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Eukaryotic Cell, February 2006, p. 238-247, Vol. 5, No. 2
1535-9778/06/$08.00+0     doi:10.1128/EC.5.2.238-247.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

MNN5 Encodes an Iron-Regulated {alpha}-1,2-Mannosyltransferase Important for Protein Glycosylation, Cell Wall Integrity, Morphogenesis, and Virulence in Candida albicans

Chen Bai, Xiao-Li Xu, Fong-Yee Chan, Raymond Teck Ho Lee, and Yue Wang*

Institute of Molecular and Cell Biology, ASTAR Biomedical Sciences Institutes, 61 Biopolis Drive, Singapore 138673, Singapore

Received 13 September 2005/ Accepted 23 November 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans {alpha}-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both {alpha}-1,2- and {alpha}-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5{Delta} mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5{Delta} mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5{Delta} mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Candida albicans, an opportunistic human fungal pathogen, frequently causes life-threatening systemic infections in immunocompromised patients (3, 35). The worldwide appearance of drug-resistant strains and the limited options of anti-C. albicans drugs underscore an urgent need to identify new antifungal targets for drug development. Several virulence traits have been described for this pathogen, such as the transition between yeast and hyphal forms of growth (3, 41, 57), the production of lytic enzymes (23), phenotype switching (42), adherence to host cells (9), and iron acquisition (37). Since the cell wall provides functions at the interface between microbial pathogens and host cells, it often critically determines the outcome of host-pathogen interactions. The general structure of the fungal cell wall is conserved, containing an inner layer of structural polysaccharides, glucans, and chitin and an outer layer enriched for mannoproteins (25). The highly glycosylated mannoproteins are involved in host cell adhesion, antigenicity, and modulation of host immune responses (10, 46, 54). Previous studies showed that mannoproteins of the outer layer mediate direct interactions of C. albicans with host cells (11, 47) and play important roles in pathogenesis (8, 48).

Studies with Saccharomyces cerevisiae have provided much of our current understanding of protein glycosylation in fungi. Protein glycosylation starts in the endoplasmic reticulum, where the first mannose is transferred to the OH group of a serine or threonine residue in O-linked glycosylation (45) or an oligosaccharide core structure is attached to the NH2 group of an asparagine residue in N-linked glycosylation (26). Then the glycoproteins move to the Golgi apparatus, where the elongation of O-linked mannans and synthesis of complex N-linked glycans take place (13, 30). Many of the glycosylation steps in S. cerevisiae have been extensively characterized at the biochemical and genetic levels (7, 16), providing valuable knowledge for the understanding of protein glycosylation in C. albicans. However, to date, only a few C. albicans genes responsible for protein glycosylation have been studied in detail, including MNT1 (8) and PMT1, -2, -4, -5, and -6 (36, 39, 48, 49). These genes are members of the MNT and PMT families specifically involved in the O-glycosylation pathway. Gene deletion experiments demonstrated that the O-linked glycans are important for virulence (8, 39, 48, 49). MNN9 was found to have a role in extending N-linked glycan outer chains and maintaining normal cell wall composition (44). MNN4 is required for mannosylphosphate transfer to the acid-labile N-mannan side chains. An mnn4{Delta} mutant exhibited drastically reduced cell wall mannosylphosphate content, but its virulence and interaction with macrophages were not affected (20). CaVRG4 and CaSRB1 encode proteins required for supplying the Golgi with the mannose donor GDP-mannose and are essential in C. albicans, indicating the importance of overall protein glycosylation to cell viability (33, 55). The Golgi GDPase CaGDA1 has also been shown to be important in transporting GDP-mannose into the Golgi. A gda1 null mutant is viable but has defects in cell wall biogenesis, hyphal formation, and O-mannosylation (19). Some metal ions are known to regulate mannosyltransferase activity. Tkacz et al. (50) showed that manganese ions are an essential cofactor of Golgi-bound mannosyltransferases. CaPmr1 was recently found to pump Ca2+/Mn2+ ions into the Golgi. A Capmr1 null mutant showed defects in both O- and N-glycosylation, growth dependence on supplemented calcium after entering stationary phase, and attenuated virulence (2).

We previously cloned a C. albicans gene encoding a Golgi {alpha}-1,2-mannosyltransferase with highest sequence homology to S. cerevisiae Mnn5 and Mnn2 and thus named it CaMNN5 (1). In budding yeast, Mnn2 and Mnn5 are responsible for the branching and extension of the side chains of N-linked mannose oligomers (38). Deletion of MNN2 or MNN5 in S. cerevisiae results in shortened {alpha}-1,2-linked mannose branches, blocking the subsequent addition of mannosylphosphate (12). CaMNN5 was initially identified for its ability to support the growth of S. cerevisiae mutants severely defective in iron acquisition under iron-limiting conditions (1). Heterologous expression in S. cerevisiae revealed that CaMnn5 could enhance iron uptake and coprecipitate with 55Fe, an activity not found for any other fungal mannosyltransferases. These intriguing features of Mnn5 prompted us to further characterize its function and role in its natural host, C. albicans.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and growth conditions. The C. albicans and S. cerevisiae strains used for this study are listed in Table 1. The strains were routinely grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose), GMM medium (yeast nitrogen base without amino acids and with 2% glucose), or GMM medium supplemented with required amino acids. For yeast growth in liquid media, all strains were grown at 30°C. Iron-depleted media were prepared by adding 100 µM bathophenanthroline sulfate (BPS). Solid, hypha-inducing media were prepared as described previously (57).


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  		TABLE 1. Yeast strains used for this study

 
CaMNN5 complementation of S. cerevisiae mnn2{Delta} and mnn5{Delta} mutants. The coding sequence of CaMNN5 was cloned into an S. cerevisiae 2µm plasmid (pRS426) (37) under the control of the ADH1 promoter and transformed into S. cerevisiae mnn2{Delta} and mnn5{Delta} mutants.

Alcian blue binding assay. The Alcian blue binding assay was carried out using the method of Herrero et al. (19). A series of solutions containing different amounts of Alcian blue (Sigma) was prepared in 0.02 N HCl, and the optical density at 600 nm (OD600) of each solution was determined. A standard curve was plotted of the OD values versus amounts of Alcian blue. To quantify Alcian blue binding to the cell surface, 500 µl exponential-phase yeast cells (OD600, 1) was centrifuged, and the cells were washed once with 2 ml of 0.02 N HCl and resuspended in 1 ml 0.02 N HCl containing 50 µg Alcian blue. The cell suspension was allowed to stand for 10 min at room temperature and then centrifuged for 2 min to pellet the cells. The OD600 of the supernatant was measured. The amount of Alcian blue was determined by using the standard curve. The amount of dye bound to the cells was calculated by subtracting the amount of dye in the supernatant from 50 µg.

Expression and purification of GST-Mnn5 fusion protein. The region of C. albicans MNN5 encoding amino acid residues 211 to 597 was cloned into pGEX-4T-1 (Amersham) in frame with the glutathione S-transferase (GST) gene and transformed into Escherichia coli strain BL21 (Amersham). Purification of the GST-Mnn5 fusion protein was performed by following standard protocols (15).

Preparation of polyclonal antibodies. Two rabbits (New Zealand White) were purchased from the Sembawang Laboratory Animals Centre (Singapore). Each rabbit was injected with 500 µg purified Mnn5 emulsified in complete Freund's adjuvant (Sigma). Booster injections using the same amount of antigen emulsified in incomplete Freund's adjuvant (Sigma) were administered every 2 weeks. The rabbits were bled 10 days after the fifth injection and after each subsequent boost. Affinity purification of Mnn5-specific antibodies was performed using the purified GST-Mnn5 fusion protein coupled to glutathione-Sepharose 4B beads. The serum was incubated with 1 ml of the beads at 4°C for 3 h. The beads were then washed extensively with cold phosphate-buffered saline (PBS) (4°C), and the bound antibodies were eluted with ImmunoPure immunoglobulin G elution buffer (Pierce).

Western blot analysis. Protein concentrations were determined by the Bradford assay (Bio-Rad). Samples for Western blot analysis were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a Hybond-C nylon membrane (Amersham). The purified anti-Mnn5 antibodies described above were used as the primary antibody. An ECL Western blot detection kit (Pierce) was used to visualize the target protein.

Mnn5 expression in Pichia pastoris and FPLC purification of Mnn5. The expression of C. albicans Mnn5 using P. pastoris was done as previously described (1). To purify the secreted Mnn5 protein, the culture was centrifuged to pellet the cells, and the supernatant was concentrated by passage through a Centriplus spin filter (molecular size cutoff, 10 kDa) and washed three times with and then resuspended in a small volume (~1/10 the original volume) of 20 mM Tris-HCl (pH 8) before being subjected to fast-performance liquid chromatography (FPLC) separation using a Mono-Q column (10 x 100 mm; Amersham). A KCl gradient, which was increased from 0 to 0.8 M from 0 to 50 min, was used to elute the protein. Fractions (4 ml/min) were collected for SDS-PAGE and Western blot analysis to locate Mnn5. The elution buffer of the fraction containing Mnn5 was replaced with 20 mM phosphate buffer (pH 6.0) by using a Centriplus filter as described above.

Assay of Mnn5 enzyme activity. The enzyme assay was carried out by using 10 µl (8 µg) of purified Mnn5 as the enzyme source in a buffer containing 50 mM Tris-Cl (pH 7.2), 10 mM MnCl2, 0.23 µM GDP-[14C]mannose (specific activity, 289 mCi/mmol), 20 mM 2- or 6-O-{alpha}-D-mannopyranosyl-D-mannopyranose acceptor (Sigma), and 5 mg/ml bovine serum albumin. Standard reactions were performed in 50 µl at 30°C for 60 min. To terminate the reaction, the mixtures were passed through 0.8 ml QAE-Sephadex anion-exchange resin to remove the unlabeled GDP-mannose. The labeled product was eluted with 0.8 ml water, and radioactivity was counted in a scintillation beta counter (Amersham).

Gene deletion. The MNN5 gene was deleted from CAI4 by using the URA blaster method (14), which is schematically illustrated in Fig. 3A. A pair of oligonucleotides (5'-CAATTGCCCAGTTCTACA-3' and 5'-AACATGCGTCTTCTTGAA-3') was used to PCR amplify the region upstream of the coding region of MNN5, and other primers (5'-CGGCGAATGATTATGAGA-3' and 5'-CGCCCAAAGAGGTTTATG-3') were used for the region downstream of MNN5. S. cerevisiae mnn2{Delta} and mnn5{Delta} mutants were derived from strain CRY2{alpha}. A gene deletion cassette was constructed by flanking LEU2 or HIS3 with two DNA fragments corresponding to the 5'- and 3'-untranslated regions of the target gene. The following pairs of primers were used to PCR amplify the 5'- and 3'-flanking fragments of MNN2 and MNN5. For the MNN2 5' fragment, 5'-GAAGCATTCGTTATCAGG-3' and 5'-CCTACCTACACTGATTTG-3' were used; for the MNN2 3' fragment, 5'-CAACGGGTCGGACTATAG-3' and 5'-GCATTCGAATTCCTTAAC-3' were used; for the MNN5 5' fragment, 5'-CTATCTCCCACATGTTGGC-3' and 5'-CCTGAACTATTAGTATATC-3' were used; and for the MNN5 3' fragment, 5'-GAGAAAAACTAATGAAGTG-3' and 5'-GAATTGCTGTGCAGAAAC-3' were used. Appropriate restriction sites were added to the PCR primers when needed. Transformants were selected on histidine- or leucine-dropout medium according to the selection marker used. Correct deletion of each gene was verified by Southern blotting (data not shown).


Figure 3
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  		FIG. 3. Chromosomal deletion of MNN5. (A) Schematic description of the strategy used to knock out (KO) the first and second copies of MNN5. The first base of the start codon ATG is designated nucleotide 1. The restriction sites for HindIII and SpeI are shown for the Southern blotting analysis below. (B) Strategy for reintegrating a copy of wild-type MNN5 into the KO mutants. A plasmid was constructed containing the MNN5 coding region with ~1,500 bp of the 5'- and 600 bp of the 3'-untranslated region and the hisG sequence. A StuI restriction site was introduced into hisG by site-directed mutagenesis. This plasmid was linearized by StuI and transformed into the mnn5{Delta} mutant for site-specific integration at the genomic locus, where hisG replaced MNN5. (C) Southern blotting confirmation of MNN5 gene deletion and reintegration. Genomic DNA samples were digested with HindIII and SpeI. The region corresponding to nucleotides –236 to –644 of the MNN5 promoter region was PCR amplified and labeled with 32P for use as a probe. Lane 1, CAI4; lane 2, CaWYBC1; lane 3, CaWYBC1.1; lane 4, CaWYBC2; lane 5, CaWYBC2.1; lane 6, CaWYBC1.2; and lane 7, CaWYBC2.2.

 
O-glycan analysis. Total cellular O-glycans were analyzed as described previously (18), with a minor modification. To radioactively label mannans, the yeast cells growing in 2 ml of YP plus 0.5% sucrose were incubated with 1.85 MBq of [2-3H]D-mannose (777 GBq mmol–1; Perkin-Elmer) at 30°C for 2 h. Cells were then pelleted, resuspended in 300 µl PBS, and lysed by bead beating as previously described (1). The cell wall was then pelleted by centrifugation and lyophilized. Equal amounts of the lyophilized cell walls from different strains were treated with 0.1 M NaOH for 24 h at room temperature for ß-elimination of O-glycans. The reaction was terminated by adding HCl to a final concentration of 0.1 M, and the mixture was then centrifuged at 80,000 rpm using a TLA-100.2 rotor (Beckman) for 2 h at 4°C. 3H-labeled glycans in the supernatant were subjected to thin-layer chromatography (TLC) on Silica Gel 60G plates (Merck) in ethyl acetate-butanol-acetic acid-water (3:4:2.5:4) with two ascents. The chromatograms were then treated with EN3HANCE reagent (Perkin-Elmer) and exposed to Kodak X-OMAT X-ray film at –70°C. To quantify each labeled species, a 1-cm by 0.5-cm area of the silica plate containing each species was cut out and subjected to scintillation counting of 3H using a scintillation beta counter (Amersham).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of MNN5 in C. albicans. Since MNN5 expression can rescue the iron acquisition defects of an S. cerevisiae ftr1{Delta} mutant (1), we first wanted to determine whether MNN5 overexpression will do the same for a C. albicans ftr1{Delta} mutant. We used several relatively strong promoters, such as the GAL1-10, ADH1, and MET3 promoters, to drive MNN5 expression from either a single copy integrated at the RP10 locus or a multiple-copy plasmid (pABSK1), but none of these constructs was able to alleviate the growth deficiency of the C. albicans ftr1{Delta} mutant (data not shown) under iron-limiting conditions. We then examined whether MNN5 expression in C. albicans cells is regulated by iron and found that neither the mRNA nor the protein level of MNN5 was affected by iron concentrations (data not shown).

CaMNN5 rescues the defect of an S. cerevisiae mnn5{Delta} mutant but not an mnn2{Delta} mutant. Since CaMnn5 shares high sequence homology with two S. cerevisiae mannosyltransferases, Mnn5 and Mnn2, we wanted to know whether CaMNN5 can functionally complement an S. cerevisiae mnn2{Delta} or mnn5{Delta} mutant. Negatively charged mannophosphate units are known to be incorporated into N-linked oligosaccharides of mannoproteins, and thus defects in N-glycosylation are often associated with a decrease in the cell's affinity for the cationic dye Alcian blue (31, 34). Both mnn2{Delta} and mnn5{Delta} mutant strains showed a significant loss of Alcian blue binding (Fig. 1) (39). When a copy of CaMNN5 driven by the ADH1 promoter in a 2µm plasmid was transformed into the two mutants, Alcian blue binding was significantly improved in mnn5{Delta} but not in mnn2{Delta} mutant cells, suggesting that CaMNN5 may be the counterpart of S. cerevisiae MNN5 and may provide similar functions in C. albicans.


Figure 1
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  		FIG. 1. CaMNN5 restores Alcian blue binding in an S. cerevisiae mnn5{Delta} mutant. The assay was done as described in Materials and Methods. The amount of Alcian blue bound to the cells of a strain is shown as a percentage of that bound to the wild-type cells (CRY2{alpha}). The strains used were CRY2{alpha}, ScWYBC8 (mnn2{Delta}), ScWYBC8.1 (mnn2{Delta} CaMNN5), ScWYBC9 (mnn5{Delta}), and ScWYBC9.1 (mnn2{Delta} CaMNN5). All assays described throughout the paper were done three times, and standard deviations are shown.

 
Mnn5 has {alpha}-1,2-mannosyltransferase activity. To analyze the enzymatic activity of C. albicans Mnn5, we used a P. pastoris expression system to produce soluble, secreted protein. Figure 2A (lane 2) shows that the culture supernatant of the P. pastoris strain expressing MNN5 contained a major 64-kDa protein that matches the predicted size of Mnn5 but was not present in the culture supernatant of the strain transformed with the empty vector (Fig. 2A, lane 3). To purify the protein, the supernatant was subjected to FPLC using a Mono-Q anion-exchange column and eluted with a KCl gradient. SDS-PAGE and Western blot analysis were performed to locate the fractions that contained Mnn5. The purified protein (Fig. 2A, lane 4) was subjected to a mannosyltransferase activity assay. Figure 2B (top) shows that the purified Mnn5 protein catalyzed the transfer of [14C]GDP-mannose to both {alpha}-D-1,6- and {alpha}-D-1,2-mannobiose, with similar activities. We also found that Mnn5 exhibited high activities at pH 6.0 and 9.0 under the assay conditions used (Fig. 2B, bottom).


Figure 2
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  		FIG. 2. Expression, purification, and enzyme activity of CaMnn5. (A) SDS-PAGE (a) and Western blot analysis (b) of Mnn5 expressed in P. pastoris. Mnn5 expression in P. pastoris and FPLC purification of Mnn5 were carried out as described in Materials and Methods. Lane 1, molecular weight markers; lane 2, 20 µl of culture supernatant of the P. pastoris strain expressing Mnn5; lane 3, 20 µl of supernatant of the P. pastoris strain (KMvec) transformed with an empty vector; lane 4, FPLC-purified Mnn5. The Western blot was probed by using an anti-Mnn5 antibody as the primary antibody. (B) Mannosyltransferase assay (a) of Mnn5. The assay was performed at pH 6.0 as described in Materials and Methods. [14C]GDP-mannose was used as the mannose donor, and {alpha}-1,2- and {alpha}-1,6-mannobiose were used as acceptors. Purified Mnn5 (8 µg) was used in each reaction. The same amount of bovine serum albumin was used as a negative control. The radioactive products were isolated for quantification in a scintillation counter, expressed as counts per minute (cpm)/mg of protein/h. The pH dependence of Mnn5 activity (b) was evaluated using the same protocol, except with reaction buffers at different pHs. The same pattern was obtained using either {alpha}-1,2- or {alpha}-1,6-mannobiose as the acceptor. (C) Test of metal ion requirement for Mnn5's enzymatic activity. The same assay protocol was used as that described for panel B, except that the reaction buffer (pH 6.0) contained a 0.1, 1, or 10 mM concentration of one of the divalent metal ions, as indicated. (D) Ferrous iron regulates Mnn5 activity in the presence of Mn2+. The assay buffer (pH 6.0) contained 10 mM Mn2+ and was supplemented with different amounts of BPS and ferrous ammonium sulfate, as indicated at the bottom of the graph.

 
Mnn5 requires Mn2+ and Fe2+ for activity. Several Golgi mannosyltransferases are known to require Mn2+ for activity (24, 38, 56). To test the metal ion requirements of Mnn5, the transferase activity was assayed in buffers supplemented with various divalent metal ions and chelators. We used purified Mnn5 as the enzyme source and {alpha}-1,2-mannobiose as the acceptor for [14C]GDP-mannose. First, a metal ion such as Mn2+, Fe2+, Mg2+, Zn2+, or Cu2+ was included in the reaction buffer at a range of concentrations from 0.1 to 10 mM. Figure 2C shows that when added alone, the only divalent metal ion supporting high enzyme activity was Mn2+, while all the other ions tested had little effect, consistent with a role of Mn2+ as an essential cofactor. Since iron was previously found to bind to Mnn5 (1), we tested whether it was required for the enzymatic activity of Mnn5 in the presence of Mn2+. Adding different amounts of the iron chelator BPS to the reaction buffer was found to inhibit the enzyme activity in a concentration-dependent manner (Fig. 2D). In the presence of 0.1, 0.2, and 0.4 mM BPS, the enzymatic activity was lowered to 50, 30, and 18% that of the nonchelated control, respectively, suggesting that iron is required for the mannosyltransferase activity of Mnn5. This observation was further substantiated by the restoration of the enzymatic activity when Fe2+ was added to the buffer containing 0.4 mM BPS. Adding Fe2+ to the reaction buffer without BPS slightly enhanced the enzyme activity when the concentration of the added iron was within 2 to 10 µM, suggesting that the reaction mix initially contained sufficient iron. The ferric iron chelator desferri-ferrichrome had no effect on Mnn5 activity (data not shown), suggesting that ferrous but not ferric iron is required. Adding other metal ions, such as Cu2+, Mn2+, and Mg2+, did not relieve the inhibition by BPS. Depleting Cu2+ by adding the chelator 2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulphonic acid (BCS) also had no effect on the enzyme activity (data not shown). Taken together, the results indicate that Mn2+ is an essential cofactor for the mannosyltransferase activity of Mnn5 and that the activity is regulated by ferrous iron, consistent with its iron-binding ability detected in our previous study (1).

Construction of MNN5 deletion mutant. To investigate MNN5's cellular functions in C. albicans, we used two different gene deletion cassettes, hisG-URA3-hisG and cat-URA3-cat, to sequentially delete the first and second copies of MNN5 from strain CAI4, as described previously (14, 22) (Fig. 3A). Figure 3B shows the strategy for reintroducing a wild-type MNN5 copy into the mutants by integration at the hisG locus. The genotypes of the heterozygous and homozygous mutants and reintegrants were verified by Southern blot analysis (Fig. 3C).

We determined the growth rate of the mnn5{Delta} mutant over a wide range of iron concentrations but did not observe a significant difference in comparison with the wild-type strain (data not shown). Also, the mnn5{Delta} mutant did not exhibit reduced 59Fe uptake under all conditions used (data not shown). The results indicate that unlike its function in the heterologous host S. cerevisiae, Mnn5 may not be involved in iron uptake in its natural host (see Discussion).

Study of Mnn5's role in N-linked and O-linked glycosylation. The S. cerevisiae mnn5{Delta} mutant is impaired in N-linked glycosylation. To test whether Mnn5 is involved in N-glycosylation, we performed an Alcian blue binding assay on mnn5{Delta} mutant cells. The assay showed drastically decreased binding of the dye to mnn5{Delta} cells compared to that for the wild-type cells (Fig. 4A). Approximately 90% of the dye coprecipitated with the wild-type cells, in contrast to only 20% with mnn5{Delta} mutant cells. Reintroducing a wild-type copy of MNN5 into mnn5{Delta} cells restored the dye binding activity. We previously showed that mutation of either one of the aspartic acid (D) residues to alanine (A) in the conserved DXD282-284 motif of Mnn5 totally abolished the mannosyltransferase activity (1). Reintroducing a mutated mnn5 gene (D282A or D284A) into the mnn5{Delta} mutant did not restore cell binding of the dye (Fig. 4A), indicating that Alcian blue binding ability is dependent on the enzyme activity of Mnn5. Taken together, the results show that the amount of cell surface mannosylphosphate is significantly reduced in the mnn5{Delta} mutant, which is most likely the consequence of blocked synthesis of N-linked mannan branches.


Figure 4
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  		FIG. 4. Investigation of Mnn5's role in N-linked and O-linked glycosylation. (A) The mnn5{Delta} mutant exhibited greatly reduced Alcian blue binding. The following strains were included in the assay, as described in Materials and Methods: SC5314, CaWYBC2 (mnn5{Delta}), CaWYBC2.2 (mnn5{Delta} MNN5), CaWYBC2.3 (mnn5{Delta} mnn5D282A), and CaWYBC2.4 (mnn5{Delta} mnn5D284A). (B) ß-Elimination of O-glycans. The mannans of SC5314 (WT) and CaWYBC2 (mnn5{Delta}) were labeled with 3H by adding [2-3H]mannose to the growth medium. The base-sensitive O-glycans were released by ß-elimination, separated by thin-layer chromatography (ethyl acetate-butanol-acetic acid-water [3:4:2.5:4]), and visualized by subjecting the TLC plate to autoradiography. The different species of mannose oligomers, mannose (M1) to pentamannose (M5), are shown. The N-glycans were base resistant and thus were retained at the origin of sample loading. (C) An area (1 cm x 0.5 cm) of the TLC plate containing each mannose species was cut out for scintillation counting. The relative amount of each mannose species (3H count) is shown as a percentage of the total count for M1 to M5.

 
To determine whether the mnn5{Delta} mutant also has defects in O-glycosylation, we grew cells in the presence of [2-3H]mannose to label the mannose units of glycosylated proteins. The O-linked mannans were released by ß-elimination and resolved by TLC. The ß-elimination condition used was previously shown to completely release O-glycans from mannoproteins (18). An autoradiograph of the TLC plate (Fig. 4B) displays five major 3H-labeled molecules containing from one to five mannose residues (M1 to M5), as previously shown by Herrero et al. (19). When the amounts of each 3H-labeled species were compared between the wild type and the mnn5{Delta} mutant, we found that the mnn5{Delta} mutant showed a markedly higher level of M1 and less M2, M3, and M5 than the wild type, suggesting an impaired ability of the mutant to extend O-linked mannan chains. The area on the TLC plate corresponding to each radioactive spot was cut out and subjected to quantification by scintillation counting of 3H (Fig. 4C). M1 was found to represent 16.6% of the total 3H count for all five species (M1 to M5) in the wild-type strain, whereas the percentage increased to 46.6% in the mutant, showing that a large fraction of the first O-linked mannose residue cannot be further extended in the absence of Mnn5. The radioactive signal at the origin of sample loading should largely represent N-glycans, which are resistant to ß-elimination and remain attached to the proteins. Quantification of the 3H-labeled glycans at the origin showed a 50% reduction in the mnn5{Delta} mutant in comparison with the wild-type strain. These results indicate that Mnn5 is involved in extending O-linked, and likely also N-linked, mannans.

The mnn5{Delta} mutant exhibits markedly reduced sensitivity to LF. Lactoferrin (LF) is a mammalian iron-binding protein abundantly present in saliva, milk, and other exocrine secretions. It constitutes an important part of innate immunity, partly because its strong iron-chelating activity effectively removes free iron from the environment that is needed for the growth of nearly all microorganisms (40). In particular, LF and some LF-derived peptides are known to inhibit the growth of C. albicans under certain cultural conditions (27, 53). We routinely test C. albicans mutants defective in iron metabolism for their sensitivity to LF. An established protocol (43) involves the treatment of C. albicans cells with 100 µg LF in 1 ml of 0.05 mM KCl for 75 min at 37°C before scoring the CFU on iron-sufficient plates. A mock treatment of each strain without adding LF was done in parallel as a control. Figure 5 shows that if the cells were pregrown in GMM medium, ~5% of the wild-type cells survived the LF treatment, whereas if they were pregrown in iron-depleting medium, ~20% of the wild-type cells survived. This observation suggests that the level of cellular iron is a determinant of a cell's sensitivity to LF. Strikingly, the mnn5{Delta} mutant exhibited markedly reduced LF sensitivity. Approximately 33% of the mutant cells pregrown in GMM and 50% of those pregrown in the iron-limiting medium survived the LF treatment. Reintroducing a mutated mnn5 gene (D282A or D284A) into the mnn5{Delta} mutant did not restore sensitivity to LF (Fig. 5), indicating that the mannosyltransferase activity is required for Mnn5-mediated LF killing of C. albicans. For comparison, reintroducing a wild-type copy of MNN5 into the mnn5{Delta} mutant restored LF sensitivity. The results reveal that Mnn5 has a role in determining a cell's sensitivity to LF which depends on cellular iron homeostasis. Is the increased resistance to LF killing a unique phenotype of the mnn5{Delta} mutant or one shared by other mannosyltransferase mutants? To answer this question, we tested the LF sensitivity of a series of mannosyltransferase mutants, including pmt1{Delta}, pmt2{Delta}/PMT2, pmt4{Delta}, pmt5{Delta}, pmt6{Delta}, mnt1{Delta}, and mnt2{Delta} mutants (8, 32, 36). We found that only the mnn5{Delta} mutant exhibited increased resistance to LF killing (Table 2), indicating a unique role of Mnn5 in mediating LF killing of C. albicans.


Figure 5
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  		FIG. 5. LF sensitivity assay. The following strains were used: SC5314, CaWYBC2 (mnn5{Delta}), CaWYBC2.2 (mnn5{Delta} MNN5), CaWYBC2.3 (mnn5{Delta} mnn5D282A), and CaWYBC2.4 (mnn5{Delta} mnn5D284A). Each strain tested was grown in iron-sufficient (GMM) or iron-limiting medium (GMM plus 100 µM BPS) to mid-log phase before being treated with 100 µg LF in 1 ml of 0.05 mM KCl (pH 7.0). The cells were incubated in a shaking water bath at 37°C for 75 min. Cells in KCl buffer without LF were included as a control. The number of viable cells was enumerated by plating the LF-treated cells on YPD plates and counting the CFU after a 2-day incubation at 30°C. LF sensitivity is presented as CFU of LF-treated cells, expressed as a percentage of the CFU of the untreated control.

 

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  		TABLE 2. Sensitivity of mannosyltransferase mutants to LF, Congo red, and hygromycin B

 
The mnn5{Delta} mutant shows a weakened cell wall. For both S. cerevisiae and C. albicans, the deletion of genes encoding mannosyltransferases has been reported to cause defects in cell wall integrity. To determine whether the mnn5{Delta} mutant has a similar defect, we tested the sensitivity of the mnn5{Delta} mutant to lyticase, an enzyme normally used to remove the fungal cell wall. Exponential-phase cells grown in YPD were diluted to an OD600 of 0.6 in a hypo-osmotic solution containing 10 mM Tris and 0.2 mg/ml lyticase. The extent of cell lysis was monitored by measuring the OD600 of the cell suspension at timed intervals. Figure 6A shows that the mnn5{Delta} mutant is much more sensitive to damage by lyticase than the wild-type strain. By 1 h, the OD600 of the mnn5{Delta} cell suspension had dropped from 0.58 to 0.2. Microscopic examination of the cell suspension revealed that >90% of the mutant cells had been lysed (data not shown). In contrast, the OD600 of the wild-type cells was 0.52 by 1 h and dropped to 0.2 by 5 h. The heterozygous MNN5/mnn5{Delta} mutant strain displayed an intermediate sensitivity to lyticase, indicating a gene dosage effect.


Figure 6
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  		FIG. 6. The mnn5{Delta} mutant is hypersensitive to lyticase, Congo red, and hygromycin B. (A) Lyticase sensitivity assay. The following strains were included in the assay: SC5314, CaWYBC1 (MNN5/mnn5{Delta}), CaWYBC1.2 (MNN5/mnn5{Delta} MNN5), CaWYBC2 (mnn5{Delta}), and CaWYBC2.2 (mnn5{Delta} MNN5). Exponential-phase yeast cells of the strains tested were diluted to an OD600 of 0.6 in 10 mM Tris-HCl (pH 7.5) containing 0.2 mg/ml lyticase. The cell suspension was incubated at 30°C, and the OD600 was taken at timed intervals. Congo red (B) and hygromycin B (C) sensitivity assays were also performed. Exponential-phase yeast cells of the strains shown were serially diluted and dropped onto agar plates containing either 200 µg/ml Congo red or 200 µg/ml hygromycin B. The plates were incubated at 30°C for 3 days.

 
We also tested the sensitivity of the mutant to the cell wall construction inhibitor Congo red and found that the mnn5{Delta} mutant exhibited very high sensitivity to Congo red. The heterozygous MNN5/mnn5{Delta} mutant already showed significantly increased Congo red sensitivity compared with the wild-type strain (Fig. 6B). This sensitivity was further increased in the homozygous mutant. Reintroducing a wild-type copy of MNN5 into the mutants partially restored the resistance to Congo red. Again, a gene dosage effect was observed, and it appears that the severity of Congo red damage is more sensitive to the cellular level of Mnn5. Previous reports described that some pmt mutants are supersensitive to the antifungal drug hygromycin B (36, 49). We found that the mnn5{Delta} mutant also showed a significantly higher sensitivity to hygromycin B than the wild type, in a gene dosage-dependent manner (Fig. 6C).

We also compared the sensitivities of the mnn5{Delta} mutant to Congo red and hygromycin B with those of other mannosyltransferase mutants and found that several mutants, such as mnn5{Delta}, pmt1{Delta}, and pmt2{Delta}/PMT2 mutants, exhibited increased sensitivity to both compounds (Table 2), suggesting that these phenotypes may be a general consequence of compromised cell wall integrity. The results of Congo red and hygromycin B sensitivity tests with the PMT and MNT mutants are consistent with previous reports (8, 32, 36).

The mnn5{Delta} mutant is defective in hyphal morphogenesis. C. albicans mutants defective in O-glycosylation, such as pmt1{Delta} and pmt6{Delta} mutants, were shown to be partially impaired in hyphal morphogenesis (48, 49). We examined the hyphal development of the mnn5{Delta} mutant in several common hypha-inducing media. The mnn5{Delta} mutant strain showed normal hyphal development in all the liquid inducing media used but no or poor hyphal growth on some solid inducing media, such as RPMI, serum, Spider, and embedding media (Fig. 7).


Figure 7
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  		FIG. 7. The mnn5{Delta} mutant is defective in hyphal growth on some solid media. Yeast cells of strains SC5314 and CaWYBC2 (mnn5{Delta}) were inoculated onto several hypha-inducing solid media, as indicated, and incubated at 37°C for 3 days.

 
MNN5 is required for C. albicans virulence. Next, we used the mouse systemic infection model to evaluate the importance of MNN5 in C. albicans virulence. Each mouse was injected through the tail vein with 1 x 106 yeast cells of either a wild-type strain (SC5314), the mnn5{Delta} mutant, or the reintegrant strain CaWYBC2.2. Previous tests had shown that all three strains grew at nearly identical rates in YPD and GMM media (data not shown). Injection with SC5314 killed all animals within 1 week, whereas 80% of the mice injected with the mnn5{Delta} mutant survived the full 35-day course of observation. The mice injected with the reintegrant strain showed a mean survival time of 10 days (Fig. 8), again indicating a gene dosage effect. The results show that MNN5 is required for the virulence of C. albicans in this infection model.


Figure 8
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  		FIG. 8. The mnn5{Delta} mutant showed markedly reduced virulence. Strains SC5314, CaWYBC2 (mnn5{Delta}), and CaWYBC2.2 (mnn5{Delta} MNN5) were grown in GMM at 30°C to exponential phase. A 200-µl volume of 1 x 106 cells of each strain in PBS was injected into each animal through the tail vein. Eight mice were used for each strain, and their survival was monitored daily for 35 days. The experiment was done twice, with similar results.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CaMnn5 shares nearly equal sequence similarities with S. cerevisiae Mnn5 and Mnn2, but our complementation test showed that CaMNN5 restored Alcian blue binding to the cell surface of mnn5{Delta} but not mnn2{Delta} mutant cells, suggesting that CaMNN5 may be the functional counterpart of MNN5. Camnn5{Delta} and mnn5{Delta} mutants share a range of similar defects, such as a reduction of cell surface binding of Alcian blue and hypersensitivity to cell wall-damaging agents. The C. albicans mnn5{Delta} mutant exhibited defects in O-linked glycosylation, as ß-elimination of O-linked glycans yielded a dramatic increase in the mannose monomer (M1) and a decrease in mannose oligomers (M2, M3, and M5) in comparison with the wild-type strain. The mnn5{Delta} mutant may also be defective in N-linked glycosylation, as evidenced by the decreased Alcian blue binding and the amount of ß-elimination-resistant glycans (Fig. 4). In addition to the defects commonly associated with mutants of mannosyltransferase genes in yeast, the mnn5{Delta} mutant also exhibited some phenotypes unique to C. albicans, such as a markedly reduced sensitivity to LF killing, a lack of hyphal growth on solid media, and attenuated virulence in mice. Of particular importance, Mnn5 activity was found to be regulated by iron, and manifestation of some of the phenotypes of the mnn5{Delta} mutant appears to be affected by the cellular iron status.

Since MNN5 was first cloned for its unanticipated ability to support S. cerevisiae growth in the presence of high concentrations of iron chelators, we were interested in knowing whether it may perform similar functions in C. albicans. Surprisingly, overexpression of MNN5 in C. albicans did not enhance cell growth, and the mnn5{Delta} mutant did not exhibit any growth defects under iron-limiting conditions. This discrepancy might be due to the different functions manifested by Mnn5 in the two different biological systems. The growth-enhancing activity of C. albicans Mnn5 found in S. cerevisiae under iron-limiting conditions appeared to be largely, if not entirely, attributable to the novel iron-binding ability of the protein, because the growth enhancement did not require mannosyltransferase activity but was abolished when the putative iron-binding sites were mutated (1). On the other hand, the effect of CaMnn5's mannosyltransferase activity on budding yeast may be quite limited, possibly because of the redundant functions provided by host mannosyltransferases such as Mnn5. Consistently, overexpression of either MNN2 or MNN5 in S. cerevisiae also had no discernible effect (1). Nevertheless, the iron-dependent growth-enhancing activity of CaMnn5 found in S. cerevisiae revealed a novel property of the protein, namely, iron binding. In this study, we established that the mannosyltransferase activity of C. albicans Mnn5 is regulated by iron, being activated at low and inhibited at high iron concentrations (Fig. 2D). Also, the observation that high concentrations of BPS (100 to 400 µM) were needed to significantly reduce the enzymatic activity of purified Mnn5 suggests a high affinity of the protein for iron, consistent with the previously observed coprecipitation of 55Fe with Mnn5 (1). It is an attractive possibility that Mnn5 may regulate certain cell functions by altering protein glycosylation in response to the cellular or environmental iron status. In further support of this hypothesis, we observed a parallel between the mnn5{Delta} mutant's resistance to LF killing and the significantly enhanced LF resistance of wild-type C. albicans cells grown under iron-limiting conditions. Iron is an essential nutrient for almost all organisms. Keeping free iron away from microbial pathogens is an important element of host innate immunity. Thus, tactics for the efficient acquisition and use of iron constitute virulence factors for many microbial pathogens, including C. albicans (37). At the moment, we do not know whether the iron regulation of Mnn5 is of specific importance for C. albicans survival in the host. Although the mnn5{Delta} mutant is much less virulent than the wild-type strain, this could be the result of many compromised cellular functions. The isolation of MNN5 mutants that have lost the response to iron may help to answer this question in future. Nevertheless, for the purpose of drug development, Mnn5 may serve as a good target.

The ability of C. albicans to switch from yeast to filamentous growth is important for pathogenicity (28, 57). The outer layer of the fungal cell wall is rich in mannoproteins, and mutations in genes responsible for constructing mannan structures have been reported to cause various degrees of filamentous growth defects in C. albicans (44, 48, 49). For example, all pmt mutations except pmt5 were reported to suppress hyphal morphogenesis under standard inducing conditions (36). In this study, we found that the loss of MNN5 function blocked hyphal formation on several solid hypha-inducing media, but not in the corresponding liquid media. Although the reasons remain unclear, it might be that hyphal growth on and penetration into solid media are much more demanding on cell wall strength and integrity than growth in liquid media or that hyphal growth on solid medium requires certain cell surface properties which are lost in the mutant. Such conditional hyphal growth defects have been described for many C. albicans mutants (4, 48, 49), underscoring the high degree of complexity of the signals that feed into the signal transduction pathways for filamentous growth. The impaired hyphal growth observed as a result of various mannosyltransferase gene mutations suggests that the defect might be a consequence of the loss of proper glycosylation of many cell surface proteins important for the construction of hyphal morphology. However, it remains possible that some members of the mannosyltransferase family may have more specific roles in regulating cell morphogenesis. Indeed, Timple et al. (48, 49) found that overexpression of genes encoding components involved in hyphal formation suppressed the hyphal growth defect of a pmt6 mutant, suggesting that Pmt6 may act upstream of the morphogenetic signaling pathways.

LF is a 77-kDa iron-binding protein present in milk and mucosal secretions. It is also released by specific granules of polymorphonuclear leukocytes during inflammation. LF has been ascribed many diverse biological functions, most of which are immunomodulatory or antibacterial (5, 51, 52). It inhibits microbial growth by effectively sequestering free iron from the environment. The protein and its N-terminal fragment (amino acids 1 to 11) are also known to have direct bactericidal and candidacidal activities (6, 21). However, the mechanisms of cell killing are not understood. Some studies have suggested that the peptide targets energized mitochondria, causing the production of ATP and reactive oxygen species. The reactive oxygen species may lead to a reduction in the cellular thiol level, whereas ATP interacts with the LF-interacting proteins in the cell envelope. These events are thought to collectively contribute to cell death (29). We demonstrated that the mnn5{Delta} mutant lost sensitivity to LF killing and that this effect depends on the cellular iron status, rendering MNN5 the first gene identified that mediates the candidacidal activity of LF. Mnn5 is an intracellular protein and thus unlikely to be the direct target of LF. It is more plausible that Mnn5 modifies some cell surface proteins which are responsible for the interaction with LF or the peptide. The failure of enzyme-dead Mnn5 to restore LF sensitivity to the mnn5{Delta} mutant supports this view. Importantly, among a total of eight C. albicans mannosyltransferase mutants tested, only the mnn5{Delta} mutant exhibited increased resistance to LF. In contrast, several mutants exhibited increased sensitivity to either Congo red, hygromycin B, or both. These observations strongly suggest that Mnn5 has a specific role in determining C. albicans sensitivity to LF, while the functions of multiple mannosyltransferases are required for resistance to cell wall-damaging agents or certain antifungal compounds. Our finding provides important information for further elucidation of the mechanisms underlying the LF killing of C. albicans. Given the potent candidacidal activity of LF, finding the LF target and designing compounds that may mimic LF activity could be a worthy strategy for developing new anti-C. albicans therapies.


   ACKNOWLEDGMENTS
 
We thank Neil A. R. Gow and Joachim F. Ernst for generously providing us the MNT and PMT mutants, respectively. We also thank members of the Wang laboratory for stimulating discussions and suggestions.

Y.W. is an adjunct associate professor in the Department of Microbiology, National University of Singapore.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore. Phone: 65-6586-9521. Fax: 65-6779-1117. E-mail: mcbwangy{at}imcb.a-star.edu.sg. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Eukaryotic Cell, February 2006, p. 238-247, Vol. 5, No. 2
1535-9778/06/$08.00+0     doi:10.1128/EC.5.2.238-247.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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