A mutation in C. neoformans mitochondrial NADH dehydrogenase results in increased virulence in mice

Abstract

Cryptococcus neoformans: (H99W) was serially passaged in the invertebrate wax moth Galleria mellonella fifteen times to study how fungal virulence evolves under selection and whether those adaptations affect virulence. The G. mellonella passaged strain (P15) and the pre-passage H99W strains were used to infect three different host models of C. neoformans: C. elegans, G. mellonella, and Balb/c mice. While there was no difference in survival in the invertebrate models, P15 killed mice faster than H99W through both intratracheal and intravenous routes of infection and mice infected intravenously with P15 showed higher fungal burden in the brain. Characterization of the major virulence factors of C. neoformans found that P15 had increased capsule size, GXM release, and melanization. Whole genome sequencing of P15 and H99W revealed two mutations in P15, an insertion in the promoter region of NADH dehydrogenase (CNAG_09000) and an insertion in the LMP1 gene (CNAG_06765). Both ATP production and metabolic rate were higher in P15 compared to H99W. Quantitative RT-PCR suggested that the increased ATP was due to increased RNA levels of NADH dehydrogenase. Thus, adaptation to growth in hemocytes resulted in increased production of ATP, increased metabolic rate, and increased virulence in mice. This was likely due to differential expression of virulence factors, which skewed the host immune response to a less efficient Th2 response, with higher levels of IL-4, IL-10, and TNF-α in the brain. Overall, serial passage experiments have increased our understanding of how this yeast evolves under innate immune selection pressure.

Keywords: Cryptococcus neoformans; Galleria mellonella; NADH dehydrogenase; serial passage.

Figure 1.

Schema of strain generation

Figure 2.

Survival data of mice infected with P15 or H99W. (a) Mice infected intratracheally with strain P15 show decreased survival compared to mice infected with the pre-passage strain H99W or the PBS control. Ten mice were infected for each group. (b) Mice infected intravenously with strain P15 show decreased survival compared to mice infected with the pre-passage strain H99W or the PBS control. Ten mice were infected for each group

Figure 3.

Fungal burden in mice. (a) Mice infected intratracheally with strain P15 have higher fungal burden in the spleen, but there is no difference in fungal burden between mice infected with strain H99W or P15 in the lungs or the brain. Error bars represent the standard deviation of the mean. Six mice are infected in each group. Zero values are not plotted for PBS. (b) Mice infected intravenously with strain P15 show increased fungal burden compared to mice infected with the pre-passage strain H99W or PBS. Error bars represent the standard deviation of the mean. Six mice are infected in each group. Zero values are not plotted for PBS

Figure 4.

Capsule size, GXM release and melanin production is increased in strain P15. (a) Bar graph showing increased capsule diameter (µm) in strain P15 compared to the pre-passage strain H99W. The graph is a representative graph of two independent experiments. Error bars represent the standard error of the mean. Numbers within bars are the numbers of cells measured per strain. (b) Bar graph showing increased GXM release in strain P15 compared to the pre-passage strain H99W. The graph depicts the average of two independent experiments. Error bars represent the standard deviation of the mean. Numbers within bars represent the sample size. (c) Line graph showing increased melanization of P15 compared to H99W. The graph depicts the average of three independent experiments

Figure 5.

ATP production and metabolism is increased in strain P15 compared to H99W. (a) P15 makes more ATP than the pre-passage H99W parent strain. The graph is representative of three independent experiments. Error bars represent the standard deviation of the mean. (b) P15 has an increased metabolic rate compared to H99W, as measured by XTT reduction assay, in minimal media. The graph is representative of three independent experiments. Error bars represent the standard error of the mean

Figure 6.

Cytokine production after intratracheal (a) and intravenous (b) infection with C. neoformans. (a) IFN-γ production in lungs of mice and TNF-α production in the brain of mice. Mice infected intratracheally with strain P15 show increased production of IFN-γ in the lungs, but decreased production of TNF-α in the brain compared to mice infected with the pre-passage strain H99W or PBS. White bars represent mice infected with H99W, light gray bars represent mice infected with strain P15, and dark gray bars represent mice infected with PBS. Error bars represent the standard error of the mean. Six mice were infected for each group. Significance was tested using a Wilcoxon Rank Sums test and ANOVA, respectively. (b) Mice infected intravenously with strain P15 show increased production of IL-4, IL-10, and TNF-α in the brain compared to mice infected with the pre-passage strain H99W. White bars represent mice infected with H99W, light gray bars represent mice infected with strain P15, and dark gray bars represent mice infected with PBS. Error bars represent the standard deviation of the mean. Six mice were infected for each group. Significance was tested using a Wilcoxon Rank Sums test