Biology subjects

Amatsu, S.

Publications and source records attributed to Amatsu, S..

5 recordsLinked to original sources

bioRxiv2026

LRP1 is an entry receptor for the botulinum toxin complex in the gut

Botulinum neurotoxin (BoNT) is an etiologic agent of food poisoning caused by Clostridium botulinum. The large progenitor toxin complex (L-PTC) crosses the intestinal epithelial barrier to deliver BoNT to target neurons; however, it is not clearly understood how BoNT enters the host. Here, we identified low-density lipoprotein receptor-related protein 1 (LRP1) as a major enterocyte transcytosis receptor for the hyper-oral-toxic L-PTC serotype B-Okra (L-PTC/BOkra). We found that hemagglutinin (HA), a neurotoxin-associated protein within the L-PTC/BOkra complex, binds to LRP1 via N-glycans. HA/BOkra co-localized with LRP1 within the internalized vesicles in cultured cells and enterocytes. LRP1 deletion inhibited the apical-to-basal transcytosis of L-PTC/BOkra in an intestinal epithelial cell line, and this effect was rescued by LRP1 re-expression. Finally, intestinal epithelial cell-specific LRP1-deficient mice displayed reduced susceptibility to toxicity caused by oral administration of L-PTC/BOkra. Taken together, these results indicate that N-glycosylated LRP1 mediates L-PTC/BOkra transcytosis via enterocytes, enabling BoNT to traverse the intestinal epithelial barrier.

microbiology↗

bioRxiv2026

An Antibiotic-Treated Mouse Model Reveals the Progression of Intestinal Botulism

Intestinal botulism, including infant botulism and adult intestinal toxemia botulism, is a life-threatening disease caused by intestinal infection with Clostridium botulinum (Cb) spores. Infants are particularly susceptible to Cb infection because of their immature gut microbiota, whereas healthy adults are generally protected by gut microbiota-mediated colonization resistance. Thus, immature gut microbiota or gut dysbiosis is thought to permit Cb colonization. However, the mechanisms underlying colonization resistance and disease progression remain poorly characterized. Here, using adult mice with antibiotic-induced dysbiosis, we established a model for studying Cb infection and characterized intestinal colonization, bacterial expansion, BoNT accumulation, and disease progression during intestinal botulism. Intestinal botulism developed in antibiotic-treated mice after intragastric administration of strain 62A spores, whereas untreated adult mice showed no symptoms. In antibiotic-treated mice, Cb expanded over time in fecal samples, followed by accumulation of BoNT/A, which correlated with the progression of botulism symptoms. Cb growth and BoNT/A accumulation occurred mainly in the cecum and colon, but not in the small intestine. Furthermore, this mouse model was applicable to the analysis of intestinal botulism caused by other Cb strains, including 7I03-H, Okra, and Osaka05. Taken together, this mouse model provides a useful platform for elucidating the pathogenesis of intestinal botulism and developing novel therapeutic strategies.

microbiology↗

bioRxiv2025

Gut mucin fucosylation dictates the entry of botulinum toxin complexes

Botulinum toxins (BoNTs) are the most potent known bacterial toxins. The BoNT complex from Clostridium botulinum B-Okra (large progenitor toxin complex (L-PTC)/BOkra, hyper-oral-toxic) exerts at least 80-fold higher oral toxicity1 in mice compared with that from serotype A1 (L-PTC/A62A, non-hyper-oral-toxic). Here, we showed that L-PTC/BOkra was predominantly absorbed through enterocytes, whereas L-PTC/A62A targeted intestinal microfold cells. Furthermore, we demonstrated that 1,2-fucosylation of intestinal mucin determined the oral toxicity of L-PTCs as well as their entry routes; more specifically, these routes were governed by the carbohydrate-binding spectrum of hemagglutinin (HA) complex, which is one of the L-PTC components. Disruption of fucosylation in fucosyltrasferase-2 (Fut2)-null mice hindered the intestinal mucin penetration of L-PTC/BOkra via HA and reduced the susceptibility to its oral intoxication. Our data establish the molecular mechanism by which the oral toxicity of BoNTs is increased after they cross intestinal mucus layers.

microbiology↗

bioRxiv2024

Development and Optimization of Genetic Manipulation Systems in Group I Clostridium botulinum

Clostridium botulinum causes the disease botulism, and nontoxigenic Clostridium sporogenes is closely related to group I C. botulinum. Despite its pathogenicity, C. botulinum remains poorly characterized. Genetic manipulation is critical for understanding bacterial physiology and disease. We compared the conjugal transformation efficiencies of seven strains, including group I C. botulinum (strains 62A, 7I03-H, Okra, Osaka05, 111) and C. sporogenes (strains JCM 1416T, ATCC 15579), and our results showed that few or no transformants were obtained in certain strains. In the present study, we demonstrate that our optimized protocol increases the efficiency of DNA transfer from E. coli donor cells to recipient strains. In addition, we developed a novel conjugal suicide vector pXMTL that contains xylose-inducible mazF as a counter-selection marker, and can be transferred into Clostridium spp. by conjugation. The allele-coupled exchange (ACE) system using pXMTL provides a rapid method for precise, markerless and scarless genome editing in group I C. botulinum and C. sporogenes. ImportanceGroup I C. botulinum and C. sporogenes exhibit low transformation efficiencies, and few or no transformants are yielded by some strains. In this study, we optimized the conjugation protocol to improve transformation efficiency. In addition, we developed a novel suicide vector pXMTL harboring a xylose-inducible mazF marker, and can be transferred into Clostridium spp. by conjugation. The combination of pXMTL and the optimized conjugation protocol provides a powerful tool for genetic manipulation of group I C. botulinum and C. sporogens.

microbiology↗

bioRxiv2019

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately [≥] 100 i.p. LD50/mg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of M2 and M4 was able to completely neutralize BoNT/B1 with a potency greater than 10,000 i.p. LD50/mg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication. IMPORTANCEBotulinum neurotoxins (BoNTs) produced by Clostridium botulinum and related species cause human botulism. Immunotherapy is the most effective treatment for botulism and equine immune serum formulations are used in cases of human botulism. However, these antisera may cause serum sickness or anaphylaxis. Additionally, the production of immune sera involves complicated and time-consuming manufacturing processes and quality management. Therefore, the development of safe, effective, and higher productive antibodies is required. Here we generated fully human monoclonal antibodies against serotype B BoNT (BoNT/B). We found that the combination of these antibodies (M2+M4) had potent and broad neutralization activity against BoNT/B, and showed therapeutic and preventive effects against botulism in mouse models. These data indicate that M2+M4 are promising candidates for the development of human therapeutics and prophylactics for BoNT/B intoxication.

microbiology↗