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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Host-pathogen protein interactions are crucial in infections but difficult to study.
• Existing methods for identifying these interactions have limitations, including abundance biases and lack of experimental flexibility.

Purpose of the Study:

• To develop and apply a novel, bias-free protein interaction detection platform for studying host-pathogen protein networks.
• To identify interaction partners and substrates of Legionella pneumophila effectors SidM and LidA.

Main Methods:

• Utilized the Nucleic Acid-Programmable Protein Array (NAPPA) platform combined with a HaloTag-Halo ligand detection system.
• Screened interactions between L. pneumophila effectors (SidM, LidA) and approximately 10,000 human proteins.
• Employed Click chemistry-based NAPPA to identify substrates for SidM's AMPylation activity.
• Validated novel targets using in vitro pull-down and in vivo cell-based assays.

Main Results:

• Identified known and novel interaction candidates for L. pneumophila SidM and LidA effectors.
• Discovered substrates for SidM's AMPylation activity.
• Confirmed a subset of novel targets through independent validation experiments.
• Gained insights into effector discrimination of host Rab GTPases.

Conclusions:

• The NAPPA-HaloTag system offers a robust, high-throughput method for identifying host-pathogen protein interactions and effector substrates.
• This platform circumvents protein purification and labeling requirements, making it broadly applicable to various pathogens.
• The findings provide a deeper understanding of L. pneumophila pathogenesis and host manipulation strategies.