Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• CarD is an essential global transcription regulator in Mycobacterium tuberculosis (Mtb).
• It interacts with RNA polymerase to activate transcription.
• Previous studies showed monomeric and domain-swapped dimeric states, but solution behavior was unclear.

Purpose of the Study:

• To determine the oligomeric state of CarD in solution.
• To investigate the potential for CarD to form amyloid-like fibrils.
• To elucidate the role of domain-swapping in CarD's aggregation.

Main Methods:

• Synchrotron-based small-angle X-ray scattering (SAXS) to assess solution structure.
• Biochemical and biophysical experiments.
• Mass spectrometry, transmission electron microscopy (TEM), and confocal imaging to visualize fibrils.
• Site-directed mutagenesis to study domain-swapping mutants.

Main Results:

• CarD exists as a homodimer in solution.
• CarD is the first identified soluble cytosolic protein in Mtb capable of forming amyloid-like fibrils in vitro and in vivo.
• Deletion of N-terminal residues involved in domain-swapping significantly inhibits fibril formation.

Conclusions:

• CarD's homodimeric state is confirmed in solution.
• CarD possesses a unique amyloidogenic property, forming fibrils through a domain-swapping mechanism.
• This discovery opens new avenues for understanding essential protein functions and potential therapeutic targets in Mtb.