Repurposed drugs block toxin-driven platelet clearance by the hepatic Ashwell-Morell receptor to clear Staphylococcus aureus bacteremia

Abstract

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a "toxin-platelet-AMR" regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

Fig. 1.. Platelets are essential for blood immunity against Staphylococcus aureus (SA) bacteremia and SA α-toxin induces thrombocytopenia to evade platelet-mediated microbicidal activity.

(A) Correlation of circulating platelet counts with leukocyte counts and duration of bacteremia in 49 consecutive patients with SA bacteremia from a tertiary medical center. Spearman’s rank correlation coefficient compared variables. (B) Mortality in patient cohort associated with different white blood cell (WBC) and platelet (PLTS) count cutoffs; Chi-square without Yates correction. (C) Washed isolated human platelets and neutrophils exposed to SA or Streptococcus pneumoniae (SPN) at MOI = 0.01 for 2 h. Samples were sonicated, serial diluted, and plated on THA plates for enumeration of bacterial colony forming units (CFU). (D) Reduction in platelet count 2 h after intravenous infection of mice with SA (n = 8) vs. non-infected littermate controls (n = 4). Two biological replicates were performed and data pooled; data represented as mean ± SEM. (E) Ex vivo killing of SA upon 2 h co-incubation with blood collected from mice 16 h after treatment with anti-CD41 antibody (n= 9) or IgG control (n= 12). (F) Mice treated with platelet depleting anti-CD41 antibody (n = 5) or IgG control (n = 4) for 16 h prior to intravenous SA infection. Organs harvested and CFU enumerated 2 h post-infection in triplicate for each sample. (G) Assessment of α-toxin production by the infecting SA isolate in 49 consecutive bacteremia cases and its association with patient platelet counts and (H) WBC counts. (I-J) Platelet counts (I) and CFUs (J) in outbred CD-1 mice intravenously challenged with wild-type SA (n = 4) or isogenic ΔHla mutant (n = 4). Blood harvested by cardiac puncture, complete blood count performed and colony forming units (CFUs) enumerated 4 h post-infection. (K) Ex vivo killing of SA by freshly isolated human platelets (2 h co-incubation) vs. isogenic ΔHla mutant. All data represented as mean ± SEM and representative of at least 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test (C-F, I, J, K) or one way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test (G,H). *P < 0.05. For floating bar graphs, + denotes the mean, whiskers represent min. to max, and floating box represents 25th to 75th percentile.

Fig. 2.. FDA-approved P2Y12 inhibitor ticagrelor blocks SA α-toxin-mediated platelet cytotoxicity.

(A) Effect of 10 μM aspirin (ASA) or 10 μM ticagrelor (TICA) (15 min pretreatment ex vivo) on human platelet killing of MRSA for 2 h (n = 9). Experiments were performed in triplicate and repeated three times. (B) Representative transmission electron microscopy image of platelets pre-treated with or without 10 μM TICA and exposed to MRSA at multiplicity of infection (MOI) 0.1 for 2 h. Scale bar = 5 μM. (C) P2Y12 inhibitor (TICA) pretreatment blocks human platelet cytotoxicity by 5 μg/ml purified α-toxin as measured by LDH release (n = 3) in a (D) dose-dependent manner. Inhibitors: P2Y12 (ticagrelor), GPIIb/IIIa (eptifibatide), COX-1 (SC560), PAR-1 (vorapaxar), and PAR-4 (ML-354). (E) TICA (blue line) treatment of human platelets reduces proteolytic cleavage of an ADAM10-specific fluorogenic substrate compared to PBS control (black line). Data representative of three independent experiments and statistical significance determined by least squares ordinary fit, *P < 0.5. (F) TICA (blue line) reduces intracellular calcium levels in human platelets loaded with 2 μM Fluo-3 dye and stimulated with 5 μg/mL recombinant α-toxin compared to PBS control (black line); calcium influx was measured every 30 sec by fluorescence. For both (E) and (F), α-toxin stimulated platelets (whether TICA or PBS treated) were normalized to their respective non-stimulated platelet controls. (G) TICA treatment of human platelets did not alter surface ADAM-10 expression as determined by flow cytometry. (H) Human platelets with or without TICA treatment were infected with MRSA at MOI = 0.1 for 90 min. Surface glycoprotein-6 (GP6) was measured by flow cytometry and perecent decrease in expression (GP6 shedding) calculated. (I) Human platelet P-selectin expression indicating platelet activation measured by flow cytometry with or without MRSA challenge (MOI = 0.1) and with or without TICA for 90 min. All data represented as mean ± SEM and are representative of at least 3 independent experiments. Statistical significance was determined by One way ANOVA with Bonferroni’s multiple comparisons test (A,C,G), unpaired two-tailed Student’s t-test (H) and two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons posttest (I). *P< 0.05, **P < 0.005. PBS, phosphate buffered saline; ns, not significant.

Fig. 3.. FDA-approved P2Y12 inhibitor ticagrelor protects against SA bacteremia.

(A and B) Outbred CD-1 mice treated with 4 mg/ml ticagrelor (TICA; n = 9) or PBS (n = 9) control every 12 h for 3 days, then challenged intravenously with 1 × 10⁸ colony forming units (CFUs) of SA; blood platelets and bacterial CFU burden enumerated 4 h post-infection. (C) Enumeration of bacterial CFU burden at 72 h in organs of mice pretreated with vehicle (PBS) or 4 mg/kg ticagrelor 12 h prior to intravenous SA and q 12 h thereafter; (n= 8). (D) Mortality curves of outbred CD-1 mice pretreated with vehicle (PBS) or 4 mg/kg ticagrelor beginning 24 h prior to intravenous SA infection then q 12 h over a 10-day observation period (n = 20). Independent experiments were repeated twice and data pooled. (E) Hematoxylin and eosin stain (H&E) of representative histological kidney sections from mice pre-treated with PBS vehicle or 4 mg/kg ticagrelor 12 h prior to SA infection and q 12 h thereafter for 72 h; (n = 8). Yellow stars denote formation of dense bacterial colonies and black arrows represent immune infiltrate. All histological sections are representative photos of at least 6 samples per two independent experiments. Where applicable, results are represented as mean ± SEM and statistical significance was determined by unpaired two-tailed Student’s t-test (B,C), and two-way ANOVA with Bonferroni’s multiple comparisons posttest (A). For survival curves, statistical significance determined by Log-rank Mantel-Cox test (D); *P < 0.05. For floating bar graphs, + denotes the mean, whiskers represent min. to max, and floating box represents 25th to 75th percentile. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 4.. SA α-toxin activates endogenous platelet sialidase activity.

(A) Percent desialylated platelets in platelet rich plasma from non-infected subjects or patients with SA or E. coli bacteremia measured by flow cytometry. (B) SA and S. pneumoniae sialidase activity assessed for over 4 h. (C) Sialidase activity examined on washed human platelets exposed to WT SA or isogenic ΔHla for 1 h or (D) sialidase activity examined on washed human platelets exposed to 5 μg/mL and 10 μg/mL recombinant α-toxin for 30 min. (E) Sialidase assay performed on washed human platelets treated with or without 10 μM ticagrelor and exposed to WT SA for 1 h. Where applicable, all data represented as mean ± SEM and are representative of at least 3 independent experiments. Statistical significance determined by one way ANOVA with Bonferroni’s multiple comparisons test (A, C, D, E). *P < 0.05. PBS, phosphate buffered saline; ns, not significant. ND, not detectable.

Fig. 5.. Inhibition of the hepatic Ashwell-Morell receptor (AMR) supports platelet-mediated defense against SA bacteremia.

(A) C57/Bl6 (n = 4) and Asgr2^−/− (n = 6) mice were challenged by intraperitoneal injection with SA, blood harvested by cardiac puncture, and platelet count enumerated. (B) 10-day mortality study with C57/Bl6 (n = 22) and Asgr2^−/− mice (n = 16) challenged by intraperitoneal injection with SA. Study performed two independent times and data pooled. (C) 8-day mortality study with C57/Bl6 treated with fetuin (n = 10) or asialofetuin (n = 10) and challenged by intraperitoneal injection with SA. (D) C57/Bl6 mice treated with asialofetuin (n = 4) or fetuin (n = 4) and challenged by intraperitoneal injection with SA, platelet count enumerated, and (E) kidneys, liver, spleen, and blood harvested 24 h post-infection for bacterial CFU enumeration. (F) C57/Bl6 and Asgr2^−/− mice challenged with wild-type MRSA or the isogenic ΔHla mutant. 4 h post-infection, blood was harvested by cardiac puncture for enumeration of platelet count. Statistical significance was determined by unpaired two-tailed Student’s t-test (E), two-way ANOVA with Bonferroni’s multiple comparisons posttest (A,D,F) or log-rank (Mantel-Cox) Test (B,C) for the survival curves. For floating bar graphs, + denotes the mean, whiskers represent min. to max, and floating box represents 25th to 75th percentile. *P < 0.05, **P < 0.005. PBS, phosphate buffered saline; ns, not significant.

Fig. 6.. FDA-approved sialidase inhibitor oseltamivir blocks AMR-mediated platelet clearance and protects against SA bacteremia.

(A) St3gal4^−/− mice that have decreased platelet sialylation and thrombocytopenia show accelerated mortality upon SA bloodstream infection (n = 10 per group). (B) circulating platelet count 4 h after IV SA challenge in WT vs. St3gal4^−/− mice (n = 10 per group). (C) Platelets isolated from Asgr2^−/− mice treated with or without oseltamavir and infected with MRSA were assessed for RCA-1 lectin binding. (D) C57/Bl6 mice were treated with oseltamavir (n = 6) or PBS control (n = 5) and infected with WT SA by intraperitoneal injection. Blood was harvested 24 h after infection and platelet counts collected. (E) 8-day mortality study conducted on C57/Bl6 mice treated with DANA (n = 16), oseltamavir (n = 16), or PBS control (n = 16). Statistical significance was determined by unpaired two-tailed Student’s t-test (B), two-way ANOVA with Bonferroni’s multiple comparisons posttest (C,D), or Log-rank (Mantel-Cox) Test (A,E). For floating bar graphs, + denotes the mean, whiskers represent min. to max, and floating box represents 25th to 75th percentile. Unless otherwise stated, *P < 0.05, **P < 0.005. PBS, phosphate buffered saline; ns, not significant.