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

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 summary The 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.

. Interestingly, as also 150 shown in Figs 1A-B and S1A, the GST-tagged full-length BteA protein of B. pertussis in 151 complex with its cognate chaperone BtcA (BteA/BtcA) exhibited similar, although, 152 somehow stronger binding of negatively charged lipids than LRT alone. Nevertheless, the 153 lipid-binding abilities of the full-length BteA protein might have been affected by the 154 presence of BtcA, which co-purified with the BteA. In contrast to LRT protein, we were 155 unable to produce soluble BteA without its cognate chaperone BtcA in Escherichia coli.

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To avoid the limitations of the protein-lipid overlay assay and experiment-to- 165 three membrane surfaces with the typical association and dissociation phases of the 166 sensograms ( Fig 1C). The binding affinities of LRT to lipid vesicles were next calculated 167 from steady-state binding data as the global fitting of the binding curves to several kinetic 168 models did not provide satisfactory results in terms of χ 2 and residual statistics. The near- 169 equilibrium values (R eq ), which were taken from the end of the association phase of the 170 individual binding curves, were plotted as a function of the LRT concentration (Fig 1D), 171 and the equilibrium dissociation constant (K D ) was determined by nonlinear least-squares 172 analysis of the binding isotherm. As shown in Fig 1D,

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The surface of the LRT domain structure displays a positively charged interface,  Table 1). Our experimental data and 365 model obtained by molecular docking of PIP2 headgroups to LRT structure (Fig 7B), 366 hence, suggest that membrane targeting by LRT is reminiscent of that of 4HBM. The L51 367 residue of the L1 region may directly penetrate the apolar membrane milieu, whereas the 368 positively charged residues at the domain tip and helix B would provide a combination of 369 electrostatic attraction to the negatively charged membrane surface and a specific 370 headgroup recognition of the membrane phospholipids ( Fig 7B). Besides, helix D of LRT 371 (Fig 7B), which is analogous to L3 of 4HBM, may provide structure-stabilizing interactions.
372 Indeed, the critical residue R100 within helix D aligns with conserved R71 residue of 373 4HBM and is positioned towards the structure interior. We, therefore, propose that the 374 LRT domain of Bordetella BteA constitutes an additional member of 4HBM family of 375 bacterial proteins that targets host plasma membrane by a basic-hydrophobic motif [7,8].
376 The proposed model of LRT interaction characterized by the membrane-penetrating 377 leucine residue within loop L1 differs from the "side-on" interaction model suggested 378 within the recent biophysical study [21]. However, similarly to this study, we identified the 379 critical role of residues R59 and K62 within helix B for membrane interaction of LRT (Fig.   380 7B). Further work will be needed to address the membrane targeting of homologous LRT-381 like domains. Importantly, we were able to locate a homologous hydrophobic leucine or 382 isoleucine residue within the L1 loops of these domains.

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The intriguing question is the differential specificity of the LRT domain of BteA as  Fig 6). It remains 413 to be established whether the LRT domain has one or more additional interacting partners 414 other than PIP2 and PS that would further shape its specificity for lipid rafts in mammalian 415 cells.

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The mechanism of cytotoxic activity of BteA inside host cells remains unknown.   582 line were then determined by the "Profile plot tool" in FIJI, and profiles were scored as 583 follows. The score "PM" (plasma membrane) was assigned to profiles that displayed two 584 peaks in grey value near the cell periphery clearly distinguishable from the other signal 585 (cytoplasmic signal), as shown by the representative plot in Fig 3C -WT. This distribution 586 was typical for wild type LRT domain. The score "PM+" was assigned to profiles that 587 displayed two peaks in grey value near the cell periphery and no or very little cytoplasmic 588 signal, as shown in Fig 3C -L51F. This score meant greater plasma membrane 589 association than the wild type. The score "PM-" was assigned to those profiles that 590 showed two peaks in grey value near the cell periphery and a predominant signal between 591 them. This score meant less plasma membrane association than the wild type (attenuated 592 plasma membrane localization). Finally, the score "Cyto" (cytoplasmic) was assigned to 593 those profiles that were characterized by a single broad peak in grey value, as shown by