Host–Pathogen Interaction Profiling Using Self-Assembling Human Protein Arrays

Abstract

Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10,000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or pre-labeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.

Elucidation of host–pathogen protein–protein interactions to uncover mechanisms of host cell rewiring

“…Regrettably, their lack of solubility and their necessity of remaining in lipid-rich environment highly impairs their interactome mapping via conventional methods such as AP-MS. To overcome these challenges, Glick Y et al introduced a screening method for HP interactions, adequate for transmembrane proteins [62] named the human Membrane Protein Array (MPA). Similarly, several studies have developed and applied Protein Micro Array technologies [63] including Nucleic Acid Programmable [64] or AVEXIS (AVidity-based Extracellular Interaction Screen) [65,66] to study soluble and transmembrane HP interactions (see supplementary table 1). …”

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

“…In addition, insufficient or biased coverage of the screened host cell proteome can impede these approaches. Recently, nucleic acid programmable protein arrays (NAPPA), which rely on in vitro transcription and translation to allow the profiling of effector interaction partners on self-assembling protein arrays, have been introduced (Yu et al 2015). These promise to provide a straightforward way of screening around 10 000 host cell proteins for interaction with an effector without the need of purifying the effector and host cell proteins.…”

“…6C). A chemical reporter for protein AMPylation has been successfully employed to profile target proteins of SidM, the Vibrio parahaemolyticus effector VopS and the human protein HYPE (Grammel et al 2011;Bunney et al 2014;Yu et al 2015). In addition, metabolic labelling of host cells with substrate analogues can also reveal the PTM of effectors by host cell enzymes.…”

Chemical reporters for exploring ADP-ribosylation and AMPylation at the host–pathogen interface

“…For example, N6-pATP can be used with recombinant AMPylators to identify potential substrates in whole cell lysates [48•]. To complement profiling of cell lysates, N6-pATP and recombinant AMPylators can also be used with n ucleic a cid- p rogrammable p rotein a rrays (NAPPA) to identify candidate substrates in vitro [49•,50•]. These studies have revealed new candidate GTPase and non-GTPase substrates for the secreted bacterial effectors VopS and IbpA [49•].…”