A fluorescent reporter system for tracking Lactobacillus casei T1 in the murine gastrointestinal tract

Fluorescent reporter systems are useful for studying probiotic colonization and host-microbe interactions. However, their use in lactic acid bacteria is still limited by relatively weak fluorescence signals, insufficient expression stability, and limited resolution during in vivo imaging. In particular, efficient strain-specific tracking systems remain scarce.

Here, we developed a red fluorescent reporter system for Lactobacillus casei T1 (L.c T1). Lactate dehydrogenase (LDH) promoters identified from the L.c T1 genome were compared with the constitutive P32 promoter to drive expression of the red fluorescent proteins mCherry and mKate. The different promoter-reporter combinations were evaluated in both Escherichia coli DH5α and L.c T1. Fluorescence expression was further examined under different environmental pH conditions. The optimized reporter strains were then evaluated by whole-body fluorescence imaging in living mice and ex vivo imaging of gastrointestinal tissues following oral administration.

Among the constructs tested, P32-mKate produced the strongest and most stable fluorescence signal in L.c T1. Fluorescence intensity was influenced by environmental pH, with higher signals observed under mildly alkaline conditions. Whole-body fluorescence imaging showed that the engineered strain could be detected in living mice following oral administration. Ex vivo imaging of gastrointestinal tissues provided clearer localization of fluorescence, with signals mainly detected in the stomach and upper small intestine.

We established a stable and efficient red fluorescent reporter system for L.c T1. The P32-mKate system enables detection of the engineered strain both in vitro and in vivo and provides a practical approach for tracking probiotic distribution and studying host-microbe interactions in preclinical animal models.