Figure 5
Role of rfs in the regulation of M. lusitanicus virulence in mice. (A) Spores (3 × 107) from the MU636, Δrfs, Δrfs + rfswt, and MU636 + rfswt strains were intraperitoneally injected into streptozotocin-induced diabetic mice, and survival was monitored daily for 15 days. The experiments were repeated two times in independent groups with n = 10. Data were statistically analyzed using the Mantel-Cox test. ***P < 0.01. When results were not considered significant, we did not provide an additional indication (P > 0.05). Mice surviving after 7 days post-inoculation were considered to (B) determine the change in the weight of mice since the initial infection. Animals were euthanized at 15 days post-inoculation, and the livers were removed to isolate nucleic acids to determine the (C) fungal burden in the mice using quantitative polymerase chain reaction (qPCR) for the expression of the tfc-1 gene from M. lusitanicus and Actb from Mus musculus. A ΔCt analysis was performed to compare the relative abundance of the fungal DNA compared with that of the murine DNA. The mRNA levels of the inflammation marker IL-6 in the liver of mice (D) infected with spores from the different strains and (E) inoculated with 200 μL of the Vo fraction obtained through a molecular weight exclusion filter (cut off 3 kDa) were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Data are presented as the average values obtained from four independent biological replicates; ± corresponds to standard error (SE). Significance testing was performed using ANOVA with Fisher's exact test, *P < 0.05; **P < 0.01; ***P < 0.001.
