Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA.

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft-targeting (LRT) domain, and a cytotoxic C-terminal domain that induces non-apoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively-charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain binding liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), and phosphatidylserine (PS) were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both PS and PIP2 were required to target the LRT domain and/or full-length BteA to the plasma membrane of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT, and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted plasma membrane localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the plasma membrane of host cells is, therefore, dispensable for effector cytotoxicity.