The Mycobacterium tuberculosis protein O-phosphorylation landscape

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacterial phosphosignalling traditionally focuses on two-component systems.
• Ser/Thr protein kinases (STPKs) are key in eukaryotes but their role in bacteria is less understood.
• Mycobacterium tuberculosis (Mtb) possesses STPKs, but their global impact is largely unexplored.

Purpose of the Study:

• To investigate the global signalling capacity of Mtb STPKs.
• To determine the extent and nature of O-phosphorylation in Mtb.
• To characterize the Mtb phosphoproteome and its regulatory network.

Main Methods:

• Utilized STPK loss-of-function and overexpression strains.
• Employed mass spectrometry-based phosphoproteomics for deep phosphoproteome analysis.
• Identified direct substrate-STPK interactions and their transcriptional effects.

Main Results:

• Discovered a deep phosphoproteome with over 14,000 unique phosphosites.
• Revealed that O-phosphorylation affects >80% of the Mtb proteome.
• Uncovered an expansive, distributed, and cooperative phosphosignalling network comparable to eukaryotic systems.
• Generated a resource of >3,700 high-confidence direct substrate-STPK interactions.

Conclusions:

• Mtb O-phosphorylation is a vastly underexplored modification with extensive regulatory roles.
• The Mtb phosphosignalling network is highly complex and interfaces extensively with the transcriptional machinery.
• This work provides signalling context for >80% of Mtb proteins, enabling pathway prediction for mycobacterial physiology.