Secreted fungal virulence effector triggers allergic inflammation via TLR4

Abstract

Invasive fungal pathogens are major causes of human mortality and morbidity^1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages^3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses^9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Extended Data Figure 1.

(a) TNF ELISA on supernatants from BMDMs infected for 24hrs with the indicated yeasts at MOI=10. n = six biologically independent samples. Significance determined by one-way ANOVA with Bonferroni test. (b) Intracellular FACS staining for iNOS after 24hrs of infection with either C. neoformans or S. cerevisiae at the indicated MOIs. n = three biologically independent samples. (c) RNA-seq heatmap depicting log₂ fold changes of the indicated pro-inflammatory genes in BMDMs following 6hrs of stimulation. (d) RNA-seq heatmap depicting log₂ fold changes of the indicated M2/tolerized genes in BMDMs following 6hrs of stimulation. (e) Representative FACS plots of Arg1 and iNOS expression in BMDMs following 24hrs of stimulation with PBS, IL-4 (40ng/ml), or LPS (100ng/ml) and IFNγ (50ng/ml). Data are presented as mean values +/− SD. ****p < 0.0001

Extended Data Figure 2.

(a) Ranked Z-scores of hits from forward genetic screen for C. neoformans Arg1 induction. (b) List of validated screen hits and gene descriptions. (c) Representative FACS histograms of GXM staining on the indicated C. neoformans strains cultured overnight in 10% Sabouraud media. (d) RT-qPCR for CPL1 mRNA expression in cultures grown to OD₆₀₀=1.0 in the indicated conditions (A.U. = arbitrary units relative to ACT1). n = three biologically independent samples. (e) Melanin production in WT or cpl1Δ strains grown at 30°C on L-DOPA plates. (f) TNF production (measured by ELISA) to the indicated stimulations. n = six biologically independent samples. Significance determined by one-way ANOVA with Bonferroni test. Data are presented as mean values +/− SD. ****p < 0.0001.

Extended Data Figure 3.

(a) Quantification by competitive ELISA of CPL1-6xHis in supernatants from the indicated strains grown in mammalian tissue culture conditions to OD=1.0. n = six biologically independent samples. (b) RT-qPCR for Arg1 mRNA in BMDMs stimulated with the indicated C. neoformans strains (OD=0.1) along with IL-4 (10ng/ml) for 24hrs. Expression normalized to Actb, a.u. = arbitrary units. n = three biologically independent samples. (c) RT-qPCR for Mrc1 mRNA in PMA-differentiated THP-1 cells stimulated with the indicated C. neoformans strains (OD=0.1) along with IL-4 (10ng/ml) for 24hrs. Expression normalized to Actb, a.u. = arbitrary units. n = six biologically independent samples (d) RT-qPCR for Mrc1 mRNA in primary human monocyte-derived macrophages stimulated for 24hrs with PBS or recombinant human IL-4 (10ng/ml) along with the indicated C. neoformans strains (MOI=0.1). Expression normalized to Actb, a.u. = arbitrary units. n = three biologically independent samples (e) RNA-seq read counts of the indicated genes in BMDMs stimulated for 24hrs with either PBS, IL-4 (10ng/ml), rCPL1 (111nM), or IL-4 + rCPL1. n = three biologically independent samples. (f) Transwell migration assay on splenic eosinophils towards supernatants from BMDMs stimulated as in (e). n = three biologically independent samples. Data are presented as mean values +/− SD.

Exended Data Figure 4.

(a) Representative FACS staining of surface IL-4Rα levels on BMDMs stimulated for 24hrs with the indicated C. neoformans strains (MOI=10). n = six biologically independent samples (b) Phospho-FACS for pSTAT3 (left) and pSTAT6(right) after 30min of stimulation with PBS, IL-4 (10ng/ml), rCPL1 (111nM), or IL-4 + rCPL1. (c) Western blot for pSTAT6 or total STAT6 on BMDMs stimulated for the indicated times with either IL-4 (10ng/ml) alone, rCPL1 (111nM) alone, or IL-4 + rCPL1. Data are representative of three independent experiments. (d) Phospho-FACS for pSTAT3 (left) or pSTAT6 (right) in BMDMs after 8hrs of stimulation with PBS, IL-4 (10ng/ml), rCPL1 (37nM), or rCPL1+IL-4. (e) Phospho-FACS for pSTAT3 in BMDMs stimulated with 111nM rCPL1 for the indicated time points. Data are presented as mean values +/− SD.

Extended Data Figure 5.

(a) Arginase-1 FACS in Myd88^+/+ or Myd88^−/− BMDMs stimulated for 24hrs with the indicated concentrations of rCPL1 alone (left) or in combination with IL-4 (10ng/ml). n = three biologically independent samples. (b) Arginase-1 FACS gated on CD45.2+ BMDMs from the indicated genotypes co-cultured with a 50:50 mix of CD45.1 BoyJ BMDMs and stimulated for 24hrs with IL-4 (10ng/ml) or IL-4 + rCPL1 (111nM). n = three biologically independent samples. (c) Arginase-1 FACS on BMDMs stimulated for 24hrs with the indicated concentrations of LPS. (d) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of LPS alone or with 10ug/ml cholera toxin B (CTB). n = three biologically independent samples. (e) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of rCPL1 alone or with 10ug/ml CTB. n = three biologically independent samples. (f) Arginase-1 FACS on BMDMs stimulated with rCPL1 that was either kept on ice or boiled at 100°C for 15min. Cells were stimulated with either rCPL1 alone (left) or in combination with IL-4 (right). n = three biologically independent samples. (g)(h) Arginase-1 FACS in in BMDMs stimulated with the indicated concentrations of rCPL1 alone (g) or in combination with IL-4 (10ng/ml) (h) that were either treated with control or polymyxinB. n = three biologically independent samples. (i) Arginase-1 FACS on BMDMs transduced with MSCV-empty or MSCV-CPL1 retrovirus and stimulated for 24hrs with the indicated concentrations of IL-4. n = three biologically independent samples. (j) Silver stain on SDS-PAGE gel of rCPL1-6xHis or rCPL1(Y160A)-6xHis purified from P. pastoris. Image is representative of three independent experiments. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Extended Data Figure 6.

(a) Arginase-1 FACS in Tlr4^+/+ or Tlr4^−/− BMDMs stimulated for 24hrs with the indicated concentrations of rCPL1-6xHis purified from C.n. supernatants alone or in combination with IL-4 (10ng/ml) (b). n = three biologically independent samples. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Extended Data Figure 7.

(a) Representative FACS gating and quantification of lung eosinophils after 10 days of intranasal infection with the indicated C.n. strains. (b) Representative FACS gating and quantification of mediastinal lymph node GC B cells. (c) Representative FACS gating and quantification of GC B cell antibody isotype. (d) Representative FACS gating and quantification of effector CD4+ T cells. (e) Representative FACS gating and quantification of cytokine production from effector CD4+ T cells after 4hrs of stimulation with PMA, Ionomycin, and GolgiSTOP. **p < 0.01 by one-way ANOVA with Bonferroni test.

Extended Data Figure 8.

(a) Quantification of YARG expression by FACS in lung interstitial macrophages in mice infected for 10 days with 5x10⁴ CFU of the indicated strains. (b) Quantification of eosinophils in mice infected for 10 days with 5x10⁴ CFU of the indicated strains. (c) Kaplan-Meier survival curve analysis of mice infected with WT, cpl1Δ, or cpl1Δ+CPL1 C.n. (N=6 mice per group); ****p < 0.0001 by Mantel-Cox test. (d) Brain CFUs from mice infected with WT, cpl1Δ, or cpl1Δ+CPL1 C.n (5x10⁴ CFU) for 14 days. (e) Lung CFUs on G418-non-resistant (left) or -resistant (right) colonies from mice infected for 10 days with a 50:50 mix of the indicated strains.

Extended Data Figure 9.

(a) Quantification of lung eosinophils in WT or Tlr4^−/− mice infected for 10 days with 5x10⁴ CFU C. neoformans. (b) Lung CFUs from Tlr4^+/− or Tlr4^−/− mice infected with wild type C.n. (5x10⁴ CFU) for 10 days (c) FACS quantification of lung eosinophils in WT (N=6 mice) or Tlr4^−/− (N=4 mice) mice sensitized intranasally with rCPL1. (d) Model of how secreted CPL1 modulates the macrophage inflammatory state (created using Biorender.com). p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Figure 1.. Cryptococcus promotes arginase-1 expression in macrophages via a soluble, capsule-independent mechanism.

(a) Volcano plot of RNA-seq data showing uniquely differentially expressed genes (filtered on genes with <2 fold change in S. cerevisiae challenge) between uninfected and 6hrs wild type C. neoformans MOI=10 infected BMDMs based on DESeq2 analysis. Three biological replicates were used for each condition. (b) Normalized RNA-seq data on BMDMs stimulated for 6hrs with either WT C.n., cap60Δ C.n., S. cerevisiae (all at MOI=10), LPS (100ng/ml), or zymosan (10ug/ml). Three biological replicates were used for each condition. (c) Intracellular FACS staining for arginase-1 after 24hrs of infection with either C. neoformans or S. cerevisiae at the indicated MOIs. n = five biologically independent samples. (d) Arginase-1 intracellular FACS on either Il4ra^+/− or Il4ra^−/− BMDMs stimulated for 24hrs with IL-4 (40ng/ml) or WT C.n. (MOI=10). n = four biologically independent samples. (e) Arginase-1 intracellular FACS with identical conditions as in (d), but with the stimuli either added directly to the BMDMs or added to the top of a 0.2um transwell insert (image created with Biorender.com). n = four biologically independent samples. (f) Arginase-1 FACS on BMDMs stimulated for 24hrs with IL-4 (40ng/ml), live WT C.n., heat killed (55C for 15min) WT C.n., or S. cerevisiae (all MOI=10). n = four biologically independent samples. Data are presented as mean values +/− SD. ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Figure 2.. Identification of CPL1 as a fungal effector by forward genetics.

(a) Genetic screening strategy to identify fungal effectors that drive arginase-1 expression. (b) Outline of CPL1 protein domain architecture. (c) Complementation assay using arginase-1 FACS on BMDMs stimulated for 24hrs with either WT, cpl1Δ, or cpl1Δ + CPL1 C.n. strains at the indicated MOIs. n = four biologically independent samples. (d) Arginase-1 FACS on BMDMs stimulated for 24hrs with WT, cpl1Δ, or pGAL7-CPL1 C.n. strains at the indicated MOIs. n = four biologically independent samples. (e) India ink staining for capsular polysaccharides in the indicated strains after overnight culture in 10% Sabouraud media. Scale bar indicates 5 microns. Data are representative of two independent experiments. (f) Arginase-1 FACS on BMDMs stimulated for 24hrs with the indicated capsule mutant strains at MOI=10. n = three biologically independent samples. (g) Spotting assay for WT vs cpl1Δ C.n. growth on YPAD plates incubated at the indicated temperatures. (h) RT-qPCR for CPL1 mRNA expression in cultures grown to OD₆₀₀=1.0 in the indicated conditions (A.U. = arbitrary units relative to ACT1). n = three biologically independent samples. (i) Representative FACS histogram for intracellular iNOS protein levels in BMDMs pre-infected with the indicated strains at an MOI=10 for 2hrs followed by 24hr stimulation with LPS (100ng/ml) and IFNγ (50ng/ml). (j) Total nitric oxide in supernatants from BMDMs treated as in (i). n = seven biologically independent samples. Data are presented as mean values +/− SD. **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Figure 3.. CPL1 potentiates IL-4 signaling via TLR4.

(a) Silver stained SDS-PAGE gel of recombinant CPL1. n = 5 independent experiments. (b) Arginase-1 FACS on BMDMs stimulated for 24hrs with mock or purified rCPL1 alone (left) or with IL-4 (right). n = 3 biologically independent samples. (c) RNA-seq heatmap of the top 50 BMDM IL-4-induced genes comparing the log₂ fold changes of indicated stimulations to PBS. (d) C. neoformans CFUs after 48hrs of incubation at 37°C, 5% CO₂ in supernatants from BMDMs stimulated for 24hrs as in (c). n = six biologically independent samples. (e) FACS for IL-4Rα levels on BMDMs stimulated for 24hrs with rCPL1 or mock. (f) Western blot for indicated proteins on BMDMs stimulated as in (c) for 8hrs. Data are representative of three independent experiments. (g) Arginase-1 FACS on STAT3^flox/flox BMDMs transduced with empty vector or iCre retrovirus and stimulated as in (c). n = five biologically independent samples. (h) Arginase-1 FACS in WT, Tlr2^−/−, Tlr4^−/−, or Tlr2^−/−Tlr4^−/− BMDMs stimulated for 24hrs with rCPL1. n = 3 biologically independent samples. (i) Same as (h) but plus IL-4. n = 3 biologically independent samples. (j) FACS for TLR4 levels on BMDMs stimulated for 1hr with LPS (100ng/ml) or rCPL1. n = 3 biological experiments. (k) Luminescence in HEK293T cells transfected with the indicated plasmids plus NF-kB luciferase and stimulated for 6hrs with either rCPL1 or LPS (100ng/ml). n = 3 biologically independent samples. (l) Arginase-1 FACS in BMDMs stimulated for 24hrs with mock, rCPL1, or rCPL1(Y160A) alone (left) or in combination with IL-4 (right). n = 3 biologically independent samples. Concentrations are IL-4 (10ng/ml) and rCPL1 (111nM) unless otherwise noted. Data are presented as mean values +/− SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Figure 4.. CPL1 promotes arginase-1 expression in pulmonary interstitial macrophages and is required for virulence.

(a) FACS subgating on CD45+YARG+ lung cells from arginase-1-YFP (YARG) mice infected intranasally with 5x10⁴ CFU WT C. neoformans for 10 days. (b) Representative histogram of YARG expression in lung interstitial macrophages from mice injected intranasally with either saline or 5x10⁴ CFU WT C.n. for 10 days. (c) Quantification of YARG expression by FACS on interstitial macrophages from mice injected intranasally with either saline (N=5 mice), WT (N=11 mice), cpl1Δ (N=14 mice), or qsp1Δ (N=5 mice) Kn99a (5x10⁴ CFU) for 10 days; statistical significance determined by one-way ANOVA with Bonferroni test. (d) Kaplan-Meier survival curve analysis of mice infected with WT (N=10mice) or cpl1Δ C.n. (N=10 mice); ****p < 0.0001 by Mantel-Cox test. (e) Representative histogram (left) and quantification of YARG expression on WT, Il4ra^−/−, or Stat6^−/− (all N=4 mice) infected for 10 days as in (a). (f) CFUs from lungs of the indicated mouse genotypes (N=6 mice for each genotype) infected for 10 days with either WT or cpl1Δ strains; statistical significance determined by one-way ANOVA with Bonferroni test. (g) Arginase-1 FACS on lung IMs from WT or Tlr4^−/− mice infected as in (a); statistical significance determined by unpaired two-sided T-test. (h) Representative FACS histograms of C.neoformans-mCherry expression in alveolar macrophages (left) or interstitial macrophages (right) after 10 days of infection. (i) Representative FACS histograms of C.neoformans-mCherry expression in interstitial macrophages from YARG mice gated on YARG-negative IMs (left) or YARG-positive IMs (right). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001