Visualizing phenotypic heterogeneity in C. difficile gene expression in situ during infection

The ability to ask biological questions is often limited not by our ideas, but by the tools we have to test them. Ever since Craig Ellermeier and David Weiss at the University of Iowa reported that the major nosocomial pathogen Clostridioides difficile heterogeneously expresses its toxin genes in vitro¹, our lab has wondered whether this heterogeneity occurs during infection of animals and whether its expression might be spatially regulated. In this project, I spent several years optimizing a reporter system that allowed us to visualize individual C. difficile cells in their native environment, the infected gut, as well as their associated toxin gene expression. This technological advance allowed us to determine that heterogeneity in toxin gene expression is unaffected by C. difficile’s proximity to the gut epithelium. While we had originally hypothesized that toxin gene expression would be spatially regulated, the tools I developed allowed us to serendipitously discover that a toxin gene overexpressing mutant adopts a filamentous morphology during acute infection. This result illustrates how some biological features only become visible once the right technology exists, highlighting the importance of investing the time to develop the right tools.

I joined Aimee’s lab in the second year of my PhD, just as Lauren Donnelly, a senior MD/PhD student, was wrapping up her thesis work. Lauren developed the first generation of dual fluorescent reporters for visualizing the sub-population of C. difficile cells that express toxin genes². However, as she was completing her thesis, she obtained data suggesting that the constitutive reporter she had developed, P_slpA::mNeonGreen, imposed a fitness cost on C. difficile, particularly in vivo.

To overcome this serious technical limitation, my initial task was to systematically analyze the fitness and visibility of different reporter strains that Lauren and I had made. My prior experience with murine infection models, gained from working as a technician in Dr. Lynn Bry’s lab, helped me quickly establish that very bright reporter strains often failed to cause disease at wild-type (WT) levels. In contrast, reporters that could still cause disease were often not bright enough to visualize in colonic sections. That realization launched what turned into a two-year optimization effort. I systematically tested many combinations of promoters and fluorescent proteins, attempting to find “Goldilocks” reporter strains that were bright enough to see in tissues, yet retained the ability to colonize the mice and cause disease.

Guided by RNA-seq data from infected mice, I focused on promoters from genes that are highly expressed during infection, including slpA, cwp2, and gluD. I paired these with a wide range of fluorescent proteins—mNeonGreen, mGreenLantern, StayGold, hfYFP, mScarlet, and mScarlet-I3^3–6—assessing their brightness, stability, and impact on C. difficile growth. Cloning these reporters was often a waiting game, with some promoters leading to such high-level reporter gene expression that it was necessary to incubate the E. coli transformations for days on my bench. Nevertheless, I did get lucky during the cloning process because one mScarlet-I3 construct picked up a single point mutation that led to a glycine-to-arginine substitution at position 228. This small change increased the fluorescent signal by ~2-fold, significantly improving my ability to reliably detect C. difficile inside mouse tissue. This finding was particularly unexpected because both targeted and random mutagenesis had been used to develop mScarlet-I3⁴. Regardless, after years of trial and error, I finally arrived at a combination of reporters with equivalent fitness, which finally allowed me to ask the biological questions that originally drove this project!

In particular, we wanted to know if, where, and how C. difficile heterogeneously expresses toxin genes during infection. Gratifyingly, I showed that we could readily detect a mixture of magenta (toxin-OFF) or green (toxin-ON) cells (Figure 1), which were no longer observed in a mutant incapable of inducing toxin gene expression (ΔtcdR)⁷, and were markedly over-expressed at the single-cell level and across the population (ΔrstA)⁸. To our surprise, mice infected with the ΔrstA strain formed striking filaments during the acute phase of infection (day 2).

At first, we worried that the fluorescent reporters themselves might be causing this filamentation. But, after a series of control experiments involving new reporter strains, some of which were suggested by the Reviewers and my thesis advisory committee, I was able to determine that the abnormal cell morphology of the ∆rstA mutant reflected a genuine biological difference rather than a fluorescent protein artifact. Intriguingly, I subsequently discovered that this morphology was only observed during acute infection (day 2) and not after disease resolution (day 14) or during in vitro growth. This underscored one of the central themes of this work: interesting biology occurs inside the host that can only be identified by visualizing bacteria in their native context.

As mentioned earlier, another unexpected finding was that toxin gene expression was unaffected by the location of C. difficile cells in the colon—it was heterogeneous to similar degrees and magnitudes, regardless of where C. difficile was found in the gut (i.e., in the lumen, mucus, or close to the epithelium). These observations suggest that heterogeneity in toxin functions as a bet-hedging strategy, with the toxin-producing subset of cells liberating nutrients from host tissue to benefit the entire population, while the remaining cells avoid the metabolic cost of toxin production and may be better positioned to survive and disseminate, for example, through sporulation.

Ultimately, this paper provides a toolkit for visualizing bacterial gene expression in situ during infection, allowing us to uncover hidden biology within the host. For me, it’s a reminder that discovery doesn’t always come from knowing what to look for; it can also come from having the tools to see what’s there.

REFERENCES

1. Ransom, E. M., Kaus, G. M., Tran, P. M., Ellermeier, C. D. & Weiss, D. S. Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile. Mol. Microbiol. 110, 533–549 (2018).
2. Donnelly, M. L. et al. Development of a Dual-Fluorescent-Reporter System in Clostridioides difficile Reveals a Division of Labor between Virulence and Transmission Gene Expression. mSphere 7, e0013222 (2022).
3. Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40, 1132–1142 (2022).
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5. Campbell, B. C. et al. mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging. Proc. Natl. Acad. Sci. 117, 30710–30721 (2020).
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