Emerging Fungal Pathogen Candida auris Evades Neutrophil Attack

OBSERVATION

Candida auris, an emerging fungal pathogen, causes nosocomial outbreaks of invasive candidiasis with mortality rates approaching 60% (1–3). Little is known about the pathogenesis of this species that has arisen in the last 10 years, and it is unclear why it is rapidly spreading in hospitals worldwide (4). C. auris is the first fungal pathogen labeled as a public health threat due to its ability to readily colonize skin, spread efficiently person to person, and cause severe disease with high fatality (5). Drug resistance is common for this species, with the vast majority of isolates exhibiting fluconazole resistance and nearly half demonstrating resistance to two or more antifungal drug classes (1). Beyond this obstacle to treatment, mortality rates are high even for patients treated with appropriate antifungals (1).

Neutrophils are critical for control of numerous invasive fungal infections, including candidiasis (6, 7). These immune cells kill fungi through phagocytosis or the release of neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity (8–10). However, unlike many fungal infections, neutropenia has not been reported as a common risk factor for C. auris infection (1, 11). This observation prompted us to explore the interaction of neutrophils with C. auris to account for the unexplained virulence of this emerging pathogen.

To determine if neutrophils effectively eradicate C. auris, we measured fungal viability after encounter with human neutrophils and included C. albicans, the most commonly isolated Candida species, for comparison (Fig. 1a). After a 4-h coculture, neutrophils inhibited C. albicans growth by 75%. In stark contrast, the burden of C. auris was not impacted and C. auris replicated beyond the initial inoculum. Evaluation of fungal death by either propidium iodide or Live-or-Dye staining similarly revealed very little killing of C. auris by neutrophils (Fig. 1b and c).

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FIG 1 

C. auris resists neutrophil attack. (a) Human neutrophils were cocultured with C. albicans or C. auris for 4 h, and fungal survival was measured by plate counts (mean with standard error of the mean [SEM] shown; *, P < 0.05 by Student’s t test). (b) C. albicans or C. auris cells were cocultured with calcein AM-labeled human neutrophils (green) for 4 h, and samples were imaged following propidium iodide (red) staining of free DNA and cells with membrane disruption. (c) Following a 4-h coculture with human neutrophils, Candida viability was accessed by the Live-or-Dye detection of dead cells (red). (d to f) RFP-tagged C. albicans (red) or calcofluor white-stained C. auris (blue) were cocultured with calcein AM-labeled human neutrophils (green) and imaged at 1 and 4 h. The number of neutrophils engaging C. albicans or C. auris at 1 h were enumerated (n = 3; mean with SEM shown; *, P < 0.05 by Student’s t test) (d), and images are shown for C. albicans (e) and C. auris (f). (g and h) Calcein AM-labeled human neutrophils (green) were added to a mixture of C. albicans (red) and C. auris (blue), both at 50% of the concentrations in panels a and d and imaged after 1 and 4 h. Neutrophils associating with C. albicans or C. auris in the mixed culture were enumerated (n = 3; mean with SEM shown; *, P < 0.05 by analysis of variance [ANOVA] with Holm-Sidak pairwise comparisons [h]), and images are shown (g). (i) Human neutrophils were cocultured with mixture of C. albicans and C. auris cells for 4 h, and fungal survival was measured by plate counts (n = 3; mean with SEM shown *, P < 0.05 by Student’s t test).

We next theorized that C. auris may resist neutrophil killing by avoiding effective engagement and phagocytosis. Using fluorescence microscopy, we analyzed neutrophil-Candida interactions (Fig. 1d to f). At 1 h, very few neutrophils (15%) were either engulfing or adherent to coincubated C. auris cells. In contrast, 50% of neutrophils were associated with C. albicans. We next performed a competition experiment and exposed neutrophils to a mixture containing both Candida species. The preference of human neutrophils for C. albicans over C. auris was even more apparent for the mixed culture (Fig. 1g and h; see Movie S1 in the supplemental material). Approximately half of the neutrophils associated with C. albicans, while C. auris appeared to be ignored, engaged by fewer than 10% of the neutrophils. To assess if the more robust recruitment and engagement of neutrophils by C. albicans correlated with fungal killing, we coincubated neutrophils with a mixed culture and measured fungal viability. Neutrophils exhibited a potent antifungal response to C. albicans, with only 5% surviving, while C. auris was strikingly resistant to neutrophil killing (Fig. 1i).

Upon examination of the neutrophils in coculture with C. albicans or C. auris over time, we were struck by the difference in neutrophil viability (Fig. 1e and f; see Fig. S1 in the supplemental material). After 4 h, the viability of neutrophils cocultured with C. albicans decreased by half, as evidenced by loss of the fluorescent calcein AM stain. Remarkably, upon exposure to C. auris, nearly all neutrophils remained viable. A similar pattern of neutrophil death was apparent with propidium iodide staining as well (Fig. 1b). As C. albicans is known to induce the formation of NETs, we suspected that neutrophils may be undergoing NETosis, the associated programmed cell death (8). This finding suggested that C. auris may be limiting NET formation.

To examine NET formation in response to C. auris, we utilized scanning electron microscopy (Fig. 2a). Imaging revealed the phagocytosis of C. albicans at 1 h, followed by the formation of web-like structures by 4 h, indicating the release of NETs, as previously described (8). In contrast, neutrophils appeared rounded upon encounter with C. auris at both 1 and 4 h. Neutrophils engaging in phagocytosis or releasing NETs were rarely observed. To quantify NET formation, we measured free DNA with Sytox green (Fig. 2b). Following a 4-h exposure to C. albicans, neutrophils released high levels of free DNA, similar to those treated with phorbol 12-myristate 13-acetate (PMA), a potent inducer of NETs. However, C. auris did not trigger free DNA release, consistent with the lack of NET production observed in microscopy experiments.

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FIG 2 

C. auris fails to trigger the formation of NETs in vitro. (a) C. albicans or C. auris were cocultured with human neutrophils and imaged by scanning electron microscopy after 1 and 4 h. The measurement bars represent 10 and 1 µm for the 2,000× and 10,000× images, respectively. (b) Human neutrophils were exposed to Candida for 4 h, and NET release was estimated by Sytox green detection of free DNA. Data from 4 experiments performed in triplicate were combined. Neutrophil responses to C. albicans and C. auris were analyzed by Student’s t test. *, P < 0.05; mean with SEM shown. (c) Neutrophils were cocultured with Candida for 4 h, fixed, immunolabeled with anti-neutrophil elastase (red) and for total DNA with Sytox green (green), and imaged. The arrow highlights extracellular neutrophil elastase. (d) Production of ROS in response to Candida was measured by fluorescence after neutrophils were prestained with the oxidative stress indicator chloromethyl 2′,7′-dichlorofluorescein (CM-DCF). The mean and SEM of 6 experiments performed in triplicate are shown. Data for C. albicans and C. auris at each time point were analyzed by Student’s t test (*, P < 0.05). (e) Neutrophils were cocultured with Candida for 4 h, fixed, immunolabeled with an anti-citrullinated H4 antibody (red), and imaged. (f) C. auris was cocultured with human neutrophils in the presence and absence of PMA (100 nM) for 4 h, and NET release was estimated by Sytox green detection of free DNA. Experiments were performed in triplicate on 3 occasions (mean with SEM shown). Data were analyzed by ANOVA with pairwise comparisons using the Holm-Sidak method (*, P < 0.05; ns, not significant). (g) Production of ROS in response to C. auris was measured by fluorescence after neutrophils were prestained with oxidative stress indicator CM-DCF and cocultured with C. auris over 4 h. Experiments were performed in triplicate on 3 occasions (mean with SEM shown). (h) Supernatants (SN) were collected from C. auris cultures propagating for 4 h. The impact of supernatants on PMA-induced NET release was assessed by Sytox green. Experiments were performed in triplicate on 3 occasions, and data were analyzed by ANOVA with pairwise comparisons using the Holm-Sidak method (mean with SEM shown; *, P < 0.05). Neu, neutrophil-only control; PMA, phorbol 12-myristate 13-acetate.

Our next experiments were designed to dissect the influence of C. auris on specific molecular aspects of NET formation. We utilized immunofluorescence microscopy to track neutrophil elastase, a granular protein that translocates to the nucleus, cleaves histones, and is then released extracellularly during NET formation (12, 13). In response to C. albicans, neutrophil elastase was detected extracellularly, colocalizing with the DNA fibers of NETs (Fig. 2c). However, extracellular release of neutrophil elastase was not observed in response to C. auris. The generation of ROS, a process integral to the translocation of neutrophil elastase and subsequent NET formation, was also blunted in response to C. auris (Fig. 2d). We next examined citrullination, the enzymatic conversion of arginine residues to citrulline, which occurs on histones during NET formation (14) (Fig. 2e). While C. albicans triggered the release of citrullinated histones, C. auris did not. Even intracellular histone citrullination, signifying an early stage in NET formation, was not observed in response to C. auris.

We also considered the possibility that C. auris may broadly inhibit NET formation and associated reactive oxygen species (ROS) production by neutrophils. To test this hypothesis, we examined the neutrophil response to C. auris in the presence of a strong NET-inducing stimulus. C. auris did not inhibit PMA-induced NET formation or ROS production (Fig. 2f and g). Similarly, supernatants produced by C. auris failed to inhibit PMA-induced NET formation (Fig. 2h). We concluded that C. auris was not actively inhibiting NET formation and ROS production, but rather evaded these defense responses. These findings are consistent with the mixing studies showing C. auris did not impair the activity of neutrophils against C. albicans (Fig. 1g to i).

To explore the clinical relevance of neutrophil interactions with C. auris in vivo, we employed a zebrafish model of invasive candidiasis, as the translucent larvae are ideal for the tracking and imaging of leukocytes (15). Following hindbrain inoculation, neutrophils were readily recruited to C. albicans by 4 h, as described previously (15) (Fig. 3a). In contrast, C. auris recruited few neutrophils, exhibiting neutrophil recruitment similar to the saline-injected controls. To examine the neutrophil response to Candida in vivo, we utilized a transgenic zebrafish line with neutrophil-specific enhanced green fluorescent protein (EGFP) expression in the cytoplasm as well as neutrophil-specific mCherry tagged to histone H2 localized to nuclear material. By 24 h after inoculation of C. albicans into the hindbrain, a subset of neutrophils produced clouds of tagged histones (Fig. 3b). These appeared in close proximity to yeast, which may have been previously ingested. The extracellular release of the structures is most consistent with the formation of extracellular traps with histone studding of DNA. Furthermore, as the EGFP is soluble in the cytoplasm, the loss of EGFP signal in these neutrophils signifies membrane permeability and cell death. In contrast, the majority of the neutrophils of C. auris-infected zebrafish remained viable with the tagged histone colocalizing in the nucleus, and NET-like structures were not observed.

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FIG 3 

Neutrophil-Candida interactions in zebrafish. (a) Zebrafish larvae were infected with Candida by hindbrain injection and neutrophil recruitment to the hindbrain was assessed after 4 h. Experiments were performed on 3 occasions with at least 10 larvae per condition, and data were analyzed by ANOVA with pairwise comparisons using the Holm-Sidak method (*, P < 0.05; ns, not significant). (b) Transgenic larvae with neutrophils expressing cytosolic EGFP (green) and mCherry-tagged histone 2B (red) were infected with Candida by hindbrain injection and imaged after 24 h. Arrowheads indicate yeast cells. Arrows indicate intact neutrophils. Single dots show condensed nuclear material consistent with dead neutrophil fragments. The dotted line outlines the cloud of tagged histone signal. The measurement bars represent 5 µm.

In summary, our studies show vastly different responses by neutrophils to C. auris and C. albicans. C. auris resists killing by human neutrophils. Upon encounter, neutrophils do not effectively engage C. auris and pathways for NET formation that are critical for host elimination of this pathogen are not triggered. Competition experiments reveal a strong preference of human neutrophils for engagement of C. albicans over C. auris, ultimately leading to 10-fold-higher inhibition of C. albicans over C. auris. A zebrafish larva infection model similarly uncovered a failure of C. auris to recruit neutrophils. We propose that this altered innate immune response contributes to the poor outcomes observed in patients with invasive C. auris infection, by allowing this species to go unrecognized by neutrophils.

Using murine models of invasive candidiasis, several groups have found C. auris to be less virulent than C. albicans, prompting the use of immunocompetent models (16, 17). It is unclear why the mice appear to control C. auris, while human neutrophils fail to recognize and kill the pathogen. One explanation may be the species difference in neutrophil receptors important for the killing of Candida (18, 19). In addition, virulence may vary by C. auris strain. For example, an evaluation of a collection of isolates by Borman et al. revealed strain-specific virulence in the Galleria mellonella model, with the most virulent C. auris strains exhibiting pathogenicity similar to that of C. albicans (20).

C. auris genotypes vary based on geography and cluster into clades (1). The C. auris strain used in the present study was isolated from a patient in India and phylogenetically placed in the South Asian or India/Pakistan clade. We selected a strain from this clade to investigate, as recent nosocomial outbreaks in both the United Kingdom and the United States have been caused by members of the South Asian clade (21, 22). However, given the genetic diversity between clades, examination of the neutrophil response to C. auris strains belonging to other clades would be of great interest.

As C. auris has only recently emerged, very little is known about virulence factors that may influence interaction with the host. Unraveling the molecular mechanisms impeding the engagement and activation of neutrophils may not only offer an explanation for the virulence of this pathogen, but also provide avenues to the development of therapeutics for this emerging global health threat.