Lane D, Sephadex G-100 fraction 1. Lane E, Sephadex G-100 fraction 2. Lane F, Sephadex G-100 fraction 3. Lane G, Sephadex G-100 fraction 4. Lane H, Sephadex G-100 fraction 5. (C) PRP was stirred in an aggregometer. Wild type and Δnt5e strains were added at the S. sanguinis 133-79-labeled arrowhead and aggregation was measured as increasing light transmission. The start of aggregation of each strain was indicated by arrow. The aggregation tracing in response to the nt5e+ strain (not shown) was indistinguishable from the wild type. (D) Response leading to aggregation was recorded as the mean lag-time to onset of aggregation±SE, N = 4; Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test for multiple comparisons. * significantly decreased compared to wt (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369921&req=5

pone-0038059-g003: Nt5e is a trypsin-cleavable surface protein of S. sanguinis and affects platelet aggregation lag time.(A) Gel filtration chromatography of 7-minute tryptic digest of S. sanguinis 133-79. 6.4mg was placed on a column of Sephadex G-100 and chromatographed as described under “Materials and methods”. (B) SDS-PAGE analysis of S. sanguinis tryptic digest fractions from gel filtration chromatography. All samples contained 15 µg of protein solubilized in 1% (w/v) SDS sample buffer. These samples were electrophoresed on a 10% gel, and stained with Coomassie Blue. Lane A, trypsin. Lane B, flow-through from void volume. Lane C, starting 7-minute crude tryptic digest. Lane D, Sephadex G-100 fraction 1. Lane E, Sephadex G-100 fraction 2. Lane F, Sephadex G-100 fraction 3. Lane G, Sephadex G-100 fraction 4. Lane H, Sephadex G-100 fraction 5. (C) PRP was stirred in an aggregometer. Wild type and Δnt5e strains were added at the S. sanguinis 133-79-labeled arrowhead and aggregation was measured as increasing light transmission. The start of aggregation of each strain was indicated by arrow. The aggregation tracing in response to the nt5e+ strain (not shown) was indistinguishable from the wild type. (D) Response leading to aggregation was recorded as the mean lag-time to onset of aggregation±SE, N = 4; Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test for multiple comparisons. * significantly decreased compared to wt (P<0.05).
Mentions: To show that Nt5e is a cell wall-associated enzyme, we characterized a cell surface protein fraction with the requisite enzyme activities. Recovery of the cell surface proteins fragments from S. sanguinis 133-79 was maximal after 7 minutes of TPCK-trypsin digestion (data not shown). The 7-minute tryptic digests (crude digest) of S. sanguinis 133-79 were chromatographed on a column of Sephadex G-100, pooled (Figure 3A), fractions resolved using SDS-PAGE (Figure 3B), and analyzed for platelet interactions (Table 2). Fraction 3 (G100-3) had the greatest ability to inhibit S. sanguinis-induced platelet aggregation, but had no effect on platelet-S. sanguinis adhesion (data not shown). After separation of Fraction 3 by two-dimensional SDS gel electrophoresis and analysis by mass spectrometry, two putative 5′-nucleotidase superfamily proteins were identified (Table 3) [13]. The crude digest and fraction G100-3 showed 5′-nucleotidase activities (Table 2). Compared with the crude digest, G100-3 had higher ATPase, ADPase and AMPase activities, which correlated with the inhibition of platelet aggregation (Table 2). In the plasma, both ADP removal and adenosine generation can inhibit platelet aggregation [24]. Taken together, 5′-nucleotidase is a protein cleaved from the surface of S. sanguinis 133-79, which appeared to metabolize adenine nucleotides to modulate platelet aggregation.

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