# Medium dependent factors govern the functionality of engineered type III secretion systems

## TL;DR

This study shows that the growth medium affects how well bacteria can use an engineered protein secretion system, possibly due to how resources are allocated in the cell.

## Contribution

The study is the first to systematically investigate how culture conditions influence the performance of engineered type III secretion systems.

## Key findings

- Secretion efficiency was consistent in glucose minimal medium but not in rich LB medium under varying IPTG concentrations.
- High IPTG concentrations in LB medium reduced PrgH abundance and SptP secretion efficiency.
- Stress-response genes were upregulated in LB medium at high IPTG, suggesting a possible link to reduced iT3SS performance.

## Abstract

The type III secretion system (T3SS) is a syringe-like machine that pathogenic bacteria use to inject effector proteins into host cells. Its ability to mediate targeted protein delivery has prompted efforts to adapt it for diverse biotechnological applications. However, the influence of bacterial host culture conditions on the performance of the T3SS-based circuits, which has never been systematically studied, is addressed in this study.

In this study, we developed and characterized an IPTG-inducible, refactored T3SS circuit (iT3SS) in Salmonella enterica, in which the prgH gene, encoding a protein of the basal body complex, was fused to the coding sequence of GFP in order to monitor the expression of the secretion system. The engineered system was shown to secrete efficiently the effector protein SptP. The dynamics of expression of the PrgH-GFP fusion was assessed in rich LB medium and in glucose minimal medium under various IPTG concentrations. Interestingly, secretion efficiency was maintained across IPTG concentrations in cells grown in glucose minimal medium, but not in cells grown in rich LB medium. In cells grown in LB medium, secretion and invasion efficiencies did not increase proportionally with increasing IPTG concentrations. Both PrgH abundance and SptP secretion efficiency were lower at high IPTG concentration than at low and medium IPTG concentrations. Since RNA-seq analysis of cells grown in LB medium revealed that the transcription of iT3SS genes increased proportionally to inducer level, this indicated that transcription was not the limiting factor for iT3SS expression. This suggested that the limiting factor might be due to a translational and/or post-translational burden of iT3SS component mRNAs. Indeed, uneven (not stoichiometric) translation of the iT3SS components and/or their imperfect folding might impair their assembly and insertion in the membrane. Consequently, one cannot exclude that the iT3SS components not properly assembled or integrated are being degraded, giving the wrong impression of a low translation level. Interestingly, RNA-seq revealed that in LB cultures at high IPTG concentration, stress-response genes were up-regulated whereas ribosomal protein-coding genes were down-regulated. This feature might contribute to limiting iT3SS translation. Several hypotheses are proposed in the discussion to explain how culture conditions could influence the functionality of iT3SS.

Our findings demonstrate that the nature of the growth medium has an impact on the performance of programmable secretion systems that might be due to host’s resource-allocation strategy that would have a negative impact on the translational efficiency of the iT3SS components, compromising their correct assembly and thus their membrane insertion. This insight provides a medium-aware framework for optimizing engineered secretion platforms for synthetic biology applications.

The online version contains supplementary material available at 10.1186/s13036-025-00600-1.