The L. pneumophila effector PieF modulates mRNA stability through association with eukaryotic CCR4-NOT

Abstract

The eukaryotic CCR4-NOT deadenylase complex is a highly conserved regulator of mRNA metabolism that influences the expression of the complete transcriptome, representing a prime target for a generalist bacterial pathogen. We show that a translocated bacterial effector protein, PieF (Lpg1972) of L. pneumophila Str. Philadelphia-1, interacts specifically with the CNOT7/8 nuclease module of CCR4-NOT, with a dissociation constant in the low nanomolar range. PieF inhibits the catalytic deadenylase subunit CNOT7 of the CCR4-NOT complex in a stoichiometric, dose-dependent manner in vitro. In transfected cells, PieF can silence reporter gene expression and reduce mRNA steady-state levels when artificially tethered. PieF demonstrates molecular similarities to another family of CNOT7-associated factors but demonstrates divergence concerning the interaction interface with CNOT7. In addition, we show that PieF overexpression changes the subcellular localization of CNOT7 and displaces the CNOT6/6L nucleases from CCR4-NOT. Finally, PieF expression phenocopies knockout of the CNOT7 ortholog in S. cerevisiae, resulting in 6-azauracil sensitivity. Collectively, this work suggests that L. pneumophila targets host mRNA stability and expression through a highly conserved host pathway not previously associated with Legionella pathogenesis.