Figure 1.
C. glabrata resides in non-matured macrophage phagosomes.
Representative DIC and fluorescence microscopy images of viable or heat killed C. glabrata 90 min post infection phagocytosed by human MDMs (left panels) showing marker proteins in red, while GFP-expressing C. glabrata are indicated in green. To detect non-phagocytosed yeasts, samples were stained with Concanavalin A (ConA, shown in yellow). Phagocytosed yeasts are labeled with a white arrow while non-phagocytosed yeasts are marked with red arrows. Co-localization with fluorescence markers was quantified for phagosomes containing viable or heat killed (Hk) C. glabrata at indicated time points (right panel). (A) Phagosomes containing viable and heat killed C. glabrata co-localize with the late endosome marker Rab7. (B) In contrast to heat killed C. glabrata containing phagosomes, compartments containing viable yeasts show low phagosomal proteolytic activity as measured by co-localization with the fluorogenic protease substrate DQ-BSA. (C) Heat killed but not viable C. glabrata containing phagosomes acquire the lysosomal tracer texas red ovalbumin (TROV). Statistical analysis was performed comparing heat killed with viable C. glabrata at indicated time points (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).
Figure 2.
Phagosome maturation arrest occurs in different macrophage differentiation or activation states and is yeast phagosome-specific.
(A) Human M1-polarized and M2-polarized MDMs do not differ in central aspects of C. glabrata-macrophage interaction: phagocytosis, phagosome acidification and killing. Phagocytosis (MOI of 5) was quantified microscopically by determining the percentage of internalized (Concanavalin A stain-negative) yeasts out of total yeasts after 90 min. Phagosome acidification was quantified microscopically by determining the percentage of LysoTracker-positive phagosomes after 90 min. Survival of C. glabrata was determined by cfu-plating of macrophage lysates after 3 h of co-incubation and comparing to yeasts incubated without macrophages. (B) Treatment with vitamin D3 (calcitriol) has no influence on the number of LysoTracker-positive viable C. glabrata containing phagosomes of human MDMs. (C) MDMs co-infected with C. glabrata and latex beads show a acidification defect specific to C. glabrata containing phagosomes (LysoTracker-negative staining; white arrow) but acidify latex bead containing phagosomes (LysoTracker-positive staining; white asterisk). Representative image 90 min post infection. GFP-expressing C. glabrata is indicated in green and non-phagocytosed yeasts stained with Concanavalin A (ConA) are in yellow (marked with red arrows). Statistical analysis was performed comparing M1-type with M2-type macrophages (A) or drug treated with untreated viable C. glabrata (B) (n≥3; *p<0.05, **p<0.01 by unpaired Student’s t test).
Figure 3.
C. glabrata does not induce MAP-kinase or NFκB signaling cascades upon phagocytosis but activates Syk.
RAW264.7 macrophages were stimulated with LPS (1 µg/ml) or infected with viable or heat killed (Hk) C. glabrata (MOI of 5) for indicated time points. (A) Cell lysates were subjected to Western Blot analyses by using antibodies detecting either the phosphorylated or unphosphorylated form (as a loading control) of p38, p44/42 (Erk1/2), SAPK/JNK, IKKαβ, IκBα and p65. Only LPS treatment induced changes in phosphorylation patterns of analyzed proteins. Data shown are representatives of three independent experiments. (B) Cell lysates were resolved on SDS-PAGE and membranes blotted for phosphorylated Syk (P-Syk) as described in [25] (C) Localization of the NFκB subunit p65 was analyzed by immunofluorescence microscopy. Representative pictures of macrophages treated with LPS or viable C. glabrata for 10 min are shown on the left site, a quantification of indicated time points on the right site. Percentage of NFκB nuclear localization was quantified for all macrophages (LPS) or for yeast-bound macrophages (viable, heat killed). While LPS induced the translocation of p65 to the nucleus, C. glabrata independent of its viability, did not. Statistical analysis was performed for C. glabrata-infected versus LPS-treated macrophages at the indicated time points (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).
Figure 4.
Effect of phagosome pH on C. glabrata survival.
(A) Viable and heat killed C. glabrata containing phagosomes acquire similar levels of V-ATPase. Representative fluorescence microscopy images of viable or heat killed C. glabrata 180 min post-infection phagocytosed by murine J774E cells expressing a V-ATPase-GFP fusion protein (left panel). V-ATPase is shown in green while non-phagocytosed yeasts (stained with concanavalin A [ConA]) are indicated in yellow (marked with red arrows). Phagocytosed yeasts are labeled with white arrows. Co-localization with V-ATPase was quantified for phagosomes containing viable or heat killed C. glabrata at indicated time points (right panel). (B) Rising phagosome pH with chloroquine but not bafilomycin A1 reduces C. glabrata survival in MDMs. Survival of C. glabrata was determined by cfu-plating of macrophage lysates after 24 h. Co-incubation samples contained no drug (untreated), chloroquine (50 µM), chloroquine plus iron nitriloacetate (20 µM, FeNTA) or bafilomycin A1 (50 nM). (C) Chloroquine or bafilomycin A1 are not toxic to C. glabrata in vitro. Growth in presence of the drugs is comparable to untreated cultures. Statistical analysis was performed comparing heat killed with viable C. glabrata at indicated time points (A) or comparing untreated and drug-treated samples (B) (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).
Figure 5.
Environmental alkalinization by C. glabrata.
(A) Viable, but not heat killed C. glabrata were able to alkalinize an acidic medium when grown with amino acids as the sole carbon and nitrogen source. 1×106 C. glabrata cells/ml were inoculated in a 24 well plate with liquid YNB medium with 1% casamino acids and 20 mg/l phenol red. Within 24 h the pH rose from pH 4 to a pH above 6.8, as indicated by a color change of the pH indicator. The mutant put3Δ was incapable of alkalinization, the mutant bcy1Δ showed an intermediate phenotype of reduced alkalinization. (B) Similar results were obtained when using a solid medium (YNB with 1% casamino acids, 2% agar and 0.01% bromocresol green).
Table 1.
Alkalinization-defective C. glabrata mutants.
Figure 6.
The influence of mannosyltransferases of C. glabrata on environmental alkalinization and acidification of phagosomes.
(A) Representative fluorescence microscopy images of wild type (wt hltΔ) C. glabrata and mnn10Δ mutant 90 min post infection, phagocytosed by human MDMs (left panels). LysoTracker staining is shown in red, while non-phagocytosed yeasts, stained with Concanavalin A (ConA), are shown in yellow. Phagocytosed yeasts are labeled with a white arrow while non-phagocytosed yeasts are marked with red arrows. (B) Co-localization with LysoTracker was quantified for phagosomes containing wild type (wt hltΔ or wt tΔ) or mutant (mnn10Δ, mnn11Δ, anp1Δ) C. glabrata at 90 min post infection. Statistical analysis was performed comparing mutant with wild type C. glabrata (n≥3; **p<0.01, ***p<0.005 by unpaired Student’s t test). (C) mnn10Δ and mnn11Δ mutants showed severe defects in environmental alkalinization in vitro, while alkalinization by the anp1Δ mutant was comparable to isogenic wild type levels. 1×106 C. glabrata cells/ml were inoculated in a 24 well plate with liquid YNB medium with 1% casamino acids and 20 mg/l phenol red and incubated for 24 h.