Legionella pneumophila phospholipase PlaB is a cell death effector of an NADase toxin

NAD+ depletion is a defense strategy of both eukaryotic cells and bacteria against viral infection. We recently uncovered the unusual mechanism which controls activity of the Legionella pneumophila phospholipase and virulence factor PlaB. Specifically, physiological concentrations of NAD+ stabilize inactive PlaB tetramers, and contrarily, low NAD+ triggers release of two active protein dimers unleashing fierce phospholipase activity. Previous work further established bacterial surface association of PlaB by a noncanonical two-stranded β-sheet. Although PlaB homologs can be widely found in environmental bacteria, it was surprising that conservation of the respective β-sheet is low (Diwo et al. 2021: https://doi.org/10.1073/pnas.201704611). Therefore, we presumed a second intrinsic function of PlaB in altruistic cell death, which is also observed in abortive phage defense, apart from acting as a surface-exposed virulence factor. Consistent with NAD+ being central for regulation of PlaB`s activation status, in gene co-occurrence studies, we found high conservation of plaB with candidate NAD+ consuming toxin and cognate antitoxin genes suggesting a functional link. Indeed, co-expression studies conducted in E. coli and L. pneumophila revealed rapid cell death when PlaB was present only when NAD+ dropped to sub-physiological levels as a consequence of toxin induction. Gene expression analysis in L. pneumophila uncovered apparent co-regulation of plaB, toxin and antitoxin genes, all of which being induced for example upon prolonged stress. Our results support a model in which PlaB together with an NADase effector module is involved in stress response of L. pneumophila eventually facilitating cell death to benefit the population. NADase toxin and antitoxin might furthermore constitute elements of Legionella anti-bacteriophage response compiling a novel phospholipase-driven abortive infection system. In summary, our data indicate that PlaB is a cell death effector of an NADase toxin found in – but not limited to - Legionella pneumophila.