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. 2021 Jun 30;6(3):e0040621.
doi: 10.1128/mSphere.00406-21. Epub 2021 Jun 23.

Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata

Mark V Horton  1   2 , Chad J Johnson  1 , Robert Zarnowski  1 , Brody D Andes  1 , Taylor J Schoen  2 , John F Kernien  1 , Douglas Lowman  3 , Michael D Kruppa  4 , Zuchao Ma  3 , David L Williams  3 , Anna Huttenlocher  2   5 , Jeniel E Nett  1   2
Affiliations

Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata

Mark V Horton et al. mSphere. .

Abstract

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

Keywords: Candida; Candida auris; Rac2; cell wall; glucan masking; immune evasion; innate immunity; mannan; neutrophil.

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Figures

FIG 1
FIG 1
C. auris evasion of neutrophil phagocytosis is demonstrated across multiple strains. Human neutrophils from healthy donors were incubated for 1 h with C. albicans SN250 or C. auris strains labeled with calcofluor white and subsequently imaged via fluorescence microscopy. The numbers of neutrophils engulfing fungal cells were counted and the percentages of total engaged neutrophils were calculated. High power fields (n = 8 to 10) were examined with neutrophils from at least two donors. *, P < 0.05 by one-way ANOVA with Holm-Sidak multiple comparisons to C. albicans.
FIG 2
FIG 2
C. auris mannosylation pathway mutants are susceptible to neutrophil attack. (A) C. auris strains were incubated with human neutrophils for 1 h and were subsequently imaged via scanning electron microscopy. Images are 10,000× magnification, measurement bars represent 1 μm. (B and C) Human neutrophils were labeled with calcein-AM (green) and cocultured with individual C. auris strains labeled with calcofluor white (blue) for 1 h and imaged via fluorescence microscopy (B). The numbers of neutrophils engulfing fungal cells were counted and the percentages of total engaged neutrophils were calculated (C); n ≥ 3, mean with standard error of the mean (SEM) shown. (D) Individual C. auris strains were cultured with human neutrophils for 4 h and viable burden was estimated by PrestoBlue metabolic activity following neutrophil lysis; n = 3, mean with standard deviation shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant by one-way ANOVA with Holm-Sidak multiple comparisons to C. auris WT.
FIG 3
FIG 3
C. auris pmr1Δ and van1Δ strains display an altered cell wall structure that contains less mannan. (A) C. auris yeast were imaged via transmission electron microscopy at 383,000× magnification, and scale bars represent 50 nm. Brackets denote distinct cell wall layers: G+C, β-glucan and chitin; M, mannan. (B) The monosaccharide compositions of cell walls were measured by gas chromatography, n = 5, mean with SEM shown, *, P < 0.05; ns, not significant by one-way ANOVA with Holm-Sidak multiple comparisons to C. auris WT. Rha, rhamnose; Rib, ribose; Ara, arabinose; Xyl, xylose; Man, mannose; Glu, glucose. (C and D) The structures of isolated mannans were analyzed by 1H NMR and COSY spectra. C shows the 1H NMR spectra for each strain following mannan isolation. In panel D, the intensities were adjusted to the resonance assigned to sidechain-linked backbone α1-6-linked mannosyl repeat units (5.07 ppm) to compare mannan structures.
FIG 4
FIG 4
C. auris pmr1Δ and van1Δ strains display increased cell surface PAMPs. (A) Cell surface β-glucan was labeled using Fc:dectin-1 protein with Alexa Fluor 488-conjugated anti-human IgG Fc antibody and imaged by fluorescence microscopy. (B) Cell surface chitin was labeled with wheat germ agglutinin conjugated to fluorescein isothiocyanate (WGA-FITC) and assessed by fluorescence microscopy. (C and D) Total surface β-glucan and chitin were quantified by plate reader measurements of fluorescence, n = 3 mean with SEM shown, *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Holm-Sidak multiple comparisons to C. auris WT; ns, not significant.
FIG 5
FIG 5
C. auris mannan mutants stimulate increased neutrophil recruitment in the larval zebrafish hindbrain. C. auris strains were injected into the hindbrains of larvae from a cross between the Tg(lyzC:RFP) and Tg(mpeg:GFP) lines at 2 days postfertilization. Fluorescence microscopy was utilized to measure recruitment of neutrophils to the hindbrain at 4, 24, and 72 h postejection. (A) At each time point, fluorescent neutrophils were manually enumerated from maximum intensity projections from z-stacks; n = 9 to 23, experiments were performed in three replicates; the mean with SEM are shown; *, P < 0.05; ns, not significant by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons to C. auris WT. (B) Representative fluorescence microscopy images of neutrophil recruitment to zebrafish hindbrain are shown (magenta = neutrophils, cyan = C. auris cells). Scale bar = 20 μm.
FIG 6
FIG 6
C. auris pmr1Δ and van1Δ strains grow to lower burdens in the larval zebrafish hindbrain in the presence of neutrophils. Wild-type (A) or transgenic zebrafish expressing a dominant Rac2D57N mutation in neutrophils (B) were inoculated with C. auris by hindbrain injection. Fungal burden was quantified at 0, 1, 3, and/or 5 days postinfection by homogenizing whole larvae and plating for CFU. n = 20 to 24; experiments were performed in three replicates; mean with SEM are shown; *, P < 0.05; ns, not significant by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons to C. auris WT.
FIG 7
FIG 7
PMR1 and VAN1 do not influence neutrophil engagement of Candida albicans or Candida glabrata. Human neutrophils were labeled with calcein-AM and cocultured with calcofluor white-labeled C. albicans (A) or C. glabrata (B) for 1 h and imaged via fluorescence microscopy. The number of neutrophils engulfing yeast cells was counted and the percentage of total engaged neutrophils was calculated, n = 3, mean with SEM are shown; analyzed by one-way ANOVA with Holm-Sidak multiple comparisons to REF, ns = not significant. Representative fluorescence microscopy images are shown for C. albicans (C) and C. glabrata (D).
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