The role of Candida albicans AP-1 protein against host derived ROS in in vivo models of infection

Abstract

Candida albicans is a major fungal pathogen of humans, causing mucosal infections that are difficult to eliminate and systemic infections that are often lethal primarily due to defects in the host's innate status. Here we demonstrate the utility of Caenorhabditis elegans, a model host to study innate immunity, by exploring the role of reactive oxygen species (ROS) as a critical innate response against C. albicans infections. Much like a human host, the nematode's innate immune response is activated to produce ROS in response to fungal infection. We use the C. albicans cap1 mutant, which is susceptible to ROS, as a tool to dissect this physiological innate immune response and show that cap1 mutants fail to cause disease and death, except in bli-3 mutant worms that are unable to produce ROS because of a defective NADPH oxidase. We further validate the ROS-mediated host defense mechanism in mammalian phagocytes by demonstrating that chemical inhibition of the NADPH oxidase in cultured macrophages enables the otherwise susceptible cap1 mutant to resists ROS-mediated phagolysis. Loss of CAP1 confers minimal attenuation of virulence in a disseminated mouse model, suggesting that CAP1-independent mechanisms contribute to pathogen survival in vivo. Our findings underscore a central theme in the process of infection-the intricate balance between the virulence strategies employed by C. albicans and the host's innate immune system and validates C. elegans as a simple model host to dissect this balance at the molecular level.

Figure 1.C. albicans induces deformed anal region (Dar) in wild-type worms. Worms were exposed to E. coli as control (uninfected) and C. albicans (shown are two examples of infected worms) and pictures were taken on Day 4 (arrow indicates the Dar region). Scale = 20 µm.

Figure 2. Different phenotypes are observed due to Candida infection. (A) Exposure to C. albicans causes intestinal distention in the worms over time (Days 2, 3 and 4). DIC (Nomarski) pictures of worms feeding on E. coli was taken as control and the DIC, RFP and merged pictures of worms infected with C. albicans are shown over 3 d. (B) Vulva swelling is observed (arrow points to the vulvar region) by Day 4 when worms are exposed to Candida compared with worms exposed E. coli as control (uninfected). Scale = 20 µm.

Figure 3. Cap1 is a virulence factor that is required for counteracting oxidative stress created by the host during fungal pathogenesis. The Dar phenotype is observed for both (A) wild-type and (B) bli-3 mutant worms (which lack the ability to produce ROS) exposed to CAP1/CAP1, cap1Δ/Δ + CAP1 and cap1Δ/Δ strains (* denotes p < 0.001; n denotes the number of worms exposed to a particular strain). Experiment was done in triplicate. (C) Survival curves for wild-type worms when exposed to CAP1/CAP1, cap1 Δ/Δ + CAP1 and cap1 Δ/Δ with E. coli OP50 as control (p < 0.01 for CAP1/CAP1 and the cap1Δ/Δ mutant) show that worms exposed to cap1 Δ/Δ survive longer than worms exposed to wild type, while (D) bli-3 mutant worms exposed to Candida strains show no difference in survival between exposure to wild-type CAP1/CAP1 and the cap1Δ/Δ mutant.

Figure 4. Cap1 is required for survival in macrophages. Macrophages were exposed to different strains of Candida in a ratio of 1:15 macrophages with and without DPI. Percentage survival was calculated by dividing the CFUs obtained for Candida grown with macrophages to Candida alone. Data were then normalized to the wild type. Percent survival of cap1Δ/Δ mutant is significantly less (**p < 0.05) than the wild type and the complemented Cap1 strain.

Figure 5. Candida virulence is reduced when Cap1 is deleted. Ten mice/strain were injected in the tail vein with 10⁶ cells of the respective Candida strain. Survival curves of mice when infected with CAP1/CAP1, cap1Δ/Δ + CAP1 and cap1 Δ/Δ strains show that mice infected with the cap1Δ/Δ mutant were able to survive significantly longer than those injected with the wild type (p < 0.01 for CAP1/CAP1 and the cap1Δ/Δ mutant).