Development and Evaluation of Exosporium-Anchored Bioluminescent and Fluorescent Reporters for Tracking...

Clostridioides difficile is an anaerobic spore-forming pathogen and a major cause of hospital-acquired and antibiotic-associated colitis. C. difficile spores are key for persistence and transmission of CDI, but methods to trace spores, including those produced during infection along the GI tract, are lacking. Here, we developed an exosporium-anchored reporter system that labels C. difficile spores during their formation by fusing NanoLuc or fluorescent proteins to the N-terminal domain of BclA1. The repaired BclA N-terminal domain (193 aa) supported stronger surface display and higher NanoLuc signal than the truncated 48 aa variant, without detectable changes in spore ultrastructure. NanoLuc-tagged spores were detectable in vitro down to ∼10³ spores in buffer and ∼10⁴ spores in fecal material, the latter reflecting matrix inhibition. In a murine model of CDI, the NanoLuc strain colonized and caused disease similar to wild-type, and fecal bioluminescence provided a qualitative readout of spore shedding once burdens exceeded ∼10⁴ spores per gram; however, luminescence did not quantitatively correlate with spore-based CFU in stools. By contrast, fluorescent reporters produced limited and unstable spore labeling, where mScarlet-i3 accumulated in a subset of sporulating cells and was largely lost in mature spores, while mNeonGreen was obscured by intrinsic autofluorescence. However, in a murine model of CDI, a small proportion of spores were fluorescently tagged. These findings establish an exosporium-anchored NanoLuc reporter as a tool for detecting C. difficile spores in vivo and highlight the technical barriers that currently limit fluorescent tagging of mature spores.