Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria.

Area of Science:

• Microbiology
• Biochemistry
• Cell Biology

Background:

• Antimicrobial peptides (AMPs) are crucial in innate immunity and exhibit diverse mechanisms of action.
• Some AMPs target intracellular bacterial components, necessitating cellular uptake.
• Quantifying AMP internalization is vital for understanding their efficacy and developing new antimicrobial strategies.

Purpose of the Study:

• To develop and validate a fluorescence-based assay for measuring bacterial internalization of AMPs.
• To differentiate between extracellular and intracellular AMP localization.

Main Methods:

• Utilizing a fluorescently labeled AMP in conjunction with the membrane-impermeant dye trypan blue.
• Employing flow cytometry to measure fluorescence intensity at the single-cell level.
• Leveraging trypan blue's quenching effect on extracellular/surface-bound AMP fluorescence.

Main Results:

• The method successfully distinguishes between internalized and extracellular AMPs.
• Trypan blue effectively quenches fluorescence of AMPs not inside intact bacterial cells.
• Flow cytometry provides quantitative data on AMP internalization rates.

Conclusions:

• This assay offers a robust and quantitative approach to assess AMP bacterial uptake.
• The method facilitates the study of AMPs with intracellular targets.
• Accurate measurement of AMP internalization is key for antimicrobial drug discovery.