Systematic Discovery of Novel Phosphoinositide-Binding Effectors in Legionella Reveals Conserved α-Helical Folds

Intracellular bacterial pathogens deploy effector proteins to hijack host membranes by targeting specific lipids. Phosphoinositides are key eukaryotic signaling molecules that govern membrane identity and dynamics, making them powerful levers for microbial interference. However, the bacterial repertoire of phosphoinositide-binding effectors remains largely undefined. In this study, we systematically identified such proteins using an expression library of 241 Legionella pneumophila effectors. We applied a combination of lipid bead pulldowns, mass spectrometry, colocalization with phosphoinositide biosensors, and protein-lipid overlay assays, leading to the validation of 18 new phosphoinositide-binding effectors. Structural predictions revealed that these proteins share compact alpha-helical folds distinct from canonical eukaryotic lipid-binding domains. Guided by this structural signature, we performed a computational screen across L. pneumophila, Coxiella burnetii, and Burkholderia pseudomallei. This analysis identified 15 additional effectors, which were experimentally validated as phosphoinositide binders. Altogether, we report 33 previously unrecognized bacterial phosphoinositide-binding effectors. While their lipid-binding profiles were diverse, many effectors showed a preference for compartments enriched in phosphatidylinositol 3-phosphate. These findings expand the known diversity of lipid-binding effectors produced by intracellular pathogens, identify new structural modules bacteria use for phosphoinositide recognition, and broaden our understanding of how pathogens exploit phosphoinositide signaling to manipulate host membranes. ImportanceIntracellular bacterial pathogens hijack host membrane trafficking by targeting phosphoinositides (PIPs), essential eukaryotic signaling lipids. Despite their importance in virulence, bacterial PIP-binding effectors remain largely uncharted because they lack recognizable sequence motifs. Using Legionella pneumophila as a model system, we systematically identified 30 novel PIP-binding effectors, doubling the known repertoire, and discovered that they employ compact -helical folds structurally distinct from canonical eukaryotic PIP-binding domains. Remarkably, these -helical modules are conserved in Coxiella burnetii and Burkholderia pseudomallei, revealing a convergent evolutionary strategy for PIP recognition across phylogenetically diverse pathogens. This work expands our understanding of bacterial lipid-targeting mechanisms and provides a structural blueprint for identifying virulence factors in emerging pathogens.