Genetic control of susceptibility to infection with Candida albicans in mice

Abstract

Candida albicans is an opportunistic pathogen that causes acute disseminated infections in immunocompromised hosts, representing an important cause of morbidity and mortality in these patients. To study the genetic control of susceptibility to disseminated C. albicans in mice, we phenotyped a group of 23 phylogenetically distant inbred strains for susceptibility to infection as measured by extent of fungal replication in the kidney 48 hours following infection. Susceptibility was strongly associated with the loss-of-function mutant complement component 5 (C5/Hc) allele, which is known to be inherited by approximately 40% of inbred strains. Our survey identified 2 discordant strains, AKR/J (C5-deficient, resistant) and SM/J (C5-sufficient, susceptible), suggesting that additional genetic effects may control response to systemic candidiasis in these strains. Haplotype association mapping in the 23 strains using high density SNP maps revealed several putative loci regulating the extent of C. albicans replication, amongst which the most significant were C5 (P value = 2.43×10(-11)) and a novel effect on distal chromosome 11 (P value = 7.63×10(-9)). Compared to other C5-deficient strains, infected AKR/J strain displays a reduced fungal burden in the brain, heart and kidney, and increased survival, concomitant with uniquely high levels of serum IFNγ. C5-independent genetic effects were further investigated by linkage analysis in an [A/JxAKR/J]F2 cross (n = 158) where the mutant Hc allele is fixed. These studies identified a chromosome 11 locus (Carg4, Candida albicans resistance gene 4; LOD = 4.59), and a chromosome 8 locus (Carg3; LOD = 3.95), both initially detected by haplotype association mapping. Alleles at both loci were inherited in a co-dominant manner. Our results verify the important effect of C5-deficiency in inbred mouse strains, and further identify two novel loci, Carg3 and Carg4, which regulate resistance to C. albicans infection in a C5-independent manner.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Kidney fungal burden, a measure of susceptibility to C. albicans infection, in 23 inbred mouse strains.

23 out of 36 commonly used inbred strains from the Jackson Mouse Phenome Database were phenotyped for susceptibility to C. albicans. 7–10 female mice per strain were infected intravenously with 5×104C. albicans blastospores and the kidney fungal load was measured 48 h post-infection. Bars represent strain mean±SD. Horizontal lines represent mean (solid line) across all 23 strains±SD (dashed lines).

Figure 2. Genome-wide association mapping using EMMA.

Using the R package implementation of EMMA, genome-wide association mapping was conducted across 23 inbred mouse strains. We obtained genotype data consisting of 132 000 SNPs from the Mouse HapMap project and rejected SNPs with MAF 10 transformed P values indicating association significance for each SNP genome-wide (A). Standardized threshold was set at P value 1.0×10−5 (solid line) and the Bonferroni multiple testing correction threshold was calculated to be 7.99×10−8 (dashed line). The most significant hits on chromosomes 2 and 11 are depicted (B and C) along with respective haplotype maps of susceptible (S) and resistant (R) strains at those locations. The conserved haplotype blocks are indicated with brackets, along with the Hc position (B) and the arrow showing the most conserved area (C). Asterisk denotes discordant strains with respect to the C5 genotype. Haplotype map colour coding: light gray (NA), gray (A/J like), and white (B6 like).

Figure 3. Phenotypic responses of discordant AKR/J mouse strain upon C. albicans infection.

In 6 mice per strain, the heart (A), brain (B), and kidney (C) fungal load was measured 48 h after a low dose (5×104, i.v.) infection with C. albicans. In all organs, fungal load was significantly lower in AKR/J mice compared to A/J (**P<0.01, ***P<0.0001). Periodic acid-Schiff staining was performed on kidney sections (D) showing the extent of fungal burden and granulocyte infiltration. Magnification 100X and 400X (insert).

Figure 4. Differential susceptibility of A/J and AKR/J mice to C. albicans infection.

6 mice per strain were challenged with the indicated dose of C. albicans and sacrificed at either 24 h or 48 h time point to determine the kidney fungal burden (A) and serum BUN levels (B). The fungal load was significantly lower in AKR/J mice compared to A/J, and was associated with lower BUN levels (*P<0.05, ***P≤0.0001, n.d. not done due to premature death of A/J mice). Bars represent group mean ± SD. For the survival study, 8 mice per strain were infected intravenously with a high dose (3×105) of C. albicans and monitored daily for clinical signs of morbidity (C). Survival curves were compared by a Log-rank test and the median survival of AKR/J (3 d) was found to be significantly different from A/J (1 d) (P<0.0001).

Figure 5. Inflammatory cytokine levels in the AKR/J mouse strain.

Serum cytokine levels in 3–6 mice of each strain were measured prior to infection and at the 48 h time point, as described in Materials and methods. Cytokine response of AKR/J mice resembled closely that of B6 strain and was significantly different from A/J mice. The standard deviation is indicated for each group. For all cytokines, infected AKR/J shows a statistically significant difference from infected A/J mice.

Figure 6. Linkage analysis in the informative [A/JxAKR/J]F2 population.

[A/JxAKR/J]F2 mice (n = 158) were infected with C. albicans (5×104, i.v.) and kidneys were harvested 48 h post infection. CFU were determined as previously mentioned and results from four separate infections are plotted along with A/J, AKR/J, and [A/JxAKR/J]F1 controls (A). Each dot represents a single mouse. Distribution of kidney fungal load is shown in the entire [A/JxAKR/J]F2 population (B), after regression of log10CFU to an experiment and gender-specific mean (set at 0). Mice were genotyped at 257 SNPs and markers across the entire genome and interval mapping was carried out using the R/qtl software package. Whole genome LOD score traces are shown for genetic effects controlling kidney fungal burden in [A/JxAKR/J]F2 mice (C), identifying linkage to chromosome 8 (LOD = 3.95) and chromosome 11(LOD = 4.59), designated Carg3 and Carg4, respectively. Results for males and females are shown separately (dashed and dotted lines) and together (solid line), with marker positions on the x-axis. Permutation testing (1000 tests) identified genome-wide thresholds at P = 0.01 and 0.05.

Figure 7. Additive effect of the Carg3 and Carg4 loci on kidney fungal burden in [A/JxAKR/J]F2 mice.

Detailed LOD score traces are shown for chromosome 11 (Carg4) (A) and chromosome 8 (Carg4) (B) loci for males, females, and both sexes combined. The shaded area designates the Bayesian 95% confidence interval for each locus and the genome-wide thresholds are indicated at P = 0.01 and P = 0.05. The additive effect of Carg3 and Carg4 loci is demonstrated by segregating the alleles of susceptible and resistant mice found at extremities (±1 standard deviation from the mean) of the distribution (C). Haplotype map colour coding: light gray (AKR/J), gray (A/J), and white (heterozygous). Each line represents a single mouse. Genome-wide association mapping results are depicted along with LOD score traces for chromosomes 11 (D) and 8 (E). The 10 Mb blocks examined for candidate genes are indicated for both loci.