Experimental Mouse Models of Disseminated Candida auris Infection

Abstract

Disseminated candidiasis is a life-threatening disease and remains the most common bloodstream infection in hospitalized patients in the United States. Despite the availability of modern antifungal therapy, crude mortality in the last decade has remained unacceptably high. In particular, Candida auris is a multidrug-resistant, health care-associated fungal pathogen and has recently emerged as the first fungal pathogen to cause a global public health threat. A reliable animal model for disseminated C. auris candidiasis is therefore needed to study the unique aspects of this little-known host-pathogen interaction. In this study, we established an inbred A/J intravenous model as an appropriate model for human disseminated C. auris infection. We found that C5 deficiency in A/J mice results in a complex phenotype characterized by rapid fungal proliferation in target organs and the development of a unique and rapidly fatal response. In contrast, C57BL/6J mice and mice deficient in neutrophil elastase (NE^-/-) survived high-dose C. auris intravenous challenge, even with cyclophosphamide (CY)-induced immunosuppression. Our study is the first to provide insight into the role of C5 in the host responses to C. auris invasive infection and establishes an inbred A/J mouse model of systemic C. auris infection without CY-induced immunosuppression.IMPORTANCE In the last decade, Candida auris has emerged globally as a multidrug-resistant fungal pathogen. Although C. auris was initially isolated from the external ear canal, it can cause outbreaks of invasive infections with very high mortality and comorbidities. Recent reports highlight the ongoing challenges due to organism misidentification, high rates of multifungal drug resistance, and unacceptably high patient mortality. The assessment of C. auris virulence in a specific genetic deficiency mouse model of invasive C. auris infection in this study contributes to the little knowledge of host defense to C. auris infection, which holds promise as a model for investigating the pathogenesis of C. auris invasive infection, exploring the immune responses elicited by the fungus, evaluating the possible induction of immunity to the infection, and targeting candidates for an antifungal vaccine.

Keywords: Candida; Candida auris; candidiasis; complement; disseminated candidiasis; immunocompromised hosts; mouse models; neutrophil elastase (NE).

FIG 1

Comparison of virulence of C. albicans and C. auris in neutrophil elastase-deficient (NE^−/−) mice and WT C57BL/6 mice. (A) NE^−/− and C57BL/6J female 8-week-old mice were infected i.v. with 5 × 10⁵ C. albicans SC5314 cells or 2 × 10⁸ C. auris 0386 yeast cells. Mice were observed twice daily for 20 days for clinical signs of illness or mortality. The results are representative of five mice per inoculum. Survival times were statistically evaluated by the Kaplan-Meier statistic (GraphPad Prism, version 7), and P values less than 0.05 were considered statistically significant. Mice challenged with C. auris AR-0386 survived significantly longer than did control mice that received C. albicans SC5314 (P = 0.0023 for NE^−/− and P = 0.0031 for C57BL/6). (B and C) In NE^−/− mice, the fungal burdens of C. albicans and C. auris in targeted organs, the kidneys (B) and brain (C), are higher than those in wild-type C57BL/6 mice (P < 0.05); however, there is no significant difference between the two mouse strains (P = 0.25). Fungal burden in the brains of both strains remained low after C. auris challenge. Only 1 out 5 mice in WT C57BL/6 and 2 out 5 in NE^−/− mice had detectable CFU in the brain. All fungal burden data were analyzed by a multivariate analysis of variance (MANOVA).

FIG 2

Immunosuppressed intravenous mouse models of C. auris disseminated infection. C57BL/6 and NE^−/− mice received a 200-mg/kg dose of cyclophosphamide (CY) i.p. on day −3. Prolonged neutropenia was maintained by giving each animal a 150-mg/kg dose of CY i.p. every 7 days after infection. On day 0, mice were intravenously infected with 2 × 10⁸ viable C. auris cells in 0.1 ml DPBS. As controls, mice without CY treatment were challenged with the same dose of C. auris cells at the same time. There is no significant difference in the survival of the two strains with and without CY-induced immunosuppression (P = 0.45). Survival times were statistically evaluated by the Kaplan-Meier statistic (GraphPad Prism, version 7), and P values less than 0.05 were considered statistically significant.

FIG 3

Evaluation of C. auris virulence in a naive and immunosuppressed A/J mouse model of disseminated candidiasis. Female 8-week-old A/J mice received a 200-mg/kg dose of cyclophosphamide (CY) i.p on day −3. Prolonged neutropenia was maintained by giving each animal a 150-mg/kg dose of CY i.p. every 7 days after infection. On day 0, mice were infected intravenously with 1 × 10⁸ or 2 × 10⁸ viable C. auris cells in 0.1 ml DPBS. As controls, age- and sex-matched A/J mice without CY treatment were challenged with the same two inoculum sizes of C. auris cells at the same time. There is no significant difference in survival between different doses or between the groups immunosuppressed with CY or without CY-induced immunosuppression. Survival times were statistically evaluated by the Kaplan-Meier statistic (GraphPad Prism, version 7), and P values less than 0.05 were considered statistically significant.

FIG 4

Influence of immunosuppression on fungal burdens in brain, heart, and kidneys in mouse models of systemic C. auris infection. (A to C) Three mouse strains, C57BL/6, NE^−/−, and A/J mice, with and without CY pretreatment before C. auris 0386 intravenous challenge (2 × 10⁸ cells), were compared for fungal burdens in targeted organs, including kidney (A), brain (B), and heart (C). Data are expressed as the mean + standard deviation (S.D.) (n = 5). *, P = 0.05; **, P = 0.01 by two-tailed Student’s t test between the indicated A/J groups (with or without CY-induced immunosuppression) compared to the appropriate C57BL/6 (wild type) groups (with or without CY-induced immunosuppression). (D) Fungal burdens in brain of each mouse in each experimental group are shown. In both C57BL/6 and NE^−/− strains, only 1 or 2 out of 5 mice for each group had detectable fungal CFU, independent of CY-induced immunosuppression. A/J mice, with or without CY treatment, had significant fungal burdens in brain compared to C57BL/6 mice.