posted on 2020-03-17, 15:38authored byEmma Roig-Molina, Manuel Sánchez-Angulo, Jana Seele, Francisco García-Asencio, Roland Nau, Jesús M. Sanz, Beatriz Maestro
Choline-binding
proteins (CBPs) from Streptococcus pneumoniae comprise
a family of modular polypeptides involved in essential events of this
pathogen. They recognize the choline residues present in the teichoic
and lipoteichoic acids of the cell wall using the so-called choline-binding
modules (CBMs). The importance of CBPs in pneumococcal physiology
points to them as novel targets to combat antimicrobial resistances
shown by this organism. In this work we have tested the ability of
exogenously added CBMs to act as CBP inhibitors by competing with
the latter for the binding to the choline molecules in the bacterial
surface. First, we carried out a thorough physicochemical characterization
of three native CBMs, namely C-LytA, C-Cpl1, and C-CbpD, and assessed
their affinity for choline and macromolecular, pneumococcal cell-wall
mimics. The interaction with these substrates was evaluated by molecular
modeling, analytical ultracentrifugation, surface plasmon resonance,
and fluorescence and circular dichroism spectroscopies. Van’t
Hoff thermal analyses unveiled the existence of one noncanonical choline
binding site in each of the C-Cpl1 and C-CbpD proteins, leading in
total to 5 ligand-binding sites per dimer and 4 sites per monomer,
respectively. Remarkably, the binding affinities of the CBMs do not
directly correlate with their native oligomeric state or with the
number of choline-binding sites, suggesting that choline recognition
by these modules is a complex phenomenon. On the other hand, the exogenous
addition of CBMs to pneumococcal planktonic cultures caused extensive
cell-chaining probably as a consequence of the inhibition of CBP attachment
to the cell wall. This was accompanied by bacterial aggregation and
sedimentation, causing an enhancement of bacterial phagocytosis by
peritoneal macrophages. In addition, the rational design of an oligomeric
variant of a native CBM led to a substantial increase in its antibacterial
activity by multivalency effects. These results suggest that CBMs
might constitute promising nonlytic antimicrobial candidates based
on the natural induction of the host defense system.