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

Recombinant protein production in bacteria is limited by costly cell lysis and multi-step purification. To bypass these limitations, leveraging native bacterial secretion systems to secrete proteins offers a promising alternative. The type 3 secretion system (T3SS) of Salmonella enterica serovar Typhimurium can secrete proteins at hundreds of milligrams per liter. While promising, higher titers from this and other natural systems are needed to be relevant commercially. A major engineering target to enhance protein secretion is the T3SS secretion apparatus. However, efforts to quantify its assembly are hindered by bottlenecks in scalability and complexity. Assembly involves [~]20 structural proteins forming with precise stoichiometry under dynamic regulation. Membrane- and periplasmic-embedded components are difficult to probe without costly, time-intensive methods. To fully realize the T3SS as a tool for scalable protein production, new tools are needed for rapid and accurate characterization of the assembled apparatus. Here, we establish a high throughput flow cytometry method in S. Typhimurium by quantifying the abundance of the needle-bound tip protein SipD as a proxy for T3SS apparatus assembly. We then adapted a super resolution microscopy method, known as Structured Illumination Microscopy (SR-SIM), to visualize the presence of SipD and validate the flow cytometry results. Applying this approach, we revealed how overexpression of key T3SS regulators hilA and hilD impact assembly, expanded the assay with a secretion-compatible fluorescent reporter to link assembly with secretion, and uncovered how a PrgI variant impacts apparatus architecture. Together, these tools enable rapid insights into T3SS assembly and advance heterologous secretion platform development. ImportanceProducing proteins in bacteria often requires expensive and complex steps. This study focuses on S. Typhimurium, a bacterium that naturally secretes proteins using a specialized system. We developed a fast and scalable method to measure assembly of the secretion machinery, ultimately accelerating the process of optimizing this system for bacterial protein production. Further, this method provides new mechanistic insights into how the changes to the system impact the ability for the secretion machinery to form. We expect the platform could be adopted to other systems displayed on the bacterial cell surface, including T3SS homologs as well as a wide variety of multi-component membrane transporters.