Fluorescent Tracking of Yeast Division Clarifies the Essential Role of Spleen Tyrosine Kinase in the Intracellular Control of Candida glabrata in Macrophages

Abstract

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage-fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata.

Keywords: Candida; Candida glabrata; carboxyfluorescein succinimidyl ester; macrophages; phagosome; spleen tyrosine kinase.

Figure 1

Carboxyfluorescein succinimidyl ester (CFSE) labels cell wall of Candida glabrata. (A) Differential interference contrast and (B) fluorescence confocal microscopy image of live C. glabrata stained with CFSE showing labeling of all yeast cells prior to cell division. Scale bar represents 5 µm.

Figure 2

Carboxyfluorescein succinimidyl ester (CFSE)-labeling marks Candida glabrata yeast division. (A) Differential interference contrast and fluorescence microscopy image of CFSE-labeled C. glabrata that have undergone division and stained with calcofluor white. Parental cells exhibit full CFSE labeling, while daughter cells have diluted CFSE labeling leading to dimmer fluorescence signal. Scale bar represents 5 µm. (B) Scatter plot of Con A-AF647 and CFSE fluorescence. CFSE-bright parental yeast population in upper right quadrant gate. Percent (scatter plot inset) of undivided CFSE-bright population was determined over time indicated. (C) Correlation of percent CFSE-bright population to OD₆₀₀ nm of proliferating yeast. Data represent a minimum of three independent triplicate experiments.

Figure 3

Spleen tyrosine kinase is required for peritoneal macrophage control of Candida glabrata. (A) Differential interference contrast (DIC) and fluorescence microscopy image of carboxyfluorescein succinimidyl ester (CFSE)-C. glabrata and macrophage after 1 h of coculture indicating complete uptake of the yeast by the macrophages. Scale bar represents 50 μm. (B) DIC and fluorescence microscopy image of CFSE C. glabrata infected macrophages 16 h post inoculation. Macrophages control C. glabrata division in CFSE-bright undivided state, while treatment with R406 impaired the macrophages’ ability in controlling C. glabrata. Scale bar represents 5 µm. (C) Flow cytometry scatter plots of Con A and CFSE showing macrophage controlling C. glabrata division and loss of control in R406-treated cells. Data represent three independent experiments.

Figure 4

Spleen tyrosine kinase (Syk) is required at an early stage for macrophage control of Candida glabrata. Macrophages were treated with chemical Syk inhibitor, R406, prior to addition of C. glabrata for 2, 4, and 8 h after infection. All wells were lysed after 16 h of co-culture and the yeast retrieved and stained with ConA-AF647. Percent of parental CFSE-bright population was assessed via flow cytometry, with R406 treatments initiated at indicated time. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Data represent a minimum of three independent experiments.

Figure 5

R406-induced spleen tyrosine kinase inhibition does not affect macrophage phagocytosis of C. glabrata. (A) Gating scheme for calculated percentage of macrophages that have phagocytosed CFSE-labeled C. glabrata by flow cytometry. (B) Percent phagocytosis of C. glabrata by macrophages over time incubated with control, R406, or cytochalasin D by flow cytometry. **p ≤ 0.01, ****p < 0.0001. Data represent three independent experiments.

Figure 6

R406 does not result in permanent macrophage response defects to fungal stimuli as measured by reactive oxygen species (ROS) production. Plated macrophages were treated with R406 for 2 h, and then washed to remove the inhibitor. ROS production measured by lucigenin after addition of heat-killed Candida. Arbitrary light unites (AU) represents total luminescence corresponding with ROS production. *p ≤ 0.05, ****p ≤ 0.0001. Data represent a minimum of three independent experiments.

Figure 7

Spleen tyrosine kinase-mediated control of Candida glabrata is independent of Dectin-1 and Dectin-2. Percent undivided carboxyfluorescein succinimidyl ester-bright C. glabrata population from peritoneal macrophages-yeast co-cultures. Macrophages were obtained from wild type, Dectin-1^−/−, and Dectin-2^−/− mice. Comparisons between all groups are non-significant. Data represent a minimum of three independent experiments.