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