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Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

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Pestalotiopsis-like fungi are widely distributed in many plants and include endophytes, pathogens and saprobes. Five strains of Neopestalotiopsis were isolated from diseased leaves of Rhapis excelsa (Principes, Palmae), Rhododendron simsii and Rho. championiae (Ericales, Ericaceae) and Erythropalum scandens (Santalales, Olacaceae) in southern China. Based on morphology and multi-gene (ITS, tub2, tef1) phylogeny, our five strains of Neopestalotiopsis represent two new species and one extant species. Descriptions, illustrations and notes are also provided for the new species.

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