Microfluidics-enabled proteomic profiling reveal iron-driven immune evasion by an antimicrobial-resistant pathogen

Area of Science:

• Microfluidics
• Proteomics
• Infectious Disease Biology

Background:

• Studying host-pathogen interactions is complex due to mixed cell populations.
• Existing separation methods limit high-resolution analysis and cell viability.

Purpose of the Study:

• To develop a label-free microfluidic chip for separating bacteria and host cells.
• To enable high-resolution proteomic analysis of host-pathogen interactions.
• To investigate bacterial immune evasion mechanisms.

Main Methods:

• Fabrication of a microfluidic chip with an optimized 1.4 μm filter.
• Co-culture of Klebsiella pneumoniae and murine macrophages on the chip.
• Label-free separation of bacteria and host cells.
• Proteomic analysis of separated bacterial populations.
• Functional assays to assess iron's role in immune evasion.

Main Results:

• The microfluidic chip enabled reproducible separation while preserving cell viability.
• Improved host protein identification and enrichment of immune-associated proteins compared to traditional methods.
• Chip-isolated bacteria showed distinct proteomes with altered metabolic and iron-binding proteins.
• Iron was found to promote bacterial survival and macrophage evasion.

Conclusions:

• The microfluidic-proteomic workflow offers a powerful tool for dissecting host-pathogen dynamics.
• An iron-dependent mechanism contributes to bacterial immune evasion.
• This approach facilitates the study of infection biology and the identification of therapeutic targets.