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 nonapoptotic and caspase-1–independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) 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, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM 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 PM 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 PM of host cells is, therefore, dispensable for effector cytotoxicity.

The classical Bordetella species, B. pertussis and B. bronchiseptica, employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector into the host cells. Upon injection, BteA localizes to the cytosolic leaflet of lipid rafts via its N-terminal lipid raft targeting (LRT) domain and induces cell death. The plasma membrane targeting and cytotoxicity mechanisms of BteA are poorly understood. Using protein-lipid overlay assay and surface plasmon resonance, we showed here that the recombinant LRT domain, which adopts a four-helix bundle topology of membrane localization domains, specifically binds negatively charged membrane phospholipids. The binding affinity for phosphatidylinositol 4,5-bisphosphate (PIP2)-containing liposomes with Kd ~450 nM was higher than for those enriched in phosphatidylserine (Kd ~1.2 μM) while both phospholipids were required for plasma membrane targeting in yeast cells. The membrane association of LRT further depended on its electrostatic and hydrophobic interactions and involved a loop L1-located leucine residue. Importantly, charge-reversal substitutions within the L1 region disrupted plasma membrane localization of 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, hence, dispensable for the effector cytotoxicity.Author summaryThe respiratory pathogens of humans and other animals, Bordetella pertussis and Bordetella bronchiseptica, produce a type III secretion system effector protein BteA. This effector consists of two functional domains, an N-terminal lipid raft targeting (LRT) domain, and a cytotoxic C-terminal domain, which induces non-apoptotic and caspase-1-independent host cell death. We found here that the LRT domain of BteA associates with plasma membrane by binding to negatively charged phospholipids. We further discovered that the mechanism of membrane association by LRT is reminiscent of the one used by three other diverse families of toxins: clostridial glucosyltransferase toxins, multifunctional-autoprocessing RTX toxins (MARTX), and Pasteurella multocida-like toxins. Intriguingly, we also report that plasma membrane targeting by the LRT domain does not contribute to cytotoxic activity of BteA during B. bronchiseptica infection. Overall, our work elucidated the mechanism of plasma membrane association by LRT, and further provided the basis for future research on cellular activities of BteA and the mechanism of BteA-induced cell death.