"A high throughput flow cytometry assay for quantifying type 3 secretion system assembly in Salmonella"

Area of Science:

• Microbiology and Molecular Biology
• Biotechnology and Synthetic Biology

Background:

• Recombinant protein production in bacteria faces limitations due to expensive cell lysis and purification processes.
• Native bacterial secretion systems, like the Type 3 Secretion System (T3SS) in *Salmonella enterica*, offer a promising alternative for protein secretion but require optimization for commercial viability.
• Quantifying T3SS apparatus assembly is challenging due to its complexity, precise stoichiometry, and the difficulty in probing its embedded components.

Purpose of the Study:

• To develop novel, high-throughput tools for rapid and accurate characterization of the T3SS secretion apparatus assembly.
• To enable the optimization of T3SS for enhanced, scalable heterologous protein production.
• To investigate the impact of key regulators and genetic variants on T3SS assembly and secretion.

Main Methods:

• Established a high-throughput flow cytometry method in *S. Typhimurium* to quantify T3SS apparatus assembly using SipD abundance as a proxy.
• Adapted super-resolution microscopy (Structured Illumination Microscopy - SR-SIM) to visualize SipD and validate flow cytometry findings.
• Integrated a secretion-compatible fluorescent reporter to link T3SS assembly with protein secretion efficiency.

Main Results:

• Demonstrated the utility of flow cytometry and SR-SIM for rapid assessment of T3SS assembly.
• Revealed the impact of overexpressing key T3SS regulators (*hilA*, *hilD*) on apparatus assembly.
• Uncovered how a specific PrgI variant affects the T3SS apparatus architecture and linked assembly to secretion using the reporter system.

Conclusions:

• The developed flow cytometry and SR-SIM tools provide rapid insights into T3SS assembly dynamics.
• These tools advance the engineering of T3SS as a scalable platform for heterologous protein secretion.
• The findings facilitate optimization of bacterial secretion systems for biotechnological applications.