There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homologue with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs.

During active multiple sclerosis (MS), red blood cells (RBCs) harvested from patients reportedly display increased osmotic fragility and increased cellular volume (macrocytosis). The cause of these abnormalities remains unknown. We have previously proposed that Clostridium perfringens epsilon toxin (ETX) may be a blood-borne trigger for newly forming MS lesions based on its tropism for blood-brain barrier vasculature and CNS myelin. Recently, Gao et al. have reported that ETX binds to and damages human RBCs, leading to hemolysis. Moreover, the authors suggest that purinergic nucleotide (P2) receptor activation amplifies the hemolytic process. Here, we confirm that ETX indeed causes human-specific RBC lysis. However, our data suggest that the hemolytic process is mediated by metal-catalyzed oxidation of the swell-induced, nucleotide-sensitive ICln chloride channel. We use spectrophotometry, flow cytometry and Western blotting to show that ETX targets human RBCs and T lymphocytes via their shared expression of Myelin and Lymphocyte protein (MAL); a protein shown to be both necessary and sufficient for ETX binding and toxicity. ETX likely triggers T cells to release redox-active heavy metals, Cu ⁺ and Fe ³⁺ , via the lysosomal exocytosis pathway, while RBCs likely release these heavy metals via ETX pore formation within the RBC membrane. Extracellular Cu ⁺ and Fe ³⁺ may then amplify hemolysis by oxidizing a previously identified heavy metal-binding site within the ICln channel pore, thus deregulating its normal conductance. Elucidating the precise mechanism of ETX-mediated hemolysis may shed light on the underlying etiology of MS, as it would explain why MS RBC abnormalities occur during active disease. IMPORTANCE: During active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily. The exact trigger(s) for these RBC abnormalities and for newly forming MS lesions remains unidentified. We have proposed that ETX, secreted by the gut bacterium Clostridium perfringens , may be an environmental trigger for newly forming MS lesions. Indeed, ETX has been shown to breakdown the BBB, enter the brain and damage the myelin sheath. Because ETX is typically spread through the circulatory system, we wished to determine how the toxin affects human blood. Provocatively, there has been a recent report that ETX produces cellular abnormalities in human RBCs, reminiscent of what has been described during active MS. In our study, we sought to elucidate the precise mechanism for how ETX causes RBC damage. In addition to triggering BBB breakdown and CNS demyelination, ETX might also explain why RBCs appear abnormal during MS attacks.