The Legionella pneumophila Methyltransferase RomA Methylates Also Non-histone Proteins during Infection

Area of Science:

• Microbiology
• Molecular Biology
• Epigenetics

Background:

• RomA, a SET-domain protein lysine methyltransferase from Legionella pneumophila, is injected into host cells.
• Previous studies identified RomA's ability to methylate histone H3 at lysine 14.

Purpose of the Study:

• To investigate RomA's substrate specificity beyond histones.
• To identify and validate novel human non-histone protein substrates of RomA.
• To understand RomA's broader role in host cell modification during infection.

Main Methods:

• Peptide array analysis to determine RomA substrate sequence preference.
• Bioinformatic screening of the human proteome for potential RomA substrates.
• In vitro methylation assays (radioactive and antibody-based) using purified proteins.
• Site-directed mutagenesis to confirm methylation sites.
• Cell-based assays in HEK293 cells and during L. pneumophila infection.

Main Results:

• RomA recognizes a G-K-X-(PA) motif within a basic amino acid context.
• Identified 34 novel peptide substrates and validated 7 full-length human proteins as RomA targets.
• Confirmed methylation of non-histone proteins, including AROS, in human cells and during infection.
• Demonstrated that RomA methylates both histones and non-histone proteins.

Conclusions:

• RomA possesses broader substrate specificity than previously known, targeting human non-histone proteins.
• RomA's methylation of non-histone proteins suggests a more complex role in Legionella pathogenesis.
• These findings highlight bacterial effector proteins as tools for studying host cell epigenetics and function.