Microbial, Mitochondrial, and Mucosal Impacts on Chemical Susceptibility in Caenorhabditis elegans

Abstract

Humans encounter a myriad of chemicals daily, and some individuals are more susceptible to chemical exposure than others, resulting in exacerbated adverse effects or disease. One highly variable biological system that can modulate chemical susceptibility is the intestinal tract. The intestinal environment is responsible for digesting food and extracting nutrients needed to sustain cellular homeostasis, but it also encounters many harmful elements such as environmental pollutants and pathogenic bacteria. The large intestine and colon house the majority of the gut microbiome, which plays important roles in metabolism, immunity, and xenobiotic metabolism. The intestinal tract is lined with a dense mucus layer, composed primarily of mucin, that supports and protects the epithelial barrier. Disruption of this mucus layer, as seen in inflammatory diseases such as ulcerative colitis, can trigger inflammatory responses from the gut microbiota. Mitochondria are also central to intestinal health, as they provide energy in the form of adenosine triphosphate (ATP) required to maintain the epithelial barrier and play key roles in cellular differentiation and apoptosis. Because mitochondria rely on metabolites produced by gut microbiota, interactions between the gut microbiome, mitochondrial function, and intestinal mucin are crucial for maintaining intestinal health. In recent years, these interactions have received increased research attention. Environmental pollutants can disrupt the gut microbiome, induce mitochondrial dysfunction, and impair mucin production, suggesting that pollutant impacts on these key components of the intestinal tract may further contribute to individual variability in susceptibility to environmental pollutants. However, the integrative roles of these systems in modulating chemical susceptibility remain poorly understood.To better understand how the intestinal environment impacts pollutant susceptibility, I used the model organism Caenorhabditis elegans, which has well-conserved mitochondrial biology and is colonized by a diverse gut microbiome. This model offers unique advantages because of its genetic tractability, short life cycle, and conserved stress response pathways. A collection of native gut bacteria isolated from wild C. elegans, referred to as CeMbio, offers additional advantages by providing a defined diet with some previous characterization, and allows future studies to build upon this work. C. elegans is particularly well suited for investigating host-microbe interactions, as nematodes are bacterivores that can be grown on a single bacterial strain or a mixture. Moreover, germ-free nematodes can be created with relative ease compared to other model organisms by using a sodium hypochlorite treatment to which embryos are resistant. In Aim 1, I used C. elegans to test how individual strains of bacteria from the CeMbio collection impact mitochondrial bioenergetics and susceptibility to known mitochondrial toxicants. In Aim 2, I examined how intestinal mucin affects redox state and susceptibility to the environmental pollutant cadmium. Together, these aims investigate how microbial influences on mitochondrial metabolism and mucin-dependent modulation of barrier function can shape organismal response to toxicants. I discuss the strengths and limitations of the C. elegans model for understanding the interactions between pollutants, the microbiome, and mucin in the Conclusion chapter. The first aim of this dissertation explored how microbial metabolites influence mitochondrial function by shifting bioenergetic pathways, thereby altering sensitivity to mitochondrial toxicants. Although the gut microbiota is known to influence mitochondria, it is not known whether changes in bioenergetics due to the gut microbiome can alter susceptibility to mitochondrial toxicants. I found that C. elegans grown on selected bacterial strains had varying levels of steady-state ATP, with a ~3 fold difference between the highest and lowest strains. Further, C. elegans grown on selected bacterial strains showed differential sensitivity to short-term exposure to chemicals that inhibit mitochondrial electron transport chain Complexes I, II, and V, and fatty acid oxidation. To test mechanistically how microbiome-mediated sensitivities could result in chemical susceptibility, I carried out follow-up experiments using the Complex I inhibitor rotenone. I found that C. elegans grown on BIGb0170 (Sphingobacterium multivorum) had markedly higher lethality after 24- and 48-hour exposures than when grown on MYb10 (Acinetobacter guillouiae), MYb11 (Pseudomonas lurida), and OP50 (Escherichia coli) strains. I also saw no differences in mitochondrial oxygen consumption rate, mitochondrial to nuclear DNA copy number, or germline differences in nematodes grown on these four bacterial strains. Since gut microbiota typically primarily interacts with mitochondria through the release of metabolites, I used metabolomics to measure differences in C. elegans grown on the four bacterial strains. Metabolomic analysis revealed that C. elegans grown on BIGb0170 had lower amounts of triglycerides and acylcarnitines, which are metabolized into acetyl-CoA and used to fuel the TCA cycle within mitochondria. To test whether these metabolite differences could be involved in the rotenone response I had seen, I supplemented bacteria with pyruvate, which is also metabolized into acetyl-CoA, and found that ATP levels were partially rescued in nematodes grown on BIGB0170 after exposure to rotenone. These findings provide mechanistic evidence that bacteria can directly influence host mitochondrial vulnerability to toxicants by altering substrate availability. This establishes a novel link between gut microbiome composition, host energy metabolism, and toxicant sensitivity. The second aim investigates how intestinal mucin impacts susceptibility to the environmental pollutants cadmium and antimycin A. Cadmium is a heavy metal that is associated with manufacturing, while antimycin A is a registered pesticide that has been historically used to control fish populations. Both chemicals alter the cellular redox tone through differing mechanisms. Cadmium can reduce levels of glutathione, the main antioxidant within cells, while antimycin A inhibits mitochondrial electron transport chain Complex III, blocking the flow of electrons and increasing reactive oxygen species. Mucin is a high molecular weight glycoprotein that lines epithelial cells and is the main component of the mucus layer that helps create a barrier. The intestine can respond to irritants by increasing the production of mucin, likely in response to reactive oxygen species. Alternatively, chemicals can impair the mucin lining, thereby increasing permeability. Therefore, I exposed C. elegans with and without the mucin-like gene mul-1 located in the intestinal tract to increasing levels of cadmium and antimycin A for 48 hours during development. Higher cadmium exposure trended towards increased redox state overall; however, at 200 µM, mul-1 mutants showed lower oxidized to reduced glutathione ratios than wildtype. No significant differences were observed in either strain following exposure to antimycin A. However, mul-1 expression increased over the course of C. elegans development. Based on this finding, I started exposures to both chemicals at the last larval stage (L4) for 24 hours. Under these conditions, mul-1 mutants consistently trended towards less oxidized to reduced glutathione ratios compared to wildtype nematodes at all cadmium doses tested, while no differences in redox state were observed with antimycin A. Given the role of mucin in maintaining the epithelial barrier, intestinal permeability was also assessed. No differences in permeability were observed between the mul-1 and wildtype strains under control conditions or after exposure to a high dose of cadmium throughout adulthood. To better understand why mul-1 nematodes had less oxidized glutathione, expression of several genes involved in stress response and glutathione metabolism was mesasured. I found a trend towards increased expression of the catalase genes and mitochondrial superoxide dismutase gene in the mul-1 nematodes. Together, these results indicate that intestinal mucin contributes to the regulation of cellular redox balance, and that loss of mucin may trigger compensatory upregulation of antioxidant defenses and thereby alter susceptibility to environmental chemicals. In conclusion, this dissertation demonstrates that the gut microbiome, mitochondrial function, and intestinal mucin can potentially alter chemical susceptibility. By elucidating how these systems interact in C. elegans, this work provides new insight into a potential reason behind the variability of toxicant responses. As environmental exposures continue to rise globally, advancing our understanding of these host–microbe–barrier interactions will be essential for improving risk assessment, guiding therapeutic interventions, and protecting public health. Ultimately, this work underscores the need for integrative approaches to predict and mitigate chemical susceptibility across diverse populations.