Sulfated glycosaminoglycans and low-density lipoprotein receptor mediate the cellular entry of Clostridium novyi alpha-toxin

Dear Editor, Clostridium novyi ( C. novyi ) is a spore-forming anaerobic bacterium and opportunistic pathogen causing severe infectious diseases in humans and animals including gas gangrene, myositis, necrotic hepatitis, and sepsis. 1,2 The alpha-toxin (Tcn α ) is the major virulence factor of C. novyi and is produced by all known pathogenic C. novyi types. 1 Tcn α belongs to the large clostridial toxin (LCT) family which also includes Clostridioides dif ﬁ cile toxin A (TcdA) and toxin B (TcdB), Paeniclostridium sordellii lethal toxin (TcsL) and hemorrhagic toxin (TcsH), and Clostridium perfringens large cytotoxin (TpeL). Like other LCTs, Tcn α binds to the surface of the target cell and enters the cell via receptor- mediated endocytosis. The enzymatic domain is then delivered into the cytosol and glucosylates small Rho-family GTPases using UDP-N-acetylglucosamine as a co-substrate, leading to cytoskele-ton disruption and cell death. 3 Recently, the cellular receptors of several LCTs, including TpeL, TcdB, TcdA, and TcsL have been identi ﬁ ed. 4 – 10 Tcn α and TcsH are the only two major members of the LCT family with their host receptors remaining unclear. To identify potential receptors for Tcn α , we conducted genome-wide CRISPR/Cas9-mediated knockout (KO) screens. HeLa cells stably expressing Cas9 (parental cell line, referred to as the wildtype (WT) thereafter) were transduced with a lentiviral single guide RNA (sgRNA) library (GeCKO v2) and subjected to three


Genes and plasmids.
The DNA sequences encoding Tcnα (Clostridium novyi A strain GD211209) and TcdB (Clostridioides difficile strain 630) were codon-optimized, synthesized by Genscript (Nanjing, China), and cloned into a modified pHT01 vector with a His-tag introduced to their C-terminus. The expression plasmid pQTEV-LRPAP1 encoding LRPAP1 was obtained from Addgene (#31327). The DNA fragments encoding the ectodomain of human LDLR (LDLR 22-788 ) and IgG1 Fc were fused and cloned into the pHLsec vector as previously described 2 . The DNA fragments encoding mouse Ldlr were cloned into the plasmid pLVX-IRES-mCherry (Miaoling Bioscience & Technology Co., Ltd, P0424).

Protein purification.
Recombinant Tcnα and TcdB were expressed in Bacillus subtilis SL401 and purified as His6-tagged proteins as previously described 3 . In brief, B. subtilis cells were cultured at 37 ℃ till OD600 reached 0.6 and induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 25 ℃ for 20 hours. LRPAP1 was expressed in Escherichia coli BL21 (DE3) and purified as His6-tagged proteins. E. coli cells were cultured at 37 ℃ till OD600
The GeCKO v2 library is composed of two sub-libraries with each contains three unique sgRNA per gene and was independently prepared and screened 4 . HeLa-Cas9 cells were transduced with sgRNA lentiviral library at a multiplicity of infection of 0.3 and selected with 2.5 μg/ml puromycin. For each CRISPR sub-library, ~8×10 7 cells were plated onto two 15-cm cell culture dishes to ensure sufficient sgRNA coverage.
These cells were exposed to Tcnα for 16 hours and then washed with phosphate buffer saline (PBS) to remove loosely attached cells. The remaining cells were cultured with toxin-free medium to ~70% confluence and subjected to the next round of screening with higher concentrations of toxins. Three rounds of screenings were performed with increasing concentrations of Tcnα (160, 400, and 750 pM). The remaining cells were collected and their genomic DNA was extracted using the Blood and Cell Culture DNA mini kit (Qiagen). DNA fragments containing the sgRNA sequences were amplified by PCR using primers lentiGP1_F (AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG) and lentiGP-3_R (ATGAATACTGCCATTTGTCTCAAGATCTAGTTACGC). The next-generation sequencing was performed by the commercial vendor (Novogene, Beijing).
The cytopathic effect of Tcnα and TcdB was analyzed using the standard cell-rounding assay. In brief, cells were exposed to Tcnα and TcdB for 10 hours, and phase-contrast images of cells were recorded (Olympus IX73; ×10 or ×20 objectives). A zone of 300×300 μm was selected randomly, containing 50-150 cells. The numbers of normal and round-shaped cells were counted manually. The percentage of round-shaped cells was analyzed using the GraphPad Prism software (ver. 9.0.0, GraphPad Software, LLC).

Competition assays with glycans, surfen, or LRPAP1.
For competition with glycans, Tcnα (7.5 nM) was pre-mixed with or without 1mg/ml

Ethics statement.
The intramuscular injection of Tcnα in mice was performed following the institutional guidelines. All animal procedures reported herein were approved by the Institutional Animal Care and Use Committee at Westlake University (IACUC Protocol #20-046-TL). To minimize the distress and pain, the mice were monitored every hour. Animals with signs of pain or distress such as labored breathing, inability to move after gentle stimulation, or disorientation were euthanized immediately. This method was approved by the IACUC and monitored with a qualified veterinarian.

Statistical analysis.
Statistical analysis was performed using GraphPad Prism software (ver. 9.0.0, GraphPad Software, LLC) and SPSS (ver. 22, IBM) software. Experimental groups were compared using the two-sided Mann-Whitney test (Fig. 1d, f, i, l, n, and r; Supplementary information, Fig. S7). All data were obtained from independent experiments.

Data availability.
All data are fully available without restriction.