Figure 1.
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.
Table 1.
Characterization of the enzymatic activities of S. sanguinis 133-79 Nt5e.
Figure 2.
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).
Table 2.
Recovery of Sephadex G100 fractions of S. sanguinis 133-79 tryptic digest.
Figure 3.
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. 6.4 mg 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).
Table 3.
Proteins in G100-3 identified by mass spectrometry.
Figure 4.
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.