A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Type III secretion systems (T3SS) are crucial for bacterial virulence, delivering effector proteins into host cells.
• T3SS expression is tightly regulated, typically repressed in nutrient-rich environments and induced during host infection.
• Understanding the molecular basis of host-specific T3SS induction is key to controlling bacterial pathogenesis.

Purpose of the Study:

• To elucidate the mechanism of host-induced regulation of T3SS expression in *Xanthomonas citri*.
• To identify the role of the ATP-dependent protease Lon in controlling the stability of the T3SS master regulator, HrpG.
• To investigate the impact of post-translational modifications on bacterial virulence factor regulation.

Main Methods:

• Phosphoproteomic analysis (Ser/Thr/Tyr) to identify host-induced phosphorylation sites on Lon protease.
• Genetic manipulation to create Lon mutants (e.g., S654A substitution).
• Biochemical assays to assess Lon protease activity and HrpG degradation rates.
• Analysis of T3SS gene expression (*hrp*/*hrc*) in *Xanthomonas citri* under different growth conditions and in the host environment.

Main Results:

• Host-induced phosphorylation of Lon protease at serine 654 was identified in the citrus host.
• Phosphorylation of Lon at S654 deactivates its proteolytic activity, preventing HrpG degradation.
• In rich medium, Lon degrades HrpG, repressing T3SS.
• Mutating Lon S654 to alanine led to constitutive HrpG degradation, T3SS repression, and reduced virulence in the host.
• Stabilized HrpG activates downstream T3SS components, enabling virulence in the host.

Conclusions:

• Host-specific phosphorylation of Lon protease is a novel mechanism for regulating bacterial T3SS.
• This post-translational modification allows *Xanthomonas citri* to control T3SS expression based on environmental cues.
• The findings provide new insights into bacterial virulence strategies and host-pathogen interactions.