A high throughput assay for measuring secreted protein based on a de novo fluorescent reporter reveals regulatory and structural insights in Salmonella type three secretion system.

Intracellular protein production in bacteria is limited by the need for lysis and costly purification. A promising alternative is to engineer the host organism for protein secretion. While the Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Type 3 Secretion System (T3SS) has been utilized for protein secretion, its study and eventual applicability for recombinant protein production are constrained by the lack of high-throughput assays to quantitatively measure secretion titer. Developing such assays is challenging, as proteins must remain unfolded for secretion, limiting the use of several common reporter proteins. In this work, we develop a high-throughput secretion assay using mini-fluorescence activating protein (mFAP). mFAP forms a chromophore only upon addition of an exogenous substrate, allowing secretion and subsequent fluorescence detection. We demonstrate mFAP secretion via the T3SS with an N-terminal secretion tag and show that the fluorescent signal in the secreted fraction is rapid and linear over three orders of magnitude. Using this assay, we screen S. typhimurium strains with secretion-enhancing mutations, identifying a constitutively active strain and revealing temporally controlled secretion dynamics. We also show that this assay may be applicable to other secretion systems, providing a universal tool for tracking heterologous protein secretion.