Figure 1.
Cross-species genetic interaction mapping to predict the function of C. neoformans genes necessary for murine infection.
A) Cross-species genetic interaction method. We created S. cerevisiae strains expressing each C. neoformans gene of unknown function at the URA3 locus under the control of the S. cerevisiae GPD1 promoter [77] and linked to nourseothricin (NAT) resistance. We used the synthetic genetic analysis (SGA) strain background, which allows for selection of the MATa mating type and, ultimately, the haploid cellular state [16], [17]. We crossed this strain to the S. cerevisiae deletion library of targeted gene deletions marked by kanamycin (G418) resistance [8]. We selected for diploids on YPAD+NAT+G418, then sporulated diploids on sporulation medium, selected for MATa haploids (+thialysine+canavanine), and then selected for the C. neoformans gene expression construct and the knockout mutations (+NAT+G418+thialysine+canavanine). See Methods and references 10–11 for description of the SGA method. B) Analysis of cross-species genetic interaction data. We scanned plates with colonies containing both the C. neoformans expression construct and the S. cerevisiae knockout mutations with a flatbed scanner. We extracted colony size information using ScreenMill [76], then normalized colony size data using the S-score method [20]. We generated an S-score for each double mutant strain (C.n. expression construct combined with knockout mutant), then computed the Pearson correlation between each such profile and genome-wide profiles available for S. cerevisiae gene deletions [19]. We converted the correlations to Z-scores and filtered out hits for which the Z-score of either the vector or the GFP control was ≥1.96 (p = 0.05). We also filtered out hits whose ratios of C. neoformans gene correlation score/control correlation score (either vector or GFP) was between 0.95 and 1.05 (∼60 profiles).
Figure 2.
Schematics of the Liv5, Liv6, Liv7, Liv13, Mep1, and Blp1 proteins. Detectable motifs are shown.
Table 1.
S. cerevisiae genes whose knockouts significantly correlate with C. neoformans bait genes.
Figure 3.
The genetic interaction profile of the C. neoformans gene LIV7 accurately predicts that Liv7 acts within the ER/Golgi in C. neoformans.
A) Pearson correlations between the genome-wide genetic interaction profiles of pGPD-LIV7 (blue) with the published genome-wide interaction profiles of S. cerevisiae knockout mutants [19]. See Table 1. B) Subcellular roles of S. cerevisiae proteins whose deletion profiles correlate with that of C. neoformans LIV7. Trs33 is a member of the TRAPP complex and involved in vesicle transport within the Golgi [37]. Tlg2 is a t-SNARE involved in vesicle fusion in the ER/Golgi [39]. Vps51 is one of four members of the GARP complex (Vps51–54) that is involved in endosome-to-Golgi transport and retrograde transport within the Golgi [38]. C) Growth curves of C. neoformans mutants in yeast nitrogen base (YNB) at 30°C. OD600 was measured every two hours over the course of the experiment. Representative data from three experiments are shown. D) Proliferation analysis. Shown is the doubling time (y-axis) of wild-type, liv7Δ, trs33Δ, liv7Δ trs33Δ, and vps52Δ strains following treatment with 40 µg/ml Brefeldin A (BFA). Data shown are the average of three experiments and error bars represent the standard deviation and p-values were calculated using Student's t-test. E) Model to explain resistance of liv7Δtrs33Δ cells to BFA treatment. Without BFA treatment, Liv7 or Trs33 alone promotes growth. With BFA treatment, cells convert to a state in which either Liv7 or Trs33 inhibits growth. This genetic behavior is analogous to that of the S. cerevisiae MAP kinase Kss1, which is converted from an inhibitor of filamentous growth to an activator via phosphorylation by the upstream MAP kinase Ste7 [43].
Figure 4.
Liv7 localizes to the ER/Golgi in C. neoformans.
A) Localization analysis. Shown are mCherry signals of cells grown under tissue culture conditions (left) (DMEM, 5% CO2, 37°C, without shaking). The untagged control population (blue) shows mCherry signal in less than 20% of cells, whereas mCherry signal is visible in ∼50% of Liv7-mCherry positive cells (yellow). We then stained these same strains with BODIPY-labeled fluorescent BFA (fBFA; green channel; localizes to the ER/Golgi [44]). Experiments were performed three times, 100 cells counted per sample, and data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. Scale bars are 5 µm. B) Untagged control cells stained with fBFA. 50 ms exposure. C) Liv7-mCherry cells stained with fBFA. 50 ms exposure. D–G) fBFA-staining of wild-type, liv7Δ, trs33Δ, and liv7Δ trs33Δ cells.
Figure 5.
Lectin staining of surface of liv7Δ trs33Δ cells reveals a role for Liv7/Trs33 in PAMP shielding.
A) GXM (left) and chitin (middle) staining of C. neoformans strains grown under tissue culture conditions (DMEM, 5% CO2, 37°C, without shaking). Scale bars are 5 µm. B) α-β-glucan staining patterns. C) Concanavalin A (conA) staining patterns. Note that liv7Δtrs33Δ and vps52Δ mutants display a massive increase in conA staining.
Figure 6.
liv7Δ but not trs33Δ cells are defective in phagocytosis evasion.
A) Phagocytosis assays. C. neoformans cells were treated either with 1× PBS (unopsonized, blue) or 100% fetal bovine serum (opsonized, yellow) for 30 min, then used to infect RAW264.6 macrophage-like cells at a multiplicity-of-infection of two C. neoformans cells to one macrophage. Data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. B) Phagocytosis assays. Association of unopsonized C. neoformans cells with the addition of control buffer (blue), with 250 µM mannose (yellow), or 250 µM laminarin (purple). Data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. C) Model. Together with our previous work, the data described in this paper suggests that there are three parallel mechanisms by which C. neoformans evades phagocytosis. First, Liv7 acts in a partially redundant fashion with Trs33 in vesicle transport, a function that prevents exposure of pathogen associated molecular patterns (PAMPs) that are recognized by the immune system and result in phagocytosis of C. neoformans by phagocytes. Liv7/Trs33 are not part of the Gat201-Gat204-Blp1 pathway because phagocytosis of liv7Δ cells can be competitively inhibited by mannose, whereas phagocytosis of gat204Δ cells cannot. The Liv7/Trs33 pathway does not act to suppress phagocytosis via capsule production since capsule-deficient mutants do not display PAMP exposure and are sensitive to opsonization.
Figure 7.
Phenotypes of C. neoformans liv6Δ cells are consistent with the endosomal function predicted by cross-species genetic interaction mapping.
A) Pearson correlations between the genome-wide genetic interaction profiles of pGPD-LIV6 (blue) with the published genome-wide interaction profiles of S. cerevisiae knockout mutants [19]. See Table 1. B) Wild-type C. neoformans cells grown under yeast culture conditions (YNB, 30°C, with shaking) stained with LysoTracker Green. We hypothesize that the dark area surrounded by staining is the vacuole, as it is in S. cerevisiae [56]. Fluorescent images were exposed for two seconds and the scale bar represents five microns. C) liv6Δ C. neoformans cells grown under yeast culture conditions (YNB, 30°C, with shaking) stained with LysoTracker Green. Fluorescent images were exposed for two seconds and the scale bar represents five microns. D) Quantification of the number of “vacuoles” per cell (one, two, or ≥3 putative vacuoles) in LysoTracker-staining C. neoformans cells. liv6Δ cells but not other mutants show an increase in the number of vacuoles per cell (p≤0.005). Data shown are the averages of three experiments. 200 cells were counted per sample. Error bars represent the standard deviation of three experiments and p-values were calculated using the Student's t-test. E) Growth analysis of wild-type and mutant C. neoformans cells on yeast medium (YNB) without drug, with 2.5 mM neomycin, or with 10 µg/ml fluconazole. Cells were spotted at 107 cells/ml in the upper spot and diluted 5-fold in each subsequent spot. Plates were incubated 48 hours at 30°C.