From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK

Melioidosis, caused by the Gram-negative bacterium
Burkholderiapseudomallei, is a potentially fatal infection that is
endemic in Southeast Asia and Northern Australia that is poorly
controlled by antibiotics. Research efforts to identify antigenic
components for a melioidosis vaccine have led to the identification of
several proteins, including subunits forming the flagella that mediate
bacterial motility, host colonization, and virulence. This study focuses
on the B.pseudomallei flagellar hook-associated protein (FlgK(Bp)), and
provides the first insights into the 3D structure of FlgK proteins as
targets for structure-based antigen engineering. The FlgK(Bp) crystal
structure (presented here at 1.8-angstrom resolution) reveals a
multidomain fold, comprising two small -domains protruding from a large
elongated -helical bundle core. The evident structural similarity to
flagellin, the flagellar filament subunit protein, suggests that,
depending on the bacterial species, flagellar hook-associated proteins
are likely to show a conserved, elongated -helical bundle scaffold
coupled to a variable number of smaller domains. Furthermore, we present
immune serum recognition data confirming, in agreement with previous
findings, that recovered melioidosis patients produce elevated levels of
antibodies against FlgK(Bp), in comparison with seronegative and
seropositive healthy subjects. Moreover, we show that FlgK(Bp) has
cytotoxic effects on cultured murine macrophages, suggesting an
important role in bacterial pathogenesis. Finally, computational epitope
prediction methods applied to the FlgK(Bp) crystal structure, coupled
with invitro mapping, allowed us to predict three antigenic regions that
locate to discrete protein domains. Taken together, our results point to
FlgK(Bp) as a candidate for the design and production of
epitope-containing subunits/domains as potential vaccine components.