Epsilon toxin–producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege
Yinghua Ma, … , Christopher E. Mason, Timothy Vartanian
Yinghua Ma, … , Christopher E. Mason, Timothy Vartanian
Published February 28, 2023
Citation Information: J Clin Invest. 2023;133(9):e163239. https://doi.org/10.1172/JCI163239.
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Epsilon toxin–producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege

Abstract

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin–producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

Authors

Yinghua Ma, David Sannino, Jennifer R. Linden, Sylvia Haigh, Baohua Zhao, John B. Grigg, Paul Zumbo, Friederike Dündar, Daniel Butler, Caterina P. Profaci, Kiel Telesford, Paige N. Winokur, Kareem R. Rumah, Susan A. Gauthier, Vincent A. Fischetti, Bruce A. McClane, Francisco A. Uzal, Lily Zexter, Michael Mazzucco, Richard Rudick, David Danko, Evan Balmuth, Nancy Nealon, Jai Perumal, Ulrike Kaunzner, Ilana L. Brito, Zhengming Chen, Jenny Z. Xiang, Doron Betel, Richard Daneman, Gregory F. Sonnenberg, Christopher E. Mason, Timothy Vartanian

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Figure 3

Characterization of patient-derived strain SHDS0050 and its comparison to environmental and laboratory ETX-producing strains.

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Characterization of patient-derived strain SHDS0050 and its comparison t...
(A) The circular map of etx+ plasmid (pSHDS0050) from a MS patient-derived SHDS0050 strain. Hypothetical and proteins of unknown function ORFs are colored black, those involved in conjugation are purple, toxin ORFs are red, DNA methylases are green, transposases and recombinases are gray, ribonucleases are pink, plasmid replication ORFs are blue, conserved etx plasmid ORFs are red, and ABC transporters are magenta. (B) GView BLAST Atlas map comparing the circular chromosomes of pSHDS0050 as the reference genome to etx+strains CN3842, FU17, NCTC8346, ATCC3626, and NCTC3110. Type D chromosomes are shades of blue, while type B are shades of red. Colored regions for genomes indicate a BLAST hit present in the reference genome. Empty slots indicate no matching hits. SHDS0050 genome is distinct from other strains. (C) Linearized plasmid map comparisons comparing pSHDS0050 with the etx plasmids of 3 type D collection strains pCN3842, pFU17etx, and pNCTC8346etx, and 2 type B strains pATCC3626etx and pNCTC3110etx. The linearized maps use the same color scheme as A, with different transposase ORFs having different pattern fillings. The 3 type D strains share the same gene content and share many of the same genes. (D) Western blot analysis for pETX production from indicated C. perfringens strains. pETX spiked into PBS or broth were used as positive controls. Broth only was used as a negative control. (E) Susceptibility of hMAL-CHO or GFP-CHO cells to activated ETX. To confirm if cell death was ETX mediated, broth was pretreated with a neutralizing anti-ETX antibody. Broth alone was used as a negative control. Cell death was determined by PI staining. n = 3. ****P < 0.0001 determined by 2-way ANOVA with Šidák’s multiple comparisons test.