A phospholipase B effector exploits membrane lipid divergence to drive inter-Bacteroides competition and enables universal genome editing

Abstract

ABSTRACT The Type VI secretion system (T6SS) mediates interbacterial competition, yet the mechanisms underlying its effector diversity and cell-targeting specificity remain poorly understood. Here, we identify Btle, a phospholipase B (PLB) effector defining a unique Tle subfamily (Tle7), encoded in approximately 18.0% of Bacteroides fragilis strains. Lipidomic profiling reveals that Btle preferentially targets Bacteroides glycerophospholipids, disrupting membrane integrity and driving strict inter-Bacteroides competition, with self-intoxication prevented by the cognate immunity protein Btli1. Guided by structural modeling and rational mutagenesis, we developed a Btle variant (BtleM) that evades Btli1 neutralization, establishing a broadly applicable counterselection marker for homologous recombination across Bacteroides. Secretion assays combined with AlphaFold structural prediction demonstrate that Btle delivery depends on a dual-effector complex, uncovering a conserved secretion mechanism within this genus. Collectively, this work expands the Tle superfamily with a calcium-dependent PLB effector, clarifies how glycerophospholipid compositional divergence shapes interbacterial targeting specificity, and establishes a universal counterselection strategy for genetic manipulation of gut Bacteroides.