Mice

Specific pathogen-free female C57BL/6J mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed under clean conditions with free access to food and water. Female mice were used to avoid unintended infections caused by male aggression and to prevent potential effects on survival outcomes, thereby ensuring reliable and consistent experimental results. Animals purchased for the study were randomly assigned to cages, with 3–5 mice per cage. No specific measures were taken to control potential confounders such as treatment order, measurement order, or cage location. Mice older than 8 weeks were used in all experiments. Animal experiments were approved by the Animal Care and Use Committee of Tokushima Bunri University (Approval No. 19-5), and procedures were performed in accordance with institutional guidelines. Institutional guidelines conformed to the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006.

This study investigates the severity of C. perfringens type A infection and the effects of its α-toxin in the host. Mice were used as a model because they are a well-established system for studying bacterial infections, and findings from this model are relevant for understanding aspects of human disease. No study protocol was prepared prior to the experiments.

Reagents and strain

The Amplex red phosphatidylcholine-specific phospholipase C assay kit and Amplex red sphingomyelinase assay kit were purchased from Thermo Fisher Scientific Inc. (MA, USA). Clindamycin was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Micafungin, caspofungin, and peptidoglycan from Bacillus subtilis were purchased from Sigma-Aldrich (MO, USA). Cell Counting Kit-8 (CCK-8) and Cytotoxicity LDH Assay Kit-WST were from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Fluorescein isothiocyanate- or phycoerythrin-conjugated specific antibodies against mouse CD11b (clone M1/70, catalog no. 557396, lot no. 4310683) or Ly-6G/6C (Gr-1; clone RB6-8C5, catalog no. 561084, lot no. 3312623) and purified rat anti-mouse CD16/CD32 (Fc Block; clone 2.4G2, catalog no. 553141, lot no. 6092754) were purchased from BD Biosciences (CA, USA). The SCREEN-WELL FDA-Approved Drug Library V2, Japan version was obtained from Enzo Life Sciences, Inc. (NY, USA). All other chemicals were of the highest grade available from commercial sources. Strain 13 was used as wild-type C. perfringens type A strain.

Drug screening methodology

The effects of primarily 50 μM of the test compounds (only one at 10 μM) on the PLC activity of 50 ng/ml α-toxin were measured using the Amplex red phosphatidylcholine-specific phospholipase C assay kit according to the manufacturer’s instructions. After one hour of incubation, the measurement was performed by detecting fluorescence intensity with excitation at 550 nm and emission at 580 nm.

The effects of micafungin or caspofungin on SMase activity were measured using Amplex red sphingomyelinase assay kit according to the manufacturer’s instructions. The measurement was performed by detecting fluorescence intensity with excitation at 550 nm and emission at 580 nm.

Purification of α-toxin

α-Toxin was purified as previously described¹². Briefly, B. subtilis ISW1214 was transformed with recombinant forms of pHY300PLK, which harbors the structural α-toxin gene, and was then cultured in Luria-Bertani broth supplemented with 50 μg/ml ampicillin at 37 °C. The culture medium containing secreted α-toxin was collected, and α-toxin was chromatographically purified.

The lethal activity of α-toxin in mice was measured using the following method. C57BL/6J mice were injected intraperitoneally with 600 ng of α-toxin and 300 µg of micafungin or caspofungin, which had been pre-mixed and diluted in phosphate-buffered saline (PBS), and the survival of mice was monitored. The experimental unit was a cage of animals. The exact number of mice per group and the total number of mice per experiment are provided in the figure and figure legend. No a priori sample size calculation was performed. Sample sizes were chosen based on previous experience with similar experiments and expected variability. No specific exclusion criteria were established, and no data points or animals were excluded from the analysis. The experiment was conducted by investigators who were aware of the group allocations, while outcome assessment was performed by a separate evaluator who was blinded to the group assignments. Humane endpoints were established such that animals exhibiting more than 20% body weight loss compared to controls, or showing persistent hunching, labored breathing, or other signs of severe distress, were immediately euthanized to minimize pain and suffering.

Cell culture

To obtain bone marrow cells (BMCs), femurs and tibias were crushed in PBS supplemented with 2% heat-inactivated fetal bovine serum (FBS). After cells were filtered through a 40-µm mesh, red blood cells were hemolyzed with ACK lysing buffer (GIBCO, NY, USA). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin.

Human umbilical vein endothelial cells (HUVECs, product code C-12208) were obtained from PromoCell (Sickingenstr, Heidelberg, Germany). They were supplied from pooled donors. HUVECs were cultivated in Endothelial Cell Growth Medium 2 (PromoCell, Sickingenstr, Heidelberg, Germany). The medium contains 2% FBS. HUVECs were incubated for 4 h in the presence or absence of α-toxin and the candidate compounds. Cell viability was measured using CCK-8. The median effective concentrations (EC₅₀) of micafungin and caspofungin for inhibiting α-toxin-mediated cell death were calculated using GraphPad Prism (9.5.1). A LDH leakage assay was performed using Cytotoxicity LDH Assay Kit-WST according to the manufacturer’s protocol.

Bacterial culture and infection

Bacterial culture and infection were performed as previously described¹². In brief, C. perfringens strain 13 was grown in tryptone, glucose, and yeast extract (TGY) medium under anaerobic conditions at 37 °C. Exponentially growing bacteria were harvested, washed, re-suspended in TGY medium, and injected into the femoral muscle of mice. Immediately after administering bacteria, caspofungin and/or clindamycin diluted in PBS was intraperitoneally administered. To quantify CFUs, residual bacteria were serially diluted, plated on brain heart infusion agar plates, and cultured anaerobically at 37 °C.

In the experiment assessing mouse survival, exponentially growing bacteria (9 × 10⁸ CFU) were harvested, washed, re-suspended in TGY medium, and injected into the femoral muscle of mice. Immediately after administering bacteria, 1 mg of caspofungin and/or 2 mg of clindamycin diluted in PBS was intraperitoneally administered, and the survival of mice was monitored. The experimental unit was a cage of animals. The exact number of mice per group and the total number of mice per experiment are provided in the figure and figure legend. No a priori sample size calculation was performed. Sample sizes were chosen based on previous experience with similar experiments and expected variability. No specific exclusion criteria were established, and no data points or animals were excluded from the analysis. The experiment was conducted by investigators who were aware of the group allocations, while outcome assessment was performed by a separate evaluator who was blinded to the group assignments. Humane endpoints were established such that animals exhibiting more than 20% body weight loss compared to controls, or showing persistent hunching, labored breathing, or other signs of severe distress, were immediately euthanized to minimize pain and suffering.

Flow cytometry analysis

Cultured BMCs were labeled with antibodies diluted 1:20 in PBS containing 2% FBS after blocking Fc receptors with purified rat anti-mouse CD16/CD32 at 1:100. Labeled cells were assessed using Guava easyCyte (Millipore, MA, USA), and data were analyzed by FlowJo software (Tree Star, OR, USA). Cells stained with propidium iodide were excluded from analyses.

ELISA

HUVECs were cultured in Endothelial Cell Growth Medium 2 in accordance with the manufacturer’s protocol. After the treatment of cells with α-toxin, peptidoglycan (PGN), and the test compounds, culture supernatants were harvested and interleukin-6 levels were measured using a human IL-6 Quantikine ELISA kit (R&D Systems, MN, USA).

Myotube morphology analysis

Exponentially growing bacteria (1 × 10⁸ CFU) were harvested, washed, re-suspended in TGY medium, and injected into the femoral muscle of mice. The experimental unit was a cage of animals. Three mice per condition were used across two experiments. C. perfringens-infected muscles were isolated 24 h after infection. Isolated tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections were cut from the tissue and stained with hematoxylin and eosin to visualize muscle fibers. Images of muscle fibers were taken using a digital camera, and the diameters of muscle fibers were measured using Image J software. The diameters of 300 muscle fibers were assessed for each condition.

Docking simulations

PDB structure file of C. perfringens α-toxin (PDBID: 1qm6) was downloaded from the RCSB repository and the PDB file was processed with UCFS Chimera to create pdbqt file (https://www.rcsb.org)^20,21. The 3D data of micafungin and caspofungin were generated by MoleView and corrected, edited, and conformation-searched by Avogadro, and converted to pdbqt files using UCFS Chimera respectively (https://molview.org)^22,23. Preliminary docking simulations of micafungin, caspofungin and phosphatidylcholine with α-toxin were performed using AutoDock vina (1.1.2) to discover potential binding sites in α-toxin^24,25. Then, the 23 amino acids (asp58, tyr62, leu64, tyr65, gln66, asp67, trp70, asp71, thr74, phe78, ser89, ile90, pro91, asp92, thr93, gln97, lys100, phe101, tyr261, asp293, pro295, lys 304, thr306) in α-toxin were selected from those that are within 3 angstroms of the ligand molecules and have residues that protrude toward the ligands, and two pdbqt files required to set rigid and flexible residues in α-toxin were created by AutoDockTools respectively. The flexible docking simulations using AutoDock vina were performed under the following conditions: center_x = −20, center_y = 95, center_z = 95, size_x = 40, size_y = 25, size_z = 35, energy_range = 3, exhaustiveness = 10, num_modes = 20. Finally, the experimental results were visualized and analyzed using UCFS Chimera. The resulting binding score for the best pose of micafungin was −9.5 and for caspofungin was −6.7.

Statistics and reproducibility

Drug screening was conducted in a single experiment; however, all subsequent in vitro assays were performed at least three times, and representative results are shown in the figures. In addition, the in vivo experiments were conducted under the same conditions in two independent rounds, and the data were combined into each single figure.

All statistical analyses were performed with Easy R (Saitama Medical Center, Jichi Medical University)²⁶. Differences between two groups were evaluated by the two-tailed Student’s t test. A one-way analysis of variance (ANOVA) followed by Tukey’s test was used to evaluate differences among three or more groups. The Log-rank test followed by a Bonferroni multiple comparison analysis was performed to assess the significance of differences in survival. Differences were considered to be significant at P