Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis

Abstract

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of β-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

Keywords: Candida albicans; extracellular vesicles; vaccines.

Fig 1

Properties and physical characterization of EVs released by C. albicans strain 90028. Transmission electron micrographs from negative contrasting EVs isolated from C. albicans strains (A). Size distribution was obtained by selecting random micrographs in which EVs were manually measured using ImageJ (B) and by NTA (C). Protein (D) and sterol (D) concentrations were measured in EVs. Results represent the average of three independent EV isolation experiments, and error bars represent the standard deviation. The protein:lipid ratio was calculated (D).

Fig 2

IL-6 production in response to C. albicans EVs is impaired in the absence of TLR4. (A) BMDCs derived from wild type (WT) and Tlr2^-/-, Tlr4^-/-, and Clec7a^-/- mice were stimulated with C. albicans EVs (strain 90028) for 24 h, and the levels of IL-6 were measured in the culture supernatants. MOCK-EVs (B and C) or treatment with PMXb (C) was also used to confirm that TLR4 activation is not mediated by LPS contaminants (B and C, respectively). LPS and P3C were used as positive controls. Error bars represent the standard deviation. Results represent the average of three independent experiments (n = 4). Statistical analysis was performed using two-way ANOVA and was analyzed by Tukey’s multiple comparisons test. ***P = 0.0007.

Fig 3

TNF-α, IL-10, and production after stimulation of BMDCs with EVs are not regulated by TLR4. WT and Tlr4^-/- BMDCs were treated with C. albicans EVs (strain 90028), and the levels of IL-6, TNF-α, and IL-10 were measured in the culture supernatants by ELISA. Error bars represent the standard deviation. Results represent the average of three independent experiments (n = 4). Statistical analysis was performed using two-way ANOVA and was analyzed by Tukey’s multiple comparisons test.

Fig 4

Antibody blockade of TLR4 hampers IL-6 production in human monocytes stimulated with C. albicans EVs. PMA-activated THP-1 cells, preincubated or not with anti-TLR4 antibodies, were stimulated with C. albicans EVs (strain 90028) for 18 h. IL-6 production was measured in the culture supernatants by ELISA. Error bars represent the standard deviation. Results represent the average of three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA and was analyzed by Tukey’s multiple comparisons test. ****P < 0.0001.

Fig 5

Properties and physical characterization of EVs released by C. albicans strain NGY152 and mutant strains (mnt1/mnt2Δ, mnnΔ⁶, and pmr1Δ). (A) Differences in N- and O-linked mannans found in the different strains are shown. (B) Transmission electron micrographs from negative contrasting EVs isolated from C. albicans strains. Size distribution was obtained by selecting random micrographs in which EVs were manually measured using ImageJ (C) and by NTA (D). Protein (E) and sterol (F) concentrations were measured in the different EVs. Results represent the average of three independent EV isolation experiments, and error bars represent the standard deviation. The protein:lipid ratio was calculated (G). Statistical analysis was performed using one-way ANOVA and was analyzed by Tukey’s multiple comparisons test. **P < 0.001 and ****P < 0.0001.

Fig 6

The impact of mannosyl residues carried by C. albicans EVs for IL-6 production by BMDCs and G. mellonella protection against lethal candidiasis. (A) WT, Tlr4^-/-, and Clec7a^-/- BMDCs were treated with C. albicans EVs released by 90028, NGY152, mnt1/mnt2Δ, mnnΔ⁶, and pmr1Δ strains, and the levels of IL-6 were measured in the culture supernatants by ELISA. Error bars represent the standard deviation. Results represent the average of three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA and was analyzed by Tukey’s multiple comparisons test. ****P < 0.0001. (B) Insects were inoculated with EVs (10 µL of EV suspensions at 100 µg/mL) released by strains NGY152, mnt1/mnt2Δ, mnnΔ⁶, and pmr1Δ. Two days after EV inoculation, the insects were infected with 10 µL of a suspension containing 2 × 10⁵ yeasts of C. albicans (strain NGY152). Mortality was monitored for 7 d (n = 10). Statistical analysis was performed using one-way ANOVA, and the difference between groups was analyzed by log-rank (Mantel–Cox) test. P = *0.0182 and *0.0116.

Fig 7

Protection induced by C. albicans EV vaccination in a systemic murine model of candidiasis is TLR4 dependent. Wild type (A) and TLR4 KO (B) mice were immunized with C. albicans EVs (strain 90028), immunosuppressed with CP, and then challenged with a lethal inoculum of C. albicans. Statistical analysis was performed using one-way ANOVA, and the difference between groups was analyzed by log-rank (Mantel–Cox) test, **P = 0.0069. Results are representative of two independent experiments (n = 7).

Fig 8

EVs produced by C. albicans strain 90028 protect animals against other strains. Insects were inoculated with EVs (10 µL of EV suspensions at 100 µg/mL) released by strain 90028 (A–F). Two days after EV inoculation, the insects were infected with 10 µL of a suspension containing 2 × 10⁵ yeasts of C. albicans clinical strains (A) OD2, (B) OD6, (C) OD7, (D) OD9, and (E) OD10 or the (F) ATCC strain SC5314. Mortality was monitored for 7 d (n = 10). (G) Survival rate and (H) weight (mean ± SD) of the mice immunized with C. albicans EVs (strain 90028), immunosuppressed with CP, and then challenged with a lethal inoculum of C. albicans ATCC SC5314. Statistical analysis was performed by log-rank (Mantel–Cox) test. *P = 0.0001 and **P = 0.0007.