Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus

Lysine acylation is a posttranslational modification
(PTM) conserved
in all domains of life and is essential for regulating diverse biological
processes. Traditional methods for investigating acylation rely on
anti-acyl-lysine antibodies, which are costly and time-consuming and
often exhibit variable affinity. To remedy these pitfalls, we developed
an antibody-free method for bacterial acylome enrichment using bioorthogonal
click chemistry coupled with tandem mass spectrometry. We applied
this approach to the pathogens Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to explore the biological significance of acylation in each
organism. We characterized the acetylome, propionylome, and butyrylome
in P. aeruginosa UCBPP-PA14 and the
acetylome and propionylome in MRSA. Comparative analyses revealed
unique PTM dynamics showing that acylation regulated a wide range
of cellular functions, including metabolism, antibiotic resistance,
virulence, and stress response. This work establishes the first antibody-free
enrichment method for defining bacterial acylomes and provides new
insight into global lysine acylation networks in pathogenic bacteria.