Candida albicans biofilms do not trigger reactive oxygen species and evade neutrophil killing

Abstract

Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a molecular Candida viability assay. Reactive oxygen species generation was assessed by measuring fluorescence of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester in preloaded neutrophils. We found that mature biofilms were resistant to leukocytic killing and did not trigger reactive oxygen species, even though neutrophils retained their viability and functional activation potential. Beta-glucans found in the extracellular matrix negatively affected antibiofilm activities. We conclude that these polymers act as a decoy mechanism to prevent neutrophil activation and that this represents an important innate immune evasion mechanism of C. albicans biofilms.

Figure 1.

Candida albicans mature biofilms are resistant to leukocytic killing compared with early biofilms and planktonic cells. Killing was assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay (A) or a real-time reverse-transcription polymerase chain reaction assay (B). Planktonic cultures, early (3-hour) biofilms, and mature (24-hour, 48-hour) biofilms were tested at 2 effector-to-target (E:T) ratios (10:1, 1:1). Results from triplicate experiments with HL-60 cells are shown/ **P < .005 compared with 3-hour biofilm and *P < .05 for a t-test comparison between planktonic cultures and mature biofilms.

Figure 2.

Biofilms fail to trigger a reactive oxygen species (ROS) response but do not compromise leukocyte function. A, The ROS production was measured after 1 hour of incubation of neutrophils with early (3-hour) biofilms, 24-hour biofilms, phorbol myristoyl acetate (PMA) (0.1 μM), or 24- hour biofilms plus PMA (0.1 μM). *P < .05 for t-test comparisons between 3-hour biofilms and 24-hour biofilms and between 24-hour biofilms and disrupted 24-hour biofilms. **P < .005 for a t-test comparison with 24-hour biofilms. B, Three-hour biofilms , 24-hour biofilms, 24-hour biofilms plus PMA (0.1 μM), or disrupted 24-hour biofilms were exposed to neutrophils at a 5:1 E:T ratio, and fungal damage was assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay. *P < .05 for t-test comparisons between 3-hour biofilms and 24-hour biofilms and between 24-hour biofilms and 24-hour biofilms plus PMA. Results represent means and standard deviations of triplicate experiments with neutrophils from 2 human donors.

Figure 3.

Leukocytic cells retain their viability after interacting with biofilms for up to 3 hours. A, Live HL-60 cells (red) labeled with Cell Tracker Orange after 3 hours of contact with early (3-hour) or late (24-hour) biofilms. Bars = 50 μm. B. Quantification of viability of HL-60 cells after 3 hours and 24 hours of contact with early (3-hour) and late (48-hour) biofilms using a Live/Dead cytotoxicity assay. Results represent means and standard deviations of triplicate experiments. *P < .005 for a t-test comparison between early and late biofilms.

Figure 4.

Mature biofilm supernatants do not inhibit killing of early biofilms. Early (3-hour) biofilms were exposed to HL-60 cells in the presence (dark bars) or absence (light bars) of supernatants from late biofilms at 5 different effector-to-target (E:T) ratios. Forty-eight-hour biofilm supernatants were added at 1:2 dilution. Killing of early biofilms was assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, and results represent means and standard deviations of triplicate experiments. *P < .05 for a t-test comparison between the presence and absence of supernatants.

Figure 5.

The effects of the biofilm matrix on neutrophil activation are β-glucan-dependent. A, Matrix from 48-hour biofilms coating green fluorescent protein (GFP)–expressing yeast organisms. To visualize presence of extracellular polysaccharides, a GFP-tagged Candida albicans strain (green) was coated with water (control), extracted biofilm matrix (in water), or negative control extract from planktonic cultures (in water). Yeast was stained with ConA-Alexa Fluor 633 or an anti-β-glucan antibody (red). Bars = 10 μm. B and C, Biofilm matrix extract reduces reactive oxygen species (ROS) activation in response to early biofilms. B, Matrix was extracted in Roswell Park Memorial Institute 1640 medium (RPMI 1640), concentrated 6-fold and used to coat 3-hour biofilms (Early Biofilm/Matrix). Concentrated RPMI 1640 (C-Medium) or planktonic extract (PE) was used as negative controls. Results shown are based on triplicate runs with neutrophils from a single human donor. *P < .05 for a t-test comparison with early biofilms. C, Three-hour biofilms were treated with glucanase (Early Biofilm/Glucanase) or coated with glucanase-treated matrix extract (Early Biofilm/Matrix/Glucanase), and ROS production was measured. Controls included heat-inactivated glucanase and DNAse I–treated matrix (not shown). Results shown are based on triplicate runs with HL-60 cells *P < .05 for a t-test comparison with early biofilms with matrix. D, Laminarin inhibits ROS activation and killing of early biofilms by HL-60 cells. Laminarin (150 μg/mL) was used to coat 3-hour biofilms, and HL-60 cells were added at 5:1 or 2:1 effector-to-target ratios. Early biofilm damage (bars) and ROS stimulation (lines) were measured after 3 hours and 1 hour, respectively. The ROS inhibition by laminarin was associated with a reduction in killing. P < .05 for a t-test comparison between the presence and absence of laminarin in each effector-to-target ratio tested.

Figure 6.

Glucanase treatment depletes biofilms of β-glucans and increases their susceptibility to killing. A and B, Effect of glucanase or heat-inactivated glucanase treatment on biofilm cell ConA-reactivity. Biofilms of a green fluorescent protein (GFP)–tagged Candida albicans strain were grown for 48 hours. Cultures were stained with ConA-Alexa Fluor 633 (red). Biofilms were examined using a fluorescence microscope (A). Basal yeast cell layer of 48-hour biofilms as seen by confocal microscopy (B). C, Effect of glucanase treatment on biofilm β-glucans. Biofilms of a GFP-tagged C. albicans strain were grown for 48 hours and stained with an anti-β-glucan antibody, followed by a Cy-3-conjugated secondary antibody (red). Bars = 20 μm. D, Glucanase treatment of biofilms increases their susceptibility to killing. Forty-eight-hour biofilms were treated with glucanase (4.5 U/mL), heat-inactivated glucanase, alpha-mannosidase (4.5 U/mL), or DNAse (353 U/mL) for 30 minutes, and HL-60 cells were added (10⁵ cells/well) for 3 hours, followed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay. P < .05 for a t-test comparison with no treatment.