Epsilon toxin-producing Clostridium perfringens colonize the gut of multiple sclerosis patients

ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination.
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What is already know
Multiple sclerosis (MS) is a complex disease of the central nervous system (CNS) and it is believed to require an environmental trigger. However, biologically plausible environmental agents responsible for lesion induction have not been identified yet. Gut dysbiosis is frequently observed in MS patients, but the specific species responsible for causing the disease remain elusive.

What this research adds
Researchers used sensitive and quantitative polymerase chain reaction (PCR) method to detect specific bacterial species involved in the pathogenesis of MS in fecal samples from patients with MS. They found a greater abundance of C. perfringens, an epsilon toxin (ETX)-producing strains, in the gut of people affected by MS compared to healthy individuals. Moreover, they used an immunization mouse model of experimental autoimmune encephalomyelitis (EAE), and discovered that ETX-induced inflammatory demyelination, which more closely resembled the lesion distribution observed in MS.

These findings imply that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination.

MS disease initiation and lesion formation, is believed to require an environmental trigger in a genetically susceptible individual. Studies have consistently found an imbalance in the gut microbiome of individuals with MS, and certain gut microorganisms can either promote or suppress autoimmunity. The neurotoxin C. perfringens ETX specifically targets the endothelial cells in the CNS and causes disruption of the blood-brain barrier integrity. Therefore, it could be a possible environmental trigger for MS. 

A recent study from Vartanian and colleagues, published in the Journal of Clinical Investigation, provides evidence that C. perfringens ETX-producing strain acts as environmental triggers for MS

The authors hypothesized that environmental triggers for MS do not occur once at disease initiation, but arise repeatedly throughout the course of disease. In line with that, ETX production occurs when C. perfringens types B or D enter log-phase growth and is thus tied to increased abundance of these strains in the gut microbiome.

By analyzing faecal samples from 62 individuals and performing mouse model experiments, Vartanian and colleagues established a link between ETX-producing strains of C. perfringens and MS in clinical samples, discovered that individuals with MS have a higher abundance of C. perfringens ETX-producing strains in their gut microbiome compared to healthy individuals, and demonstrated that ETX alone can cause multifocal, inflammatory demyelination in the presence of active immunization.

Overview of C. perfringens 

C. perfringens is a gram-positive anaerobe that can produce up to 6 toxins, with type B and D strains carrying the etx gene. These strains can be found in the small intestine of mammals, including humans, and are present in a wide range of ecological niches. C. perfringens can persist in various environments due to its spores’ resistance to heat, chemicals, radiation, and pressure. Colonization of the small intestine by C. perfringens following environmental exposure and oral ingestion is believed to depend on host genetics, microbiome composition, and other factors, such as prior antibiotic use.

Several ruminants harbor C. perfringens type D in their small intestine, but usually microorganism numbers are small and clinical disease does not occur unless the intestinal microbial balance is disrupted. When large quantities of fermentable carbohydrates are fed to these animals, undigested starch passes into the small intestine providing a substrate for C. perfringens to proliferate. This is followed by production of large amounts of the ETX toxin, which binds to CNS endothelial cells causing permeability of the blood-brain barrier and is absorbed into the systemic circulation. The bacteria are passed by feces into the environment and can survive for several months in the soil. 

The gut microbiome of MS patients harbors ETX-producing C. perfringens strains

PCR screening revealed an occurrence of etx gene in 61% of fecal microbiota samples harvested from MS patients and 13% of controls, and that ext expression was significantly associated with disease status. Moreover, the increase in the alpha toxin gene (cpa) in the MS group compared to healthy subjects, suggested that people with MS may have a more suitable gastrointestinal environment for the survival and growth of C. perfringens. These findings showed that people with MS are more likely to be colonized by EXT-producing strains of C. perfringens in their gut microbiome than healthy people.  

ETX induces multi-focal demyelination

An active immunization mouse model of EAE was used, where animals are immunized with a myelin antigen, typically a myelin specific protein or peptide. Immunized animals do not develop clinical CNS disease unless they also receive another toxin which targets CNS endothelial cells at the blood-brain barrier. The authors investigated the effects of EXT-producing C. perfringens and found that EAE mice treated with EXT developed multifocal demyelination typical of MS and had many lesions in the cerebellum, and in the corpus callosum. ETX-induced EAE showed mononuclear infiltrates that correlated with demyelination, infiltrating CD4+ lymphocytes in the cerebellum and in the thalamus. Overall, ETX induced multifocal, inflammatory demyelination in a neuroanatomic distribution consistent with MS, and with a strong correlation between immune infiltrates and demyelination in the ETX-EAE model.

In conclusion, these findings demonstrate a strong clinical association between a specific bacterium, its toxin, and a diagnosis of MS. In addition, the abundance of ETX-producing strains of C. perfringens is significantly elevated in the MS gut microbiome. However, it would be interesting to conduct a clinical trial to neutralize ETX or eliminate C. perfringens in the human host and observe the consequences.