Structural and functional investigation of the role of Gcn5-related N-acetyltransferase (GNAT) activity in the host manipulation strategies of pathogenic bacteria of order Legionellales

Abstract

The pathogenic strategy of intracellular parasite Legionella pneumophila relies on the translocation of over 330 proteins, called effectors, into the eukaryotic host cell during infection. The collective action of these effectors modulates host biochemistry to facilitate survival and proliferation of the pathogen. Among the ~18,000 effectors of genus Legionella, a subset of 8 effectors is conserved in all members sequenced to date. This universal group of effectors include a putative Gcn5-related N-acetyltransferase (GNAT) termed VipF. GNAT enzymes catalyze the acetylation of a broad range of small molecule and protein substrates, however to our knowledge this is the first instance of a secreted effector with homology to GNAT acetyltransferases. Here, using structural, biochemical, and in vivo strategy, we show VipF is a GNAT acetyltransferase with homology to bacterial Rim-family acetyltransferases showing preference for N-terminal acetylation of peptide substrates in vitro. We identify host interactors of VipF using affinity purification coupled to mass spectrometry and confirm by binary interaction in yeast 2-hybrid the physical interaction of VipF with the K subunit of human translation initiation factor 3 (eIF3) complex. We further characterize the interaction interface between these proteins and demonstrate residues critical for VipF interactions with eIF3K. Despite significant diversification in sequence in representatives of VipF in Legionella, we demonstrate that several selected members of this effector family interact with eIF3K.