Phage-Derived Protein Induces Increased Platelet Activation and Is Associated with Mortality in Patients with Invasive Pneumococcal Disease
Authors: Tunjungputri Rahajeng N. , Mobegi Fredrick M. , Cremers Amelieke J. , van der Gaast-de Jongh Christa E. , Ferwerda Gerben , Meis Jacques F. , Roeleveld Nel , Bentley Stephen D. , Pastura Alexander S. , van Hijum Sacha A. F. T. , van der Ven Andre J. , de Mast Quirijn , Zomer Aldert , de Jonge Marien I. 
Published: January 2017

To improve our understanding about the severity of invasive pneumococcal disease (IPD), we investigated the association between the genotype of
Streptococcus pneumoniae
and disease outcomes for 349 bacteremic patients. A pneumococcal genome-wide association study (GWAS) demonstrated a strong correlation between 30-day mortality and the presence of the phage-derived gene
pblB
, encoding a platelet-binding protein whose effects on platelet activation were previously unknown. Platelets are increasingly recognized as key players of the innate immune system, and in sepsis, excessive platelet activation contributes to microvascular obstruction, tissue hypoperfusion, and finally multiorgan failure, leading to mortality. Our
in vitro
studies revealed that
pblB
expression was induced by fluoroquinolones but not by the beta-lactam antibiotic penicillin G. Subsequently, we determined
pblB
induction and platelet activation by incubating whole blood with the wild type or a
pblB
knockout mutant in the presence or absence of antibiotics commonly administered to our patient cohort.
pblB
-dependent enhancement of platelet activation, as measured by increased expression of the α-granule protein P-selectin, the binding of fibrinogen to the activated αIIbβ3 receptor, and the formation of platelet-monocyte complex occurred irrespective of antibiotic exposure. In conclusion, the presence of
pblB
on the pneumococcal chromosome potentially leads to increased mortality in patients with an invasive
S. pneumoniae
infection, which may be explained by enhanced platelet activation. This study highlights the clinical utility of a bacterial GWAS, followed by functional characterization, to identify bacterial factors involved in disease severity.

IMPORTANCE
The exact mechanisms causing mortality in invasive pneumococcal disease (IPD) patients are not completely understood. We examined 349 patients with IPD and found in a bacterial genome-wide association study (GWAS) that the presence of the phage-derived gene
pblB
was associated with mortality in the first 30 days after hospitalization. Although
pblB
has been extensively studied in
Streptococcus mitis
, its consequence for the interaction between platelets and
Streptococcus pneumoniae
is largely unknown. Platelets are important in immunity and inflammation, and excessive platelet activation contributes to microvascular obstruction and multiorgan failure, leading to mortality. We therefore developed this study to assess whether the expression of
pblB
might increase the risk of death for IPD patients through its effect on enhanced platelet activation. This study also shows the value of integrating extensive bacterial genomics and clinical data in predicting and understanding pathogen virulence, which in turn will help to improve prognosis and therapy.