Of 1 male mice

Male OF-1 mice weighing 30 g (Charles River, Barcelona, Spain) were used as host models for virulence assays for their reliability in previous Mucoralean virulence assays [22 (link),44 (link)]. The mice were immunosuppressed by intraperitoneal injection of cyclophosphamide (200 mg/kg of body weight), 2 days before the infection and once every 5 days thereinafter. Groups of ten were challenged intravenously by injecting 1 × 10⁶ spores in the retroorbital venous sinus [20 (link)]. The assay was done with two independent mutants overexpressing wex1 (MU637 and MU638, Figure 5 and Figure S1, respectively). To ensure animal comfort, the mice were anesthetized with isoflurane via inhalation and monitored until they recovered from the anesthesia. Vi and Av strains were also injected as a positive and negative virulence control, respectively. Mice were housed under established conditions with free food and autoclaved water. Animal welfare was monitored twice a day for 20 days, and those mice meeting the criteria for discomfort were euthanized by CO₂ inhalation. Survival rates during this time were plotted in a Kaplan—Meier curve, and differences were considered statistically significant with a p-value ≤ 0.05 in a Mantel—Cox test.

Male OF-1 mice weighing 30 g and two months old (Charles River, Barcelona, Spain) [30 (link),32 (link),33 (link)] were used as animal models for virulence assays. The mice were immunosuppressed by intraperitoneal administration of cyclophosphamide (200 mg/kg of body weight), 2 days prior to infection and then once every 5 days. Eight-mouse groups were challenged intravenously by retroorbital injection of 1 × 10⁶ spores [32 (link)]. The assay was repeated twice using two independent mutants of mcwc-1a (MU242 and MU243). During this procedure, the mice were anesthetized using isoflurane via inhalation and monitored until they recovered from the anesthesia. Mice were housed under established conditions with free food and autoclaved water. The animal welfare was checked twice a day for 20 days, and those mice following the criteria for discomfort were euthanized by CO₂ inhalation. Survival rates during the time were plotted in a Kaplan–Meier curve (Graph Pad Prism), and differences were considered statistically significant with a p-value ≤ 0.05 in a Mantel–Cox test.

Male OF1 mice (Charles River Laboratories, Saint-Germain-Nuelles, France) were anesthetized with 2% isoflurane in 100% oxygen, and the inner faces of both hindlimbs were carefully shaved. After local disinfection, an about 1-cm skin incision was made on the left hindlimb from the inguinal region to the bifurcation region of the femoral artery into the saphenous and popliteal artery. The femoral artery and vein were dissected from the nerve. The femoral artery/vein block was ligated proximally by two 8–0 ties placed just distally from the superficial epigastric artery, and distally by two 8–0 ties placed just proximally from the bifurcation of the femoral artery into the saphenous and the popliteal artery. After cutting the femoral artery/vein block between the two proximal and between the two distal ties, the femoral artery/vein block was removed. When necessary, major branches such as the lateral circumflex femoral artery were ligated to avoid bleeding.
Thereafter, subcutaneous tissue and skin were closed with non-resorbable sutures or clamped with titanium micro clips (WDT, Garbsen, Germany). Postoperative care included pain management by injection of buprenorphine (Buprenovet, Bayer; 0.1 mg/kg) once directly after surgery or flunixin meglumine (2.5 mg/kg twice daily) during 3 days and daily local wound care with an antiseptic healing cream (Dermaflon, Pfizer).