Fig 1.
C. albicans biofilms resist killing by neutrophils and impair release of NETs.
(A) Planktonic and biofilm C. albicans were co-cultured with human neutrophils at an effector:target ratio of 1:1 and fungal inhibition was estimated by an XTT assay after neutrophil lysis. Neutrophils have very little activity against biofilms, but strongly inhibit planktonic Candida, n = 11. (B) The rate of NET release was estimated by Sytox Green detection of free DNA. Planktonic Candida generated high fluorescence, representing NET release, comparable to the levels induced by PMA. In contrast, biofilms did not produce fluorescence, similar to the neutrophil only control, n = 3. (C) Following co-culture with neutrophils for 4 h, the neutrophil response to planktonic and biofilm C. albicans was visualized by immunofluorescence using an anti-citrullinated H4 antibody. NETs were observed in response to planktonic Candida, but were rarely produced in response to biofilm. (D) By scanning electron microscopy, thread-like NETs covered planktonic cells after a 4 h co-culture with neutrophils. In contrast, neutrophils exposed to biofilm appeared rounded, with few NETs released. Measurement bars represent 20 μm and 2 μm for 2,000x and 10,000x images, respectively. *P<0.05 Error bars represent SEM.
Fig 2.
Neutrophils adhere to C. albicans biofilm.
(A) Fluorescently-labeled neutrophils were added to microfluidic channels with C. albicans biofilms, which had been propagated on the sidewall, or to planktonic C. albicans. Over the course of 90 min, neutrophils migrated to the biofilm, adhered, and extended over the surface of the hyphae. In contrast, neutrophils engulfed planktonic C. albicans and did not appear to elongate. (B) Neutrophil interactions with C. albicans were examined by scanning electron microscopy. In response to biofilms, neutrophils initially adhered to hyphae, then elongated with extended filopodia, and ultimately appeared rounded. Upon co-culture with planktonic cells, NETs developed over this 4 h time period. Measurement bars represent 2 μm for 10,000x images. Representative data are shown for experiments performed with neutrophils from at least 3 different donors on different days.
Fig 3.
NET inhibition by C. albicans is dependent on intact biofilm architecture and is not due to a soluble factor.
(A) Planktonic, biofilm, and partially dispersed biofilms were co-cultured with human neutrophils for 4 h and NET release was estimated by Sytox Green detection of free DNA. Dispersed biofilms induced NETs, marked by elevated Sytox Green, n = 6. (B) By scanning electron microscopy, NETs were triggered in response to disrupted biofilm, indicating intact structure is necessary for NET inhibition. The measurement bars represent 20 μm and 2 μm for the 2,000x and 10,000x images, respectively. (C) C. albicans biofilms and PMA (an inducer of NETs), alone and in combination were incubated with neutrophils for 4 h and NET release was estimated by Sytox Green. While PMA alone generated a robust NET response, the combination of PMA and biofilm did not trigger DNA release. (D) Supernatants from biofilms (24 h) were collected at 2 h and added to Sytox Green assays in combination with PMA. Biofilm supernatants alone did not block PMA induction of NETs, n = 5. (E) Neutrophils were pre-stimulated (pre-stim) with PMA to induce NETs for 90 min prior to addition to biofilms. While pre-stimulation increased NET release, biofilms still inhibited NETs compared to the PMA only control, n = 4. (F) The activity of pre-stimulated neutrophils against biofilms was estimated by XTT assay following lysis of neutrophils. Neutrophils pre-stimulated to induce NETs inhibited biofilms n = 4. *P<0.05 compared to reference, **P<0.05 compared to *. Error bars represent SEM.
Fig 4.
Biofilm inhibition of NETs is dependent on C. albicans mannosylation.
(A) NET release was estimated by Sytox Green after neutrophils were exposed to C. albicans biofilms for 4 h. Five mutants with disruption of mannan pathways triggered NET release in response to neutrophils from multiple donors, n = 4. (B) By scanning electron microscopy, neutrophils exposed to the reference strain biofilm appeared rounded, while the pmr1Δ/Δ biofilm elicited NETs. Measurement bars represent 2 μm for 10,000x images. (C) Complementation of the pmr1Δ/Δ mutant mostly restored the NET inhibition phenotype n = 4. (D) NET release was measured in response to planktonic C. albicans. In contrast to the response to biofilms, no significant difference was observed between pmr1Δ/Δ and the reference strain, n = 6. *P<0.05 compared to reference. Error bars represent SEM.
Fig 5.
C. albicans biofilm extracellular matrix inhibits neutrophil production of ROS and protects against neutrophil activity.
(A-D) Neutrophils were pre-labeled with CM-H2DCFDA and exposed to C. albicans biofilms in the presence or absence of PMA and ROS was measured by fluorescence. (A) Neutrophils produced higher levels of ROS in response to planktonic cells over the course of 3 h, n = 4, representative data with SD shown. (B) Dispersion of biofilms increased neutrophil production of ROS n = 5, SEM shown. (C) Neutrophils generated increased ROS in response to the C. albicans pmr1Δ/Δ mutant biofilm, several fold above the reference strain, n = 9, SEM shown. (D) C. albicans biofilms inhibited ROS production in response to PMA stimulation n = 11, SEM shown. (E-F) C. albicans biofilms and planktonic cells were co-cultured with human neutrophils and fungal killing was estimated by an XTT assay after neutrophil lysis. Neutrophils exhibited increased activity against the pmr1Δ/Δ mutant biofilm compared to the reference strain (E) but no difference was observed for planktonic cells (F), n = 6 and 4, SEM shown. *P<0.05 compared to reference.
Fig 6.
C. albicans biofilm mannosylation impairs NET release and contributes to virulence in vivo.
(A) C. albicans biofilms were formed in rat venous catheters in vivo and imaged by scanning electron microscopy. While both the reference strain and the pmr1Δ/Δ mutant formed biofilms in vivo, the pmr1Δ/Δ mutant biofilm was associated with increased host cells and overlying fibrillary material. On higher magnification, the web of fibrils appeared to extrude from host cells. (B) The contribution of mannosylation by PMR1 to biofilm immune protection was assessed by rat catheter viable burden. Disruption of PMR1 was associated with a 70% decrease in burden compared to the reference strain. *P<0.05.