Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors.

Christopher Price,Matthew B Lawrenz,Juanita Von Dwingelo,Yousef A Kwaik,Ashley Best,Snake Jones,Michael Merchant,Nawsad Alam,Ora Schueler-Furman

doi:10.3389/fcimb.2017.00054

Abstract

FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function.

Highlights

Exploitation of host post-translational machineries by translocated bacterial effector proteins is a growing paradigm for effector sub-cellular localization and function in the host cell
L. pneumophila translocates at least 11 ankyrin repeat domain-containing (ARD) proteins by its Dot/Icm type IVB translocation system into the host cell upon invasion, including those that contribute to intracellular replication (AnkB, H, J) (Al-Khodor et al, 2008; Habyarimana et al, 2008; Pan et al, 2008; Price et al, 2011)
Our search for bacterial effectors that are potentially modified by host-asparaginyl hydroxylation was based upon the canonical factor inhibiting HIF1 (FIH) hydroxylation motif that was defined by using sequence data derived from the HIF1 and ARD hydroxylation sites (Hewitson et al, 2002; Lando et al, 2002a,b; Cockman et al, 2009)

Summary

Introduction

Exploitation of host post-translational machineries by translocated bacterial effector proteins is a growing paradigm for effector sub-cellular localization and function in the host cell. HIF1 is hydroxylated by FIH on an asparagine residue and this modification acts as a molecular switch to prevent interaction with its co-activator p300/CBP, blocking transcription of hundreds of HIF1-regulated genes involved in oxygen homeostasis, energy production and immune responses (Hewitson et al, 2002; Lando et al, 2002a,b). Asparaginyl hydroxylation acts as a molecular switch to promote or reduce protein-protein interactions between HIF1-p300/CBP and ASPP2-Par (Hewitson et al, 2002; Lando et al, 2002a,b; Janke et al, 2013). The biological consequence of asparaginyl hydroxylation of eukaryotic proteins largely remains unclear

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Conclusion