Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity

The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.


Introduction
Candida albicans is a common fungal microorganism that colonizes the oral, genital and gastrointestinal surfaces of most healthy individuals. The maintenance of colonization is the result of a complex balance between fungal proliferation and host immune recognition. Despite host immune defenses aimed at clearing pathogens, C. albicans has developed numerous strategies to evade host immune detection [1]. In immunocompromised patients, C. albicans may disseminate into bloodstream, causing life-threatening systemic candidiasis [2,3]. The associated mortality rates of systemic infection are reported to be greater than 30%, highlighting the potential critical impact of C. albicans on global health burden [4][5][6].
The mature cell wall of C. albicans is a complex structure of cross-linked polysaccharides and glycosylated proteins. The cell wall is not only required for maintaining cell shape and stability, but also is critically related to immunogenicity and virulence of C. albicans. The outer layer of the cell wall is comprised of glycosylated mannoproteins that are post-translationally modified by N-and O-linked mannosides [7]. N-linked mannan chains are specifically required for cell morphology, phagocytosis, and immune recognition of C. albicans by host dendritic cells [8]. The core structure of N-mannan is a dolichol pyrophosphate anchored oligosaccharide comprised of three glucose, nine mannose and two N-acetylglucosamine residues (Glc 3 Man 9 GlcNAc 2 ). The outer chain branched mannan is attached to the N-mannan core through an α-1,6-backbone. Addition of the first α-1,6-mannose is catalyzed by mannosyltransferase Och1. Notably, Och1 mutant strains of C. albicans demonstrate attenuated virulence in animal models with systemic infection [9]. Extension of α-1,6-mannose backbone by mannose residues is performed by the enzyme complexes mannan polymerase I (M-Pol I) and II (M-Pol II) [10]. The α-1,6-backbone is then further modified with additional α-1,2-mannose units by Mnn2 family and Mnn5, which similarly, are critical for C. albicans virulence in mice or Galleria mellonella [11,12]. The outer side chains are further capped with either α-1,3-mannose or β-1,2-mannose units via Mnn1 family and β-1,2-mannosyltransferases (BMTs). The C. albicans MNN1 gene family contains six members, of which only MNN14 represent a critical factor for pathogenicity in vivo [13]. Bmt1 and Bmt3, which are required for the addition of the first and second β-1,2-mannose units respectively, are not associated with the virulence of C. albicans [14]. Although a variety of C. albicans mannosylation mutants have been found to be less pathogenic in vivo, the mechanisms of host clearance associated with abnormal mannan structures remains unclear.
Mnn10, an important subunit of cis Golgi mannan polymerase, was identified as an α-1,6-mannosyltransferase which is responsible for mannan backbone extension in non-pathogenic fungal species such as Saccharomyces cerevisiae and Kluyveromyces lactis [32,33]. In the present study, we first characterized the role of α-1,6-mannose backbone in C. albicans pathogenicity. We demonstrated that inhibition of α-1,6-mannose backbone extension can block the development of invasive C. albicans infection, and suggested α-1,6-mannose backbone extension is essential for the evasion of host Dectin-1 mediated immune response towards C. albicans.

Results
Mnn10 possesses α-1,6-mannosyltransferase activity, and is required for α-1,6-mannose backbone extension in C. albicans To analyze enzymatic activity, we used a bacterial expression system to produce MBP-fused Mnn10 protein from a pMAL-p5X vector and purified the protein by amylose magnetic beads (S1 Fig). The purified proteins were subjected to a mannosyltransferase assay system, which required α-1,6-linked mannobiose as an acceptor and GDP-mannose as a donor for proper mannosyltransferase activity [12,34]. Our results indicated that Mnn10 catalyzes the transformation of GDP-mannose to α-1,6-mannobiose to form mannotriose and mannotetraose ( Fig  1A). Consistently, the reaction products could be digested to mannose via α-1,6-mannosidase treatment ( Fig 1B). Thus our results demonstrate that Mnn10 is able to transfer mannose from the donor onto the acceptor substrates to form α-1,6-linked oligomannose.
To further confirm the role of MNN10 in α-1,6-mannose backbone extension, we generated mnn10Δ/Δ null mutant strain and mnn10Δ/Δ::MNN10 revertant strain using the homologous recombination method. The genotype was confirmed by PCR and the expression of MNN10 was determined by quantitative RT-PCR (S2 Fig). When compared to the parental strain SN152, transmission electron microscopy (TEM) analysis noted a significantly shortened external layer of mannan fibril surrounding the cell wall in mnn10Δ/Δ. Notably, MNN10 gene rescue was sufficient to restore the expected length of mannan fibril (Fig 1C).
The phosphomannan of C. albicans cell wall, characterized by Alcian Blue dye binding, is attached to the branched mannan from the α-1,6-mannose backbone [35]. Therefore, the content of phosphomannan reveals the length of α-1,6-mannose backbone. The Alcian Blue assay The reaction of expressed MBP-fused Mnn10 protein or MBP protein incubated with α-1,6-mannobiose (Man 2), GDP-mannose (GDP-Man) or controls. The reaction products were labeled with ANTS and separated by fluorophore-assisted carbohydrate gel electrophoresis (FACE). Man 3, mannotriose; Man 4, mannotetraose. (B) α-1,6-mannose assay. The reaction products of (A) were treated with or without α-1,6-mannosidase treatment, and then subjected to FACE. (C) Representative cell wall ultrastructures of SN152, mnn10Δ/Δ::MNN10 and mnn10Δ/Δ strains were observed by transmission electron microscopy. The scale bar represents 0.2 μm. (D) Alcian Blue binding assay. The cells were incubated with Alcian Blue for 10 min and the amount of dye bound to the cell wall were calculated. Data represent the mean amount of dye bound per cell ± SD from triplicates of one representative experiment of three. (E) Cell surface hydrophobicity of the indicated C. albicans strains was measured by water-hydrocarbon two-phase assay. Data are means ± SD of triplicates of one representative experiment of three. **, P < 0.01 [One-way ANOVA with Bonferroni post-test (D, E)]. demonstrated significantly decreased binding of the dye to mnn10Δ/Δ as compared to the parental strain, which could be rescued by reintegration of MNN10 into mnn10Δ/Δ, confirming the role of MNN10 in α-1,6-mannose backbone extension (Fig 1D). Inhibition of α-1,6-mannose backbone extension via deletion of MNN10 also affected the surface property of C. albicans such as hydrophobicity ( Fig 1E). Furthermore, the change of cell wall structures impaired the resistance of mnn10Δ/Δ to various stresses including calcofluor white, fluconazole, miconazole, and caspofungin (S3 Fig). Taken together, our results suggest that Mnn10 protein possesses α-1,6-mannosyltransferase activity and is crucial in α-1,6-mannose backbone extension in C. albicans.
MNN10 is required for cell wall polysaccharides biosynthesis and organization in C. albicans The cell wall polysaccharides of C. albicans consist of an inner layer of chitin and β-glucan, and an outer fibrillar layer of mannan. To examine the effects of MNN10 deletion on cell wall polysaccharides biosynthesis and organization, C. albicans yeasts or hyphae were stained with Concanavalin A (ConA), anti-β-glucan antibody and calcofluor white (CFW). We observed and quantified the polysaccharides by confocal laser scanning microscopy and flow cytometry, respectively. ConA staining indicated that mnn10 mutant, either in yeast cells or hyphae, had a markedly decreased fluorescence intensity which is suggested lower mannose content as compared to the parental or revertant strain (Figs 2A, 2D and S4A). β-(1,3)-glucan, a well-characterized PAMP of C. albicans, is buried underneath the outer layer of the cell wall. We found a remarkable exposure of β-(1,3)-glucan on the cell surface of mnn10 mutant (Figs 2B, 2E and S4B). However, no significant difference of CFW binding was visualized in mnn10 mutant strain, suggesting normal chitin content (Fig 2C and 2F).
The highly glycosylated cell wall proteins (CWPs) of C. albicans often act as virulence factors and contribute to cell wall integrity, promote biofilm formation, mediate adherence to host cells, and promote invasion of epithelial layers [36][37][38]. To further evaluate the effect of α-1,6-mannose extension in C. albicans CWPs anchorage, we analyzed CWPs extraction by LC-MS/MS. Twenty representative CWPs were identified in the cell wall of the parental and mnn10 mutant strain (S1 Table). We found that MNN10 deletion had almost no effect on the anchorage of CWPs. Notably several of the proteins are considered to mediate specific roles in the development of invasion, such as cell-surface antigens involved in virulence (Tdh3p, Eno1p, Met6p, Hsp70p), proteins involved in cell wall biosynthesis and assembly (Pga4p, Utr2p, Crh11p), and proteins involved in adhesions and cell wall morphogenesis (Sap9p, Pga24p, Ecm33p) ( Fig 2G).
Taken together, our results demonstrate that inhibition of α-1,6-mannose backbone extension by MNN10 deletion resulted in abnormal cell wall polysaccharides biosynthesis and organization, but had no effect on the anchorage of CWPs including virulence factors.
Inhibition of α-1,6-mannose backbone extension has no effect on the invasive capacity of C. albicans The composition and organization of the cell wall in C. albicans plays an important role in the initiation and maintenance of invasive infections. In the context of MNN10 deletion, we investigated the virulence of mnn10 mutant strain in vitro. Multiplication and yeast-to-hypha transition are the prerequisites of C. albicans invasive disease [39]. The growth curves obtained demonstrated that MNN10 did not impact the growth of C. albicans in vitro (Fig 3A). Moreover, mnn10 mutant also did not show defective filamentation in either liquid (RPMI1640, 10% serum liquid medium) or solid medium (Spider and Lee's agar medium) favoring hyphal growth (Figs 3B and S5A).  Adhesion to the epithelium, such as oral and intestinal epithelial cells, and hyphal penetration are the first steps of C. albicans invasion [40]. Our results indicate that deletion of MNN10 did not affect the ability of C. albicans to adhere Caco-2 intestinal epithelial and KB buccal epithelial cells (P > 0.05, S5B Fig). Following adhesion, we used scanning electron microscopy (SEM) to evaluate the ability of hyphal penetration and invasion of mnn10 mutant strain. After a 2 h co-culture, we observed hyphal forms, of all the indicated strains, penetrated Caco-2 and KB cells at the apical face and microvillis attached to the hyphae at the point of penetration, indicating that MNN10 gene deletion had no effect on the invasion ability of C. albicans in or on spider agar media for 5 days at 37°C to induce hyphal form. (C) Representative micrographs of scanning electron microscope of Caco-2 and KB cells invaded or penetrated by SN152, mnn10Δ/Δ::MNN10 and mnn10Δ/Δ after 2 h co-incubation (MOI = 1). (D) Epithelial cells damage was determined by assaying LDH release. Relative LDH release from Caco-2 or KB cells was measured after 12 h incubation with C. albicans (MOI = 0.1). Data are represented as means ± SD from triplicates of one representative experiment of three. P > 0.05 (One-way ANOVA with Bonferroni post-test). (E) Extracellular phospholipase and hemolytic activity assays. The phospholipase activity was examined by spotted C. albicans on egg yolk agar at 37°C for 3 days and observed the width of the zone of precipitation around each colony (top panel). The hemolytic activity was assessed by growing the indicated C. albicans strains on sugar-enriched sheep blood agar at 37°C for 3 days to observe the presence of a distinct translucent halo around the colonies (lower panel). Representative images are shown from one of three independent experiments. doi:10.1371/journal.ppat.1005617.g003 α-1,6-Mannose Backbone of Candida albicans and Host Antifungal Immunity vitro ( Fig 3C). Moreover, no significant difference, in epithelial cell damage, was observed among strains (P > 0.05, Fig 3D).
Phospholipases and hemolysins secreted by C. albicans can also induce host cells damage [41,42]. We screened extracellular phospholipase and hemolytic activity by growing C. albicans strains on either egg yolk agar or sheep blood agar. As compared to the parental and revertant strains, mnn10 mutant induced similar zones of precipitation or clearance around the colonies, indicating that MNN10 deletion was not significantly associated with phospholipases and hemolysins secretion ( Fig 3E).
As such, our results suggest that MNN10 deletion did not affect the invasive capacity of C. albicans in vitro.
MNN10 gene is required for normal pathogenicity of C. albicans in a murine systemic candidiasis model To examine the effects of α-1,6-mannose backbone of C. albicans on host infection in vivo, we compared the pathogenicity of SN152, mnn10Δ/Δ::MNN10 and mnn10Δ/Δ in a murine systemic candidiasis model. Mice infected with mnn10 mutant strain had a much higher survival rate than those infected with the parental or revertant strain at a lethal dose (5×10 5 CFU). Over a 30-day observation period, only one mouse infected with the mnn10 mutant strain died. By contrast, all of the mice infected with the parental or revertant strain died within 30 days (P < 0.01, Fig 4A). The median survival analysis demonstrated that the 50% survival limit was attained at 10 days for mice infected with the parental strain and 18 days for the revertant strain, respectively. At day 2 or day 5 post-infection, mice infected with mnn10 mutant had significantly lower fungal burdens in the kidneys and livers as compared to those infected with either the parental or revertant strain (P < 0.01 and P < 0.05, respectively; Figs 4B and S6A). Moreover, Hematoxylin and eosin (H&E) staining revealed that during prolonged infections, inflammatory influx and tissue necrosis of the kidneys were aggravated in mice infected with either parental or MNN10 revertant strains ( Furthermore, we performed a flow cytometry analysis to detect cellular inflammation in the kidneys of infected mice. Time-course analysis revealed that mnn10Δ/Δ infected mice recruited more SSC high CD11b + Ly-6C + Ly-6G + neutrophils and SSC high CD11b + Ly-6C + Ly-6Gmonocytes in the kidney than mice infected with the SN152 strain at day 2 and day 3, respectively (Figs 4E and S8). The cellular inflammation in the kidneys of mnn10Δ/Δ infected mice reached a peak at day 3 post-infection, when compared to SN152 infected mice. With decreased fungal burden, neutrophils and monocytes were reduced in the kidney of mnn10Δ/Δ infected mice at day 5 post-infection as compared with SN152 infected mice ( Fig 4E).
Taken together, the results suggest that inhibition of α-1,6-mannose extension by MNN10 deletion significantly impacted the pathogenesis of C. albicans by enhancing the host antifungal defense in vivo.
MNN10 deletion does not affect the pathogenicity of C. albicans in athymic nude mice To further explore whether host antifungal defense was crucial for the clearance of mnn10 mutant in vivo, we investigated the pathogenicity of mnn10 mutant strain in BALB/c mice and α-1,6-Mannose Backbone of Candida albicans and Host Antifungal Immunity athymic nude mice (BALB/c background). BALB/c mice infected with mnn10 mutant displayed significantly lower fungal burdens in the kidneys and livers as compared to those infected with either the parental or revertant strain (P < 0.05 and P < 0.01, respectively; Fig  5A). However, the results of kidneys and livers fungal burdens indicated mnn10 was not required for the C. albicans pathogenesis in athymic nude mice ( Fig 5B). To expand upon these findings, longer time course and survival experiments were performed. At day 10 post-infection, no significant difference in the levels of fungal burden of kidneys was observed between mice infected with mnn10 mutant strain versus those infected with the parental or revertant strains ( Fig 5C). Furthermore, there was no significant survival difference among these strains infected mice ( Fig 5D).
To confirm the role of elevated IFN-γ and IL-17 in the infected mice ( Fig 4D) in clearing mnn10 mutant strain, we performed an adoptive immunotherapy experiment. The combination of IFN-γ and IL-17 treatment significantly improved the survival of athymic nude mice infected with mnn10Δ/Δ (P < 0.01, Fig 5E). Moreover, mnn10 mutant infected mice treated with cytokines had a markedly higher survival rate than SN152 infected mice with the same treatment modality (P < 0.05, Fig 5E). Furthermore, we used neutralizing antibodies to IL-17 and/or IFN-γ to elucidate the importance of these cytokines in C57BL/6 mice infected with mnn10Δ/Δ. Compared with mice treated with isotype antibody rat IgG1, mice receiving anti-IFN-γ, anti-IL-17A, or both antibodies exhibited significantly higher fungal burdens in the kidneys and livers (S9 Inhibition of α-1,6-mannose backbone extension in C. albicans induces enhanced host innate immune response Our results suggested that the enhanced host antifungal immunity was the main factor that contributed to the diminished virulence of mnn10 mutant strain. Thus, we further investigated the myeloid cell recognition and response to mnn10 mutant using a macrophages-C. albicans interaction model. We found that mnn10 mutant yeast cells induced more nuclear translocation of NF-κB (p65), phosphorylation of Syk and IκBα, together with IκBα degradation in thioglycolate-elicited peritoneal macrophages ( Fig 6A). Consistently, mnn10 mutant yeast cells also induced more ERK phosphorylation, p38 phosphorylation, and JNK phosphorylation in macrophages than the parent parental or revertant strains ( Fig 6C). We also detected significantly higher levels of inflammatory cytokines including TNF-α, IL-6, IL-1β and IL-12p40 in macrophages induced by mnn10Δ/Δ yeast cells ( Fig 6E). However, no differences in NF-κB and MAPK signaling activation were observed in macrophages stimulated by hyphal forms of the parental, mnn10 mutant and revertant strains, the same as the production of proinflammatory cytokines (Figs 6B, 6D and S10).
We performed a macrophage phagocytosis assay to investigate whether mnn10 mutant was differentially taken up from the parental or revertant strain. Our results suggest that no significant difference of the macrophage phagocytosis was visually appreciated between mnn10 mutant, and the parental or revertant strains in the initial infection stage (Figs 6F and S11). However, we found that thioglycollate-elicited peritoneal neutrophils could produce kidneys from C57BL/6 mice infected with indicated C. albicans at day 5 (n = 8 per group). The cytokine levels were normalized to burden of infection in each individually kidney as fg/g tissue/CFU. Data are means ± SD and are representative of three independent experiments. (E) The cellular inflammation in the kidneys of SN152 or mnn10Δ/Δ infected mice. SSC high CD11b + Ly-6C + Ly-6G + neutrophils and SSC high CD11b + Ly-6C + Ly-6Gmonocytes in the kidneys were detected at Day 3 and Day 5 by flow cytometry. Data are representative images of five mice. *, P < 0.05; **, P < 0.01 [Log-rank test (A) and Kruskal-Wallis nonparametric One-way ANOVA with Dunns post-test (B, D)].
doi:10.1371/journal.ppat.1005617.g004 significantly more ROS and thus destroy mnn10Δ/Δ more efficiently (Fig 7A and 7B). While neutrophils can target pathogens in modalities such as myeloperoxidase (MPO), no significant difference in the intracellular MPO activity of neutrophils stimulated by mnn10 mutant versus parental or revertant strain was observed (Fig 7C). To determine whether the enhanced killing was dependent on ROS production, we scavenged ROS by 2 mM L-ascorbic acid in the co-culture medium, and found that the ability of neutrophils to eliminate mnn10Δ/Δ was diminished with decreased ROS (Fig 7D and 7E). However, we found that removal of mnn10 mutant hyphae by neutrophils was similar in parental or revertant strains (Fig 7F).

Enhanced host antifungal immunity induced by mnn10 mutant is Dectin-1 dependent
Several PRRs, such as TLRs and CLRs, are involved in host defense during C. albicans infection. We hypothesized that the enhanced host immune response induced by mnn10 mutant may be attributed to the cell wall β-(1,3)-glucan exposure. We stimulated thioglycollate-elicited peritoneal macrophages from wild-type or Dectin-1-deficient mice with mnn10 mutant yeasts, and found that activation of NF-κB and MAPK signaling was defective in Dectin-1-deficient macrophages ( Fig 9A). Consequently, the mnn10 mutant yeasts could not significantly increase the production of inflammatory cytokines such as TNF-α and IL-6 in Dectin-1-deficient macrophage cells (Fig 9B). The removal of mnn10 mutant by neutrophils from Dectin-1-deficient mice was similar in parental or revertant strains (S12A Fig). Moreover, no significant difference in the inflammatory cytokines, including IL-6, GM-CSF, IFN-γ and IL-17, were detected between the kidneys of mnn10Δ/Δ and SN152 infected Dectin-1-deficient mice (Figs 9C and S12B). The survival curves indicated that mnn10 mutant strain presented similar pathogenicity with the parental strain SN152 in Dectin-1-deficient mice (Fig 9D). Dectin-1-deficient mice had similar kidney or liver fungal burdens when infected with the parental SN152 or mnn10Δ/ Δ strain (P > 0.05, Figs 9E and S6B).
However, our results demonstrated that other PRRs involved in antifungal immunity did not contribute to the enhanced immune responses elicited by mnn10 mutant. By example, mnn10 mutant strain presented similar pathogenicity in Dectin-2 deficient mice when compared to SN152 in the wild type mice (P < 0.05, Figs 9F and S6C). Dectin-2 deficiency had no effect on the inflammatory cytokines production such as TNF-α and IL-6 in macrophages, and TLR2 or TLR4 deficiency had no effect on the activation of NF-κB and MAPK signaling in macrophages when challenged with mnn10 mutant (Fig 9G and 9H).
Taken together, these data suggest that the enhanced immune response induced by α-1,6-mannose backbone inhibition in C. albicans was Dectin-1 dependent.

Discussion
During C. albicans infection, both yeast cells and hyphae can be found in infected organs or tissues, and innate immune cells discriminate them using different PRRs to elicit a protective immune response [43]. Previous studies have shown that the mannan structure of C. albicans yeasts (MOI = 5) (C) or hyphae (MOI = 1) (D) for the indicated times. The total cell lysates were subjected to immunoblotting with the indicated antibodies of MAPK signaling. Numbers between blots indicate activity (Act) of phosphorylation of MAPK or NF-κB pathways, as measured by densitometry. (E) ELISA results for cytokines TNF-α, IL-6, IL-1β and IL-12p40 in cell supernatants of thioglycollate-elicited peritoneal macrophages, which were stimulated with the indicated C. albicans yeasts (MOI = 5) for 6 h. Usti, unstimulated. Data are means ± SD of triplicates from one representative experiment of three. Usti, unstimulated. *, P < 0.05; **, P < 0.01 (One-way ANOVA with Bonferroni post-test). (F) Phagocytosis of C. albicans by thioglycollate-elicited peritoneal macrophages. Live C. albicans was co-cultured with the macrophages grown on coverslips in multiwell plates for 90 min. After staining with CFW (1 μg/ml) and PSA-FITC (20 μg/ml) for 10 min, the samples were viewed by confocal laser scanning microscope directly. Scale bar represents 10 μm. Arrows indicate the internalized C. albicans cells inaccessible to staining with CFW. Bright field (BF), fluorescein isothiocyanate-conjugated pisum sativum agglutinin (PSA-FITC), calcofluor white (CFW) and overlay are shown individually.  plays an important role in the development of invasive infection. Here we first determined that the cell wall α-1,6-mannose backbone maintained the pathogenicity of C. albicans by preventing host, Dectin-1 mediated, recognition of β-(1,3)-glucan. These results highlight a previously unappreciated relationship between cell wall mannan structure and pathogenicity of C. albicans.
Several genes are involved in the biosynthesis of the cell wall mannan in C. albicans. Cell wall mannan structure mutant strains, induced by deletion of several genes including OCH1, MNN2 and MNN5, often represent a less pathogenic strain in vivo [9,11,12]. Herein, we determined that Mnn10 has α-1,6-mannosyltransferase activity, and is responsible for α-1,6-mannose backbone biosynthesis in C. albicans (Fig 1). We also highlighted the role of MNN10 in pathogenicity of C. albicans. Our studies using mnn10 null mutant strain demonstrated that MNN10 is required for C. albicans pathogenicity during a systemic candidiasis model in mice (Fig 4).
Infection is mediated by the interplay between a pathogen's ability to invade host, versus the host attempts to recognize and destroy the pathogen. Several cell wall proteins on the surface of C. albicans act as virulence factors to invade host [39,44]. Cell wall proteomic analysis indicated that MNN10 deletion had no effect on virulence factors of C. albicans (Fig 2G). Several steps, including adhesion to the epithelium, epithelium penetration and invasion by hyphae, vascular dissemination, and endothelial colonization, are involved in the development of invasive candidiasis [2]. However, the data from our study in vitro indicated that the mnn10 mutant was not defective in its invasive capacity (Fig 3). Therefore, we hypothesize enhanced immune recognition of the mutant strain by the host, rather than decreased virulence, contributed to the attenuated pathogenicity. The normal pathogenicity of mnn10 mutant in athymic nude mice (BALB/c background) further confirmed our hypothesis (Fig 5B, 5C and 5D). The difference of mnn10 mutant clearance in BALB/c mice versus athymic nude mice may be attributed to thymus, which is an important organ for the differentiation and maturation of T lymphocytes. Both Th1 and Th17 cells mediate host protection against C. albicans infection [19,45]. IFN-γ and IL-17 are the key cytokines produced by Th1 and Th17 cells, which recruit neutrophils and macrophages to destroy the pathogen. IFN-γ and IL-17 elevation, and corresponding neutrophil responses were observed in the kidneys of mnn10 mutant infected mice, indicating that the mnn10 mutant could stimulate stronger antifungal response (Fig 4D and 4E). Although the source of IFN-γ and IL-17 can be from innate lymphocytes, our results suggest that elevated levels of IFN-γ and IL-17 elicited by mnn10 mutant in vivo are likely not derived from innate lymphocytes such as NK cells, NKT cells and γ/δ T cells (Fig 8D and 8E). Intracellular cytokine staining analysis revealed that mnn10Δ/Δ infected mice induced more IFN-γproducing and IL-17A-producing α/β T cells than SN152 infected mice (S13 Fig). Therefore, our study suggested that inhibition of α-1,6-mannose backbone extension in C. albicans induced enhanced T lymphocyte mediated immune response in vivo.
The host innate immune cells involved in invading pathogens recognition are predominantly monocytes and neutrophils in circulation and macrophages in infected tissues. Inflammatory cytokines and chemokines can recruit innate immune cells to infected tissues. In a peritoneal infection model, we demonstrated that mnn10 mutant strain could recruit more neutrophils and monocytes by inducing cytokines and chemokines including IL-6, MCP-1, GM-CSF, MIP-1α and G-CSF in the peritoneal cavity (Fig 8A, 8B and 8C). IL-6 and G-CSF can promote neutrophil production and activation against C. albicans infection [46]. GM-CSF Data shown are means ± SD of triplicates from one representative experiment of three. Usti, unstimulated. **, P < 0.01 (One-way ANOVA with Bonferroni post-test).
doi:10.1371/journal.ppat.1005617.g007  [47,48]. MCP-1 is a crucial mediator to recruit monocytes in inflammation in vivo [49]. Neutrophils contribute to the initial step to kill fungi, and are especially important in neutropenic and immunosuppressed individuals [50]. Our data also determined that neutrophils mainly eliminated mnn10 mutant strain in a ROS-dependent manner (Fig 7). These results suggest that inhibition of C. albicans α-1,6-mannose backbone extension by MNN10 deletion could enhance host innate immune recognition.
Immune recognition could render antigen presenting cells competent to prime T cells, and thereby drive the adaptive Th1 and Th17 immune response. After encountering pathogens, the host macrophages secrete several cytokines, leading to the induction of Th cell differentiation [3]. Our study suggests that C. albicans mnn10 did not play an important role in the initial phagocytosis stage of macrophages (Figs 6F and S11). By contrast, mnn10 mutant yeast of S. cerevisiae was poorly taken up by primary macrophages, as compared to the parental strain [51]. We hypothesize that the differential macrophage phagocytosis of mnn10 mutant might be attributed to the pathogenicity and immunogenicity differences between C. albicans and S. cerevisiae. However, we demonstrated that mnn10 mutant strain elicited enhanced recognition by macrophages. The increased cellular responses of macrophages were associated with NF-κB and MAPK pathway activation, and inflammatory cytokine productions including TNF-α, IL-6, IL-1β and IL-12p40 (Fig 6A, 6C and 6E). TNF-α was involved in the innate immune response against Candida infection through promotion of neutrophil production and activation [52]. IL-6 and IL-23 (consisting of IL-12p40 and p19) contributed to Th17 differentiation induced by C. albicans and Staphylococcus aureus, and IL-1β was essential pro-inflammatory regulators of Th17 cells both at priming and effect phase [53]. Therefore, we suggest that the enhanced recognition by innate immune cells could promote Th cell response and thus contributes to host clearance of mnn10 mutant strain in vivo.
The special PAMPs on the surface of C. albicans could be recognized by PRRs of innate immune cells to initiate the host immunity. The skeletal component of C. albicans cell wall is based on a core structure of β-(1,3)-glucan that is covalently linked to β-(1,6)-glucan, chitin, and an outer layer of mannoproteins [3]. Recognition of β-(1,3)-glucan by Dectin-1 has been reported to be important in host antifungal defense [22]. However, β-(1,3)-glucan on the surface of C. albicans was normally shielded by the outer mannan layer from being recognized by Dectin-1 on innate immune cells [2,27]. Deletion of certain C. albicans genes, such as the phospholipids phosphatidylserine synthase gene CHO1 and the histidine kinase gene CHK1, could unmask β-glucans of C. albicans, specifically recognized by Dectin-1 and leading to more host immune responses [30,31]. Antifungal compounds, such as caspofungin and gepinacin, can also cause the exposure of β-(1,3)-glucan in C. albicans and elicit a stronger host immune response [29,54]. Our results demonstrate that inhibition of α-1,6-mannose backbone extension by MNN10 deletion could unmask the concealed β-(1,3)-glucan in either yeast or hyphal form (Figs 2B, 2E and S4B). The exposure of β-(1,3)-glucan may be due to the fact that the outer structure of cell surface do not adequately conceal the inner layer. The enhanced inflammatory responses stimulated by mnn10 mutant, including inflammatory signaling activation and cytokine secretion, were markedly down-regulated in Dectin-1-deficient macrophages, suggesting that they were Dectin-1 dependent (Fig 9A and 9B). Moreover, our study indicates that mnn10 mutant restored its pathogenicity in Dectin-1-deficient mice, further confirming our hypothesis (Fig 9D and 9E). While mnn10 mutant strain also stimulated enhanced flow cytometry. Data are representative images of five mice. MCP-1, chemokine CCL2; MIP-1α, chemokine CCL3; GM-CSF, granulocyte-monocyte colonystimulating factor; G-CSF, granulocyte colony-stimulating factor. *, P < 0.05; **, P < 0.01 [Mann-Whitney nonparametric t-test (B, C)].
Previous studies have reported that deficiency of Och1 or Mnn2 family involved in mannan biosynthesis of C. albicans could also lead to β-glucan exposure and decreased mannan [9,11]. However, Och1 and Mnn2 mutant elicited a host reduced immune response. As we know, PRRs can bind to short oligosaccharides and the precise carbohydrate epitopes to elicit antifungal immunity. We believe these discrepancies may be attributed to the differential effect of these genes on the mannan structure and the precise exposed carbohydrate epitopes of β-glucan. In addition, these previous studies concluded that decreased mannan, rather than β-glucan exposure, was the major PAMP recognized by host immune system in Och1 and Mnn2 mutant [9,11]. The present study not only indicates that deletion of MNN10 in C. albicans results in decreased mannose content and a more remarkable exposure of β-(1,3)-glucan on the cell surface, but also suggests that β-(1,3)-glucan of mnn10 mutant strain is the major PAMP and induced enhanced Dectin-1 dependent immune response.
In conclusion, we first identified that Mnn10 as an α-1,6-mannosyltransferase, which is involved in the cell wall α-1,6-mannose backbone extension and maintained pathogenicity of C. albicans by evading host Dectin-1 mediated antifungal immunity. In addition, there are no mammalian homologs of Mnn10 protein, thus our results provide a new potential antifungal therapeutic strategy for modulating the host immune response to C. albicans. generations on the C57BL/6 background) and Dectin-2-deficient (Clec4n -/-) mice were kindly provided by Dr. Yoichiro Iwakura (C57BL/6 background) [55,56].
To construct MNN10 null mutant strain (mnn10Δ/Δ), the entire open reading frame of MNN10 was deleted from the parental strain SN152 by homologous recombination of auxotrophic markers HIS1 and LEU2 using a fusion-PCR-based strategy as previously described [57,58]. To construct MNN10 revertant strain (mnn10Δ/Δ::MNN10), the fusion fragment containing MNN10 ORF and C. albicans SAT1-flipper cassette was transformed into mnn10Δ/Δ and the SAT marker was subsequently looped out as described previously [59]. All of the strains and the primers used in this study were listed in S2 and S3 Tables.

Expression and purification of Mnn10 protein
The non-transmembrane region of C. albicans MNN10 encoding amino acid residues 70 to 335 was cloned into pMAL-p5X (NEB) including MBP tag. And then the plasmid was transformed into BL21 (DE3) pLysS cells for expressing MBP-fused Mnn10 protein. The transformants were cultured overnight at 37°C and diluted 1:100 in fresh LB culture. When the medium OD 600 was up to 0.6 at 37°C, IPTG at a final concentration of 0.1 mM was added and the cells were grown overnight at 16°C. MBP-fused Mnn10 protein was purified by amylose resin (NEB) according to the protocols as previously described [60]. The supernatant was passed through a 0.45 μm filter and bound to amylose resin by gravity flow. Unspecific proteins were washed off by applying 10 column volumes (CVs) of column buffer. The protein of interest was then eluted by elute buffer (column buffer added with 10 mM maltose). The eluate was finally dialyzed against buffer (25 mM Tris-HCl, pH7.5) for 2 h at room temperature.

Alcian Blue binding assay
The Alcian Blue binding assay was carried out as described previously [35]. 1.5×10 7 exponentially growing C. albicans cells were washed with 0.02 M HCl, and then incubated with 30 μg/ml Alcian Blue for 10 min at room temperature. The supernatant was measured at OD 600 and the concentration of Alcian Blue was determined by reference to a standard curve. The amount of dye bound to C. albicans cells were calculated by subtracting the amount of dye in the supernatant.

Cell surface hydrophobicity
Exponentially growing C. albicans cells were washed and resuspended in PBS buffer (OD 600 = 1.0), and 0.75 ml cyclohexane was then added to the above 3 ml cell suspension. The mixtures were vortexed for 3 min and settled for 20 min at room temperature, the OD 600 of the aqueous phase was measured. The relative hydrophobicity was measured as [(OD 600 of the control minus OD 600 after octane overlay)/OD 600 of the control] × 100% [63].

Isolation and analysis of cell wall proteins (CWPs)
The covalent and non-covalent CWPs were isolated as previously described [64,65]. The major types of covalently linked CWPs are glycosylphosphatidylinositol anchored proteins (GPI-APs) and proteins with internal repeats (Pir proteins). GPI-CWPs were released by resuspending the cell wall debris in undiluted HF-pyridine and incubated at 0°C for 3 h. Pir proteins were specifically released by incubating cell wall debris with 30 mM NaOH at 4°C for 16 h. The non-covalent CWPs of C. albicans were extracted by SDS buffer (50 mM Tris-HCl, 2% SDS, 100 mM EDTA, and 10 mM DTT, pH 8.0). The whole CWPs were then mixed and further digested by trypsin for the analysis of LC-MS/MS on high-resolution instruments (LTQ-Orbitrap XL and Velos, Thermo Fisher). Raw files were processed by MaxQuant soft for peptide/ protein identification and quantification.

Confocal laser scanning microscopy
To stain β-(1,3)-glucan of the cell wall, exponentially growing C. albicans yeast cells were washed in PBS (for hyphal form assays, 1×10 6 C. albicans cells were cultured in RPMI 1640 medium at 37°C for 3 h on a microscope slide in a six-well plate), and then incubated with anti-β-(1,3)-glucan antibody overnight at 4°C and then stained by Cy3-labeled antibody for 1 h at 30°C. To stain mannan and chitin of the cell wall, C. albicans yeast cells or hyphae were washed in PBS and incubated in the dark with 50 μg/ml ConA to stain for α-mannopyranosyl or 30 μg/ml CFW for chitin for 30 min. The above stained cells were washed and scanned at 63 × magnification with confocal laser scanning microscope (TCS SP5; Leica). Micrograph pictures were then acquired and analyzed by LAS AF Lite program.
Transmission electron microscopy 5×10 7 exponentially growing C. albicans cells were washed in PBS and then fixed in 4 ml fixative solution (3% paraformaldehyde, 3.6% glutaraldehyde, pH 7.2) for 24 h at 4°C. After postfixation of samples with 1% phosphotungstic acid for 2 h, they were washed by distilled water, block-stained with uranyl acetate, dehydrated in alcohol, immersed in propylenoxide, and embedded in glycide-ether. Ultrathin sections were observed under a transmission electron microscope (Hitachi H-800, Japan) at 120 kV.

Assessment of virulence in vitro
To measure the growth curve of C. albicans, exponentially growing cells were washed and resuspended in fresh YPD broth (OD 600 = 0.1), and then the optical density was determined at the indicated time point. To observe the hyphal growth, C. albicans cells were sub-cultured at 37°C in either RPMI 1640 medium plus 10% (vol/vol) heat-inactivated fetal calf serum (FCS) or spider solid medium.
Phospholipase activity and hemolytic activity of C. albicans strains were screened as described previously [66,67]. Briefly, the suspension of yeast cells were spotted on egg yolk agar or sugar-enriched sheep blood agar and incubated at 37°C for 3 days. The phospholipase activity of each strain was observed by measuring the width of zone of precipitation around the colony. The presence of a distinct translucent halo around the colony indicated positive hemolytic activity.

C. albicans invasion and epithelial cell damage assays
The Caco-2 or KB cells were grown as approximate 80%-90% confluent monolayer in MEM medium with 20% (vol/vol) heat-inactivated FCS. For SEM, the cells were grown on 8 mm diameter glass coverslips. Each coverslip was infected with 1×10 6 live C. albicans yeast cells. After 2 h of infection, the cells were gently washed with PBS prior to 1% OsO 4 and then examined using a XL-30 scanning electron microscope (Philips, Holland) as described previously [68].
For the cell damage assay, 80%-90% confluent monolayer of Caco-2 or KB cells was infected with 1×10 5 live C. albicans yeast cells for 12 h, respectively. Lactate dehydrogenase (LDH) in the medium released from control or infected epithelial cells was determined by LDH Assay kit (Beyotime, China) according to the manufacturer's instructions. Maximal LDH release was obtained by adding 0.1 ml of 1% Triton X-100 to each well and vigorously disrupting the epithelial layers 1 h before the end of incubation period. The relative LDH activity was measured as [(OD 490 of infected cells minus OD 490 of the control)/(OD 490 of maximal LDH release minus OD 490 of the control)] × 100%.

Neutrophils killing assay in vitro
The killing assay was carried out as described previously [70]. Thioglycollate-elicited peritoneal neutrophils were mixed with live C. albicans [multiplicity of infection (MOI) = 1:20] in a 24-well plate, and were kept for 1 h at 4°C to settle the cells before being transferred to 37°C for another 1 h. Control plates were kept in parallel at 4°C during the incubation. Then the cells were mixed and plated on SDA agar for counting live C. albicans colonies for 48 h at 30°C.
For analysis of reactive oxygen species (ROS), the inflammatory cells were co-cultured with C. albicans (MOI = 1) in RPMI medium containing 10 μM dihydrorhodamine 123 for 1 h at 37°C. After incubation the fluorescent intensity of the oxidized dihydrorhodamine 123 was measured by a multi-mode microplate reader (excitation wavelength, 485 nm; emission wavelength, 538 nm). Cells loaded with dihydrorhodamine 123 but not treated with C. albicans were used to assess background of ROS production. For analysis of myeloperoxidase (MPO), the neutrophils were lysed by 1% Triton X-100 for 10 min and the MPO activity in neutrophil lysates was measured using an enzyme assay as described previously [71].
Macrophage-C. albicans interaction C. albicans yeast cells or hyphae were exposed to four doses of 100,000 μjoules/cm 2 in a CL-1000 Ultraviolet Crosslinker (UVP), with agitation between each dose to treat cells evenly [29]. The thioglycollate-elicited macrophages were stimulated with the UV-inactivated C. albicans yeasts (MOI = 5) or hyphae (MOI = 1) for the indicated time. Macrophage phagocytosis assay was performed as previously described, exponentially growing C. albicans cells were washed in PBS buffer and added to the monolayer macrophages (MOI = 5) at the indicated time (30 min, 60 min, 90 min and 120 min). CFW staining was performed for C. albicans and PSA-FITC staining was performed for macrophages. CFW/PSA-FITC stained samples were scanned immediately at 63 × magnification with confocal laser scanning microscope. Micrograph pictures were acquired and analyzed by LAS AF Lite program.

Western blotting
The cells were lysed in lysis buffer (250 mM NaCl, 50 mM HEPES, 1 mM EDTA, 1% NP-40, protease inhibitors, pH 7.4) for total cell lysates. For nuclear extracts, cells were lysed in lysis buffer (10 mM KCl, 10 mM HEPES, 0.1 mM EDTA, 0.4% NP-40, protease inhibitors, pH 7.9). The nuclear pellets were harvested, washed with the lysis buffer and resuspended in the extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, protease inhibitors, pH 7.9), and then incubated with vortexing at 4°C for 30 minutes. The cell lysates were subjected to SDS-PAGE, blotted with the indicated primary antibodies and secondary antibodies, and then developed with the chemiluminescence method according to the manufacturer's instructions (Millipore) using the ECL detection system (GE Healthcare). The densitometry of indicated blot was quantified using Image J software (National Institutes of Health, USA).

Murine systemic candidiasis model
For the C. albicans infection in vivo, groups of C57BL/6 female mice or BALB/c female mice (6-8 weeks) were injected via lateral tail vein with 200 μl of a suspension containing indicated live C. albicans (5×10 5 cells for C57BL/6 mice and 3×10 5 cells for BALB/c mice) in sterile saline. Mice were monitored daily and were killed after 2 days or 5 days of infection. The kidneys and livers were removed, and then homogenized in 0.5 ml PBS for fungal burdens measurement or fixed in 10% neutral formalin for H&E and PAS staining. Supernatants of kidney and livers homogenates were harvested and stored at -80°C for the measurement of cytokine production.
Cellular inflammation assay in the kidney C57BL/6 mice were injected with 5×10 5 CFU C. albicans and sacrificed at 12 h, 1 day, 2 days, 3 days, 4 days or 5 days post-infection and the kidneys were removed. The kidneys were minced into tissue pieces and digested for 1 h at 37°C. Then the digested tissues were passed through a 70-mm filter, washed and centrifuged in a 40%/70% percoll gradient for leukocytes isolation [72]. The leukocytes at the interphase were then analyzed by flow cytometry (BD FACS).

Murine peritoneal infection model
C57BL/6 mice were injected intraperitoneally with 5×10 5 CFU C. albicans and were killed after 4 h or 2 days. The peritoneal infiltrate was collected by lavage with ice-cold PBS containing 0.5 mM EDTA, and then the red blood cells were lysed. The inflammatory cells were counted and blocked with PBS containing 5% heat-inactivated FCS and 1 mM sodium azide at 4°C. The populations of the cells were analyzed by flow cytometry to determine the leukocyte composition as described before [22].

Statistical analysis
At least three biological replicates were performed for all experiments unless otherwise indicated. Log-rank test was used to evaluate the equality of survival curves. The two-tailed Student's t-test was used for analysis of two groups and multiple groups were analyzed by one-way analysis of variance with Bonferroni post-tests. For analysis of nonparametrically distributed data, the Mann-Whitney test or Kruskal-Wallis test was used. Statistical significance was set at a p-value in the figures as: Ã , P < 0.05; ÃÃ , P < 0.01. in the spleen of SN152 or mnn10 mutant strain infected mice. C57BL/6 mice were infected with 5×10 5 CFU of parental strain SN152 or mnn10 mutant strain via lateral tail vein (n = 5 per group). Intracellular cytokine IFN-γ and IL-17 from α/β or γ/δ T cells were analyzed after gated on CD3 + T cells, and intracellular cytokine signals from NK cells were analyzed after gated on CD3 -T cells. (TIF) S1