Ecto-5′-Nucleotidase: A Candidate Virulence Factor in Streptococcus sanguinis Experimental Endocarditis

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 (log10CFU, 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

Recovery of Sephadex G100 fractions of S. sanguinis 133-79 tryptic digest.

a<p>PRP was preincubated with the indicated fraction at a final concentration of 0.1 mg/ml.</p>b<p>Proteins were incubated with 50 µM of AMP, ADP or ATP for 15 minutes at 37°C at a final concentration of 10 µg/ml.</p

Nt5e affects vegetation weight and bacterial load in a <i>S. sanguinis</i> rabbit endocarditis model.

<p>(A) Aortic valve of a rabbit infected with <i>S. </i><i>sanguinis</i> 133-79 Δ<i>nt5e</i>. 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 <i>S. sanguinis</i> 133-79 <i>nt5e</i>+, with one vegetation in the center leaflet (white circle) and 3.3×10⁸ CFUs were recovered. (C) Aortic valve of a rabbit infected with <i>S. sanguinis</i> 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.</p

Nt5e is a trypsin-cleavable surface protein of <i>S. sanguinis</i> and affects platelet aggregation lag time.

<p>(A) Gel filtration chromatography of 7-minute tryptic digest of <i>S. sanguinis</i> 133-79. 6.4<b> </b>mg was placed on a column of Sephadex G-100 and chromatographed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038059#s4" target="_blank">Materials and methods</a>”. (B) SDS-PAGE analysis of <i>S. sanguinis</i> 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 Δ<i>nt5e</i> strains were added at the <i>S. sanguinis</i> 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 <i>nt5e</i>+ 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).</p

Characterization of the enzymatic activities of <i>S. sanguinis</i> 133-79 Nt5e.

ab<p>Enzyme parameters were calculated by nonlinear curve fitting using GraphPad Prism. K_m was represented as mean.</p><p>±SE µM and V_max was represented as mean±SE nmoles Pi/min per 10⁶ cells.</p>c<p>Statistical analysis was performed using ANOVA, and followed by Bonferroni test. Significant differences were only. obtained when comparing wt and Δ<i>nt5e,</i> P<0.01.</p

Characterization of Nt5e activity on <i>S. sanguinis</i> 133-79 whole cells.

<p>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 <i>S. sanguinis</i> 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).</p

Proteins in G100-3 identified by mass spectrometry.

<p>Proteins in G100-3 identified by mass spectrometry.</p

<i>nt5e</i> confers Nt5e activity on <i>S. sanguinis</i> SK36 whole cells.

<p>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). Δ<i>nt5e</i>: 5′-nucleotidase deletion mutant; Δ<i>nucH</i>: extracellular nuclease deletion mutant; Δ<i>cnp</i>: cyclo-nucleotide phosphodiesterase deletion mutant; and Δ<i>rad3:</i> DNA repair ATPase deletion mutant.</p

Keratinocyte-specific stat3 heterozygosity impairs development of skin tumors in human papillomavirus 8 transgenic mice tumors in human papillomavirus 8 transgenic mice.

Human papillomaviruses (HPV) of the genus β are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of β-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell–autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3+/−:HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3+/+:HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3+/−:HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer. Cancer Res; 70(20); 7938–48. ©2010 AACR