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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 affects vegetation weight and bacterial load in a S. sanguinis rabbit endocarditis model.(A) Aortic valve of a rabbit infected with S.sanguinis 133-79 Δnt5e. The aortic valve is composed of three leaflets, no visible vegetations were found and 2.5×103 CFUs were recovered. (B) Aortic valve of a rabbit infected with S. sanguinis 133-79 nt5e+, with one vegetation in the center leaflet (white circle) and 3.3×108 CFUs were recovered. (C) Aortic valve of a rabbit infected with S. sanguinis 133-79 wt, with two vegetations on center and right leaflets (white circle), respectively and 3.4×109 CFUs were recovered. (D) Plot of vegetation bacterial load (total CFU) versus vegetation mass. All vegetations on the aortic valve of each rabbit were pooled to obtain the vegetation weight and bacterial load (on TH plate). When no vegetations were found, the valves were scraped with a blade and plated to determine the valve bacterial load. R2 = 0.66 (n = 31) indicated that there is a correlation between the bacterial load and vegetation masses. (E) Bacterial loads in the rabbit endocarditis model, enumerated as log10 total CFU 4 days after infection. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test. Horizontal bars represent mean CFUs in each cohort.
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pone-0038059-g004: Nt5e affects vegetation weight and bacterial load in a S. sanguinis rabbit endocarditis model.(A) Aortic valve of a rabbit infected with S.sanguinis 133-79 Δnt5e. The aortic valve is composed of three leaflets, no visible vegetations were found and 2.5×103 CFUs were recovered. (B) Aortic valve of a rabbit infected with S. sanguinis 133-79 nt5e+, with one vegetation in the center leaflet (white circle) and 3.3×108 CFUs were recovered. (C) Aortic valve of a rabbit infected with S. sanguinis 133-79 wt, with two vegetations on center and right leaflets (white circle), respectively and 3.4×109 CFUs were recovered. (D) Plot of vegetation bacterial load (total CFU) versus vegetation mass. All vegetations on the aortic valve of each rabbit were pooled to obtain the vegetation weight and bacterial load (on TH plate). When no vegetations were found, the valves were scraped with a blade and plated to determine the valve bacterial load. R2 = 0.66 (n = 31) indicated that there is a correlation between the bacterial load and vegetation masses. (E) Bacterial loads in the rabbit endocarditis model, enumerated as log10 total CFU 4 days after infection. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test. Horizontal bars represent mean CFUs in each cohort.

Mentions: To investigate the contribution of Nt5e to S. sanguinis virulence in infective endocarditis, rabbits with experimental heart valve injury were infected by intravenous inoculation with 1×109 CFU S. sanguinis 133-79 wt, Δnt5e, or nt5e+. Growth curves in TH broth were performed for all three strains to test for changes in cell growth or fitness. Although the nt5e deletion mutant showed a slight delay in entry to log-phase, all strains displayed similar growth rates in TH broth maintained in air with 5% CO2 (not shown). The resulting vegetations ranged from non-apparent (Figure 4A) to macroscopic lesions (Figures 4B and 4C).


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 affects vegetation weight and bacterial load in a S. sanguinis rabbit endocarditis model.(A) Aortic valve of a rabbit infected with S.sanguinis 133-79 Δnt5e. The aortic valve is composed of three leaflets, no visible vegetations were found and 2.5×103 CFUs were recovered. (B) Aortic valve of a rabbit infected with S. sanguinis 133-79 nt5e+, with one vegetation in the center leaflet (white circle) and 3.3×108 CFUs were recovered. (C) Aortic valve of a rabbit infected with S. sanguinis 133-79 wt, with two vegetations on center and right leaflets (white circle), respectively and 3.4×109 CFUs were recovered. (D) Plot of vegetation bacterial load (total CFU) versus vegetation mass. All vegetations on the aortic valve of each rabbit were pooled to obtain the vegetation weight and bacterial load (on TH plate). When no vegetations were found, the valves were scraped with a blade and plated to determine the valve bacterial load. R2 = 0.66 (n = 31) indicated that there is a correlation between the bacterial load and vegetation masses. (E) Bacterial loads in the rabbit endocarditis model, enumerated as log10 total CFU 4 days after infection. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test. Horizontal bars represent mean CFUs in each cohort.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369921&req=5

pone-0038059-g004: Nt5e affects vegetation weight and bacterial load in a S. sanguinis rabbit endocarditis model.(A) Aortic valve of a rabbit infected with S.sanguinis 133-79 Δnt5e. The aortic valve is composed of three leaflets, no visible vegetations were found and 2.5×103 CFUs were recovered. (B) Aortic valve of a rabbit infected with S. sanguinis 133-79 nt5e+, with one vegetation in the center leaflet (white circle) and 3.3×108 CFUs were recovered. (C) Aortic valve of a rabbit infected with S. sanguinis 133-79 wt, with two vegetations on center and right leaflets (white circle), respectively and 3.4×109 CFUs were recovered. (D) Plot of vegetation bacterial load (total CFU) versus vegetation mass. All vegetations on the aortic valve of each rabbit were pooled to obtain the vegetation weight and bacterial load (on TH plate). When no vegetations were found, the valves were scraped with a blade and plated to determine the valve bacterial load. R2 = 0.66 (n = 31) indicated that there is a correlation between the bacterial load and vegetation masses. (E) Bacterial loads in the rabbit endocarditis model, enumerated as log10 total CFU 4 days after infection. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post-test. Horizontal bars represent mean CFUs in each cohort.
Mentions: To investigate the contribution of Nt5e to S. sanguinis virulence in infective endocarditis, rabbits with experimental heart valve injury were infected by intravenous inoculation with 1×109 CFU S. sanguinis 133-79 wt, Δnt5e, or nt5e+. Growth curves in TH broth were performed for all three strains to test for changes in cell growth or fitness. Although the nt5e deletion mutant showed a slight delay in entry to log-phase, all strains displayed similar growth rates in TH broth maintained in air with 5% CO2 (not shown). The resulting vegetations ranged from non-apparent (Figure 4A) to macroscopic lesions (Figures 4B and 4C).

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