Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Area of Science:

• Microbiology
• Cell Biology
• Biochemistry

Background:

• Type three secretion systems (T3SSs) deliver bacterial effector proteins into host cells, crucial for infection.
• Visualizing these effectors is vital for understanding host-pathogen dynamics but is technically challenging.
• Existing methods like fluorescent fusion proteins often fail due to secretion interference.

Purpose of the Study:

• To provide a detailed protocol for site-specific fluorescent labeling of bacterial secreted effectors using genetic code expansion (GCE).
• To enable super-resolution imaging of these labeled proteins within host cells.
• To facilitate the study of host-pathogen interactions.

Main Methods:

• Utilized genetic code expansion (GCE) to site-specifically incorporate non-canonical amino acids (ncAAs) into Salmonella secreted effectors.
• Employed bio-orthogonal click chemistry with tetrazine-containing dyes for fluorescent labeling.
• Performed direct stochastic optical reconstruction microscopy (dSTORM) for super-resolution imaging in HeLa cells.

Main Results:

• Successfully demonstrated site-specific fluorescent labeling of bacterial secreted proteins using GCE.
• Achieved visualization of subcellular localization of labeled effectors within host cells.
• Validated GCE combined with bio-orthogonal labeling as a robust method for imaging.

Conclusions:

• Genetic code expansion offers a powerful solution for labeling and visualizing bacterial secreted proteins.
• This protocol enables detailed super-resolution imaging of host-pathogen interactions.
• The method is broadly applicable to studying bacterial, viral, and host processes.