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J Proteome Res. 2015 Apr 3;14(4):1920-36. doi: 10.1021/pr5013015. Epub 2015 Mar 18.

Host-pathogen interaction profiling using self-assembling human protein arrays.

Author information

1
†Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, United States.
2
‡Unit on Microbial Pathogenesis, Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, United States.
3
§The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States.

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.

KEYWORDS:

AMPylation; LidA; Rab1; SidM; interactome; nucleic acid programmable protein array (NAPPA)

PMID:
25739981
PMCID:
PMC4467460
DOI:
10.1021/pr5013015
[Indexed for MEDLINE]
Free PMC Article

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