Immunoprotection against Cryptococcosis Offered by Znf2 Depends on Capsule and the Hyphal Morphology

Abstract

Systemic cryptococcosis is fatal without treatment. Globally, this disease kills 180,000 of the 225,000 infected people each year, even with the use of antifungal therapies. Currently, there is no vaccine to prevent cryptococcosis. Previously, we discovered that Znf2, a morphogenesis regulator that directs Cryptococcus yeast-to-hyphal transition, profoundly affects cryptococcal interaction with the host-overexpression of ZNF2 drives filamentous growth, attenuates cryptococcal virulence, and elicits protective host immune responses. Importantly, immunization with cryptococcal cells overexpressing ZNF2, either in live or heat-inactivated form, offers significant protection to the host from a subsequent challenge by the otherwise lethal wild-type H99 strain. We hypothesize that cellular components enriched in ZNF2^oe cells are immunoprotective. Here, we discovered that serum from protected animals vaccinated with inactivated ZNF2^oe cells recognizes cryptococcal antigens that reside within the capsule. Consistently, capsule is required for immunoprotection offered by ZNF2^oe cells. Interestingly, the serum from protective animals recognizes antigens in both wild-type yeast cells and ZNF2^oe cells, with higher abundance in the latter. Consequently, even the heat-inactivated wild-type cells become immunoprotective with an increased vaccination dose. We also found that disruption of a chromatin remodeling factor Brf1, which is important for initiation of filamentation by Znf2, reduces the antigen level in ZNF2^oe cells. Deletion of BRF1 drastically reduces the protective effect of ZNF2^oe cells in both live and heat-killed forms even though the ZNF2^oebrf1Δ strain itself is avirulent. Collectively, our findings underscore the importance of identifying the subset of cryptococcal surface factors that are beneficial in host protection. IMPORTANCE Cryptococcosis claims close to 200,000 lives annually. There is no vaccine clinically available for this fungal disease. Many avirulent mutant strains do not provide protection against cryptococcosis. We previously discovered that hyphal ZNF2^oe strains elicit protective host immune responses both in the live and heat-inactivated forms. Here we seek to understand the mechanism underlying the host protection provided by ZNF2^oe cells. We discovered increased accumulation of antigens located within the caspusle of ZNF2^oe cells and consequently the requirement of the capsule for ZNF2^oe strain-elicited host protection. Furthermore, genetically blocking the ability of ZNF2^oe cells to grow in the hyphal form significantly reduces antigen accumulation and impairs the ability of ZNF2^oe strain to provide host protection. Our findings highlight the importance of identifying the Znf2-regulated capsular surface factors that are fundamental in host protection.

Keywords: Cryptococcus neoformans; antigens; capsule; immunofluorescence; morphogenesis; vaccination.

FIG 1

ZNF2^oe cells are recognized stronger by the host serum based on immunofluorescence. (A–C) Wild-type H99 (bottom images) or ZNF2^oe (upper images) cells from an overnight culture in YPD medium were probed using sera collected from mice vaccinated with heat-killed ZNF2^oe cells. All images were taken at the same exposure (A), from mice vaccinated with heat-killed H99 cells (B), or from naive mice (C). (D) Quantification of the relative fluorescence intensity of cells (artificial unit based on calculation detailed in the method).

FIG 2

Dose-dependent vaccination effect of heat-killed H99 and ZNF2^oe cells. (A) Schematic representation of the vaccination regimens using live or heat-killed ZNF2^oe cells. The lung fungal burdens at DPI 12 of the (5 mice) control mice without vaccination and of mice vaccinated with (10 mice) live or (10 mice) heat-killed ZNF2^oe cells at the indicated doses are shown. (B) Schematic representation of the vaccination regimen using heat-killed H99 cells at two different doses. The lung fungal burdens at DPI 14 of the 5 mice vaccinated with heat-killed H99 cells at the typical dose of 1 × 10⁷ cells/animal or the higher dose of 5 × 10⁷ cells/animal are shown. (C) Schematic representation of the vaccination regimen using heat-killed H99 cells and heat-killed ZNF2^oe cells. The lung fungal burden at DPI 14 of the 5 mice vaccinated with these heat-killed cells at the higher dose of 5 × 10⁷ cells/animal are shown. The group without vaccination was included as the control.

FIG 3

The antigens are localized within the capsule and outside of the cell wall. (A) The ZNF2^oe strain and the wild-type H99 were cultured in YPD medium at 30°C overnight (upper panel) or in DMEM medium at 37°C for 2 days in ambient air (lower panel). The cells were then used for immunofluorescence and probed with sera from mice vaccinated with HK-ZNF2^oe cells (green). The cells were co-stained with calcofluor white to reveal chitin in the cell wall (blue). All images were taken at the same exposure. (B) The wild-type H99 cells were cultured in RPMI medium for 2 days. The cells were then used for immunofluorescence and labeled with sera from mice vaccinated with HK-ZNF2^oe cells (green). The cells were co-stained with calcofluor white to reveal chitin in the cell wall (blue). Then the cells were negatively stained with Indian ink to reveal the capsule (white halo surrounding the yeast cells).

FIG 4

The host protection effect caused by ZNF2 overexpression requires capsule. (A) Images of cells of wild-type H99, LW10 (ZNF2^oe), NE644 (cap59Δuge1Δ), and KH35 (ZNF2^oecap59Δuge1Δ) when cultured in YPD medium (1st column) and RPMI medium (2nd column with Indian ink staining); and colony immunoblot of these colonies probed with HK-ZNF2^oe-vaccinated serum. (B) The relative transcript level of ZNF2 in wild-type H99, NE644 (cap59Δuge1Δ) and KH35 (ZNF2^oecap59Δuge1Δ) after overnight culture in YPD. The transcript level of TEF1 of each sample was used as the internal control. (C-D) Two independent animal experiments testing the vaccination effect of heat-killed ZNF2^oe cells in the wild-type background or in the acapsular mutant background using two slightly different vaccination regimens. The acapsular mutant cap59Δuge1Δ was included as a control in both experiments. The no vaccination group was included in (C) and the group vaccinated with heat-killed H99 cells was included in (D) as additional controls.

FIG 5

Brf1 is critical for Znf2-controlled filamentation and it contributes to the protection by ZNF2^oe. (A) Immunofluorescent images of wild-type H99, ZNF2^oe, brf1Δ and ZNF2^oebrf1Δ when probed with serum from protected animals vaccinated with HK-ZNF2^oe cells. (B) The relative transcript level of ZNF2 in wild-type H99, the ZNF2^oe strain, the brf1Δ mutant, and the ZNF2^oebrf1Δ strain after overnight culture in liquid YPD medium as measured by RT-PCR. The transcript level of TEF1 in each RNA sample was used as the internal control. The error bar indicates the standard derivations among three biological replicates. n.s.: non-significant. **: P < 0.01. (C) Quantification of the relative fluorescence intensity of cells probed with serum from HK-ZNF2^oe cells vaccinated animals. (n =30). *: P < 0.05, ***: P < 0.001, ****: P < 0.0001. (D) Colony immunoblot of H99, ZNF2^oe, brf1Δ and ZNF2^oebrf1Δ probed with sera collected from mice immunized with heat-killed ZNF2^oe cells.

FIG 6

Brf1 is critical for the protective effect elicited by ZNF2 overexpression. (A) Phenotypical analyses of H99, ZNF2^oe, brf1Δ, and ZNF2^oebrf1Δ on thermo- or CO₂- tolerance (YPD at 30°C, 30°C + 10% CO₂, and 37°C) and melanization (L-Dopa medium at 22°C). (B) The lung fungal burden at day 12 postinoculation with live H99 (5 mice), ZNF2^oe (5 mice), brf1Δ (5 mice), or ZNF2^oebrf1Δ (5 mice) at the inoculum of 1 × 10⁴ cells/animal. (C) Animal survival was monitored for 60 days after inoculation with live H99 (10 mice) or ZNF2^oebrf1Δ (10 mice) at 1 × 10⁴ cells/animal. The survival rate was plotted against the days postinoculation. (D) The lung fungal burden of the surviving mice inoculated with live ZNF2^oebrf1Δ (10 mice) at the time of termination (DPI 60) compared the lung fungal burden of mice infected with live H99 (10 mice) at time of euthanization when they reached defined clinical endpoints. (E) Survival of mice vaccinated with live ZNF2^oebrf1Δ (10 mice) or ZNF2^oe (10 mice) after challenge with live H99. (F) Survival of mice vaccinated with heat-killed ZNF2^oebrf1Δ (10 mice) or ZNF2^oe (10 mice) after challenge with live H99.