Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2022 Mar 25;221(1):iyac048. doi: 10.1093/genetics/iyac048

The Mnn10/Anp1-dependent N-linked outer chain glycan is dispensable for Candida albicans cell wall integrity

Neta Dean 1,✉, Rachel Jones 2, Justin DaSilva 3, Gregory Chionchio 4, Henry Ng 5
Editor: A Mitchell
PMCID: PMC9071539  PMID: 35333306

Abstract

Candida albicans cell wall glycoproteins, and in particular their mannose-rich glycans, are important for maintaining cellular integrity as well as host recognition, adhesion, and immunomodulation. The asparagine (N)-linked mannose outer chain of these glycoproteins is produced by Golgi mannosyltransferases (MTases). The outer chain is composed of a linear backbone of ∼50 α1,6-linked mannoses, which acts as a scaffold for addition of ∼150 or more mannoses in other linkages. Here, we describe the characterization of C. albicans OCH1, MNN9, VAN1, ANP1, MNN10, and MNN11, which encode the conserved Golgi MTases that sequentially catalyze the α1,6 mannose outer chain backbone. Candida albicans och1Δ/Δ, mnn9Δ/Δ, and van1Δ/Δ mutants block the earliest steps of backbone synthesis and like their Saccharomyces cerevisiae counterparts, have severe cell wall and growth phenotypes. Unexpectedly, and in stark contrast to S. cerevisiae, loss of Anp1, Mnn10, or Mnn11, which together synthesize most of the backbone, have no obvious deleterious phenotypes. These mutants were unaffected in cell morphology, growth, drug sensitivities, hyphal formation, and macrophage recognition. Analyses of secreted glycosylation reporters demonstrated that anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ strains accumulate glycoproteins with severely truncated N-glycan chains. This hypo-mannosylation did not elicit increased chitin deposition in the cell wall, which in other yeast and fungi is a key compensatory response to cell wall integrity breaches. Thus, C. albicans has evolved an alternate mechanism to adapt to cell wall weakness when N-linked mannan levels are reduced.

Keywords: Candida albicans, Golgi, mannosyltransferase, N-linked glycosylation, cell wall, OCH1, MNN9, VAN1, ANP1, MNN10, MNN11

Introduction

As the interface between the fungus and its host, the fungal cell wall plays a major role in virulence. The outermost surface of the wall is composed of mannose-rich glycoproteins (“mannan”) that are implicated as critical ligands for Candida albicans recognition by host immune cells and for eliciting antigenicity (Shibata et al. 1992, 1995; Keppler-Ross et al. 2010; Hall and Gow 2013). The wall is also vital for maintaining cell shape during growth, division and during the switch between ovoid yeast and elongated hyphal or pseudohyphal forms. These morphological transitions involve alterations of wall composition as well as changes in surface ligand exposure. Cell wall remodeling is hypothesized to influence pathogenicity by attenuating or stimulating recognition of fungal cells by the host immune cells (Southard et al. 1999; Gow et al. 2011; Yadav et al. 2020). Thus, a better understanding of mannoprotein biosynthesis is important for understanding C. albicans pathogenicity.

A majority of cell wall mannose is attached to protein via N-linked glycans. N-linked glycosylation begins in the endoplasmic reticulum (ER) with assembly of the lipid-linked core Glu3Man9GlcNAc2 oligosaccharide and its transfer to asparagine in the amino acid consensus N–X–T/S (threonine or serine) of nascent proteins. After additional ER processing, glycoproteins are transported to the Golgi where these glycans are extensively modified. Most yeast and fungal N-linked glycans are extended by Golgi mannosyltransferases (MTases) to produce long, heavily branched oligosaccharides (for review see Dean (1999) and Mora-Montes et al. (2009)]. Synthesis of this Golgi “outer chain” begins with elongation of a linear backbone of ∼40 to 50 α1,6-linked mannoses. This backbone is conserved among most yeast and fungi, although it is branched and capped by sugars in ways that vary in a species-specific way. In Saccharomyces cerevisiae, branches include α1,2-linked mannoses and phosphomannoses capped with α1,3-linked mannose (Lussier et al. 1997, 1999; Rayner and Munro 1998; Jigami and Odani 1999). In C. albicans, α1,2 mannose and phosphomannoses are capped with α1,3- and in some serotypes β1,2-linked mannoses (Gemmill and Trimble 1999; Shibata et al. 2007).

Consistent with the structural conservation of the α1,6 mannose backbone, α1,6-MTase orthologs are encoded by most ascomycetes. These enzymes are well characterized in S. cerevisiae, where extensive genetic, biochemical, and structural studies of N-linked glycosylation have been performed (Ballou 1990). In S. cerevisiae, OCH1, ANP1 (a.k.a. MNN8), MNN9, MNN10, MNN11, and VAN1 encode the α1,6 MTases required for initiation and sequential elongation of the backbone [for review, see Dean (1999)]. Och1 adds the first initiating α1,6-mannose to the core oligosaccharide. Therefore, och1 mutants completely lack the outer chain (Nakayama et al. 1992; Nakanishi-Shindo et al. 1993; Bates et al. 2006). Further extension of the α1,6 backbone is catalyzed by sequential activity of enzymes in 2 MTase complexes, M-Pol I and M-Pol II (Hashimoto and Yoda 1997; Jungmann and Munro 1998; Stolz and Munro 2002). M-Pol I complex contains Mnn9 and Van1, which add up to 10 α1,6-linked mannoses onto the glycan (Jungmann and Munro 1998; Rodionov et al. 2009). Further elongation of the α1,6 backbone by another ∼40 mannoses is polymerized by M-Pol II, which contains Mnn9, Anp1, Mnn10, and Mnn11 (Jungmann and Munro 1998; Jungmann et al. 1999). Though these MTases reside in multimeric complexes, each MTase has a distinct function, with Anp1 and Mnn10 catalyzing the majority of the backbone (Chapman and Munro 1994; Dean and Poster 1996; Jungmann and Munro 1998). Other branching MTases add more mannoses to the backbone in α1,2, α1,3, phosphomannose, and in C. albicans, β1,2-linkages [reviewed in Dean (1999), Munro et al. (2005), and Martinez-Duncker et al. (2014)]. The end product of all these reactions is a heterogeneously branched glycan with ∼200 mannoses per N-linked chain. N-glycan synthesis therefore represents a significant investment of resources by the yeast cell. This metabolic expense is hypothesized to be required for the selective advantage of a robust cell wall (Levin 2011). In support of this idea, S. cerevisiae mannan mutants have severe growth defects associated with a weakened wall. However, these mutants are not inviable because S. cerevisiae has evolved a compensatory response to wall damage, which induces synthesis of other carbohydrate components to bolster the weakened wall (Levin 2011; Gow et al. 2017).

Although structural information about the C. albicans N-linked outer chain is emerging, details of its biosynthesis remain undefined. To gain a better understanding of N-linked glycosylation in C. albicans, we constructed strains deleted for each of the α1,6 MTases that function in backbone synthesis and studied their phenotypes. As expected based on our knowledge of this process in S. cerevisiae, these C. albicans mutants accumulate glycoproteins with highly truncated N-linked outer chains. Unexpectedly, C. albicans anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ mutants display none of the severe cell wall phenotypes seen in S. cerevisiae, nor do they display stress responses associated with wall weakness. These results demonstrate that in contrast to S. cerevisiae, the highly extended ANP1-, MNN10-dependent α1,6 backbone is not required for maintenance of C. albicans cell wall integrity.

Material and methods

Media and growth conditions

Yeast were grown on YPAD (1% yeast extract, 2% Bacto peptone, 50 mg/l of adenine sulfate, 2% glucose) or a synthetic medium (SD) (0.67% yeast nitrogen base without amino acids, 2% glucose) supplemented with the appropriate amino acids, purines, or pyrimidines. For growth of C. albicans uracil auxotrophs, media was supplemented with 75 µg/ml uridine. Transformation of S. cerevisiae and C. albicans used the lithium acetate procedure (Gietz and Schiestl 2007) except C. albicans transformations were incubated 12–15 h at 30°C, heat shocked at 44°C, and contained ∼107 cells and 5–10 µg of DNA per transformation.

To induce hyphal formation in C. albicans, cultures were grown overnight at 30°C in SD (+uridine), diluted the following day to an OD600 of 0.4 in SD (+Uri) containing 20% bovine calf serum, and incubated at 37°C for various lengths of time.

Strain construction

Yeast strains and their relevant genotype are listed in Table 1. All mutations were verified by DNA sequence analysis. Saccharomyces cerevisiae SEY6210 and C. albicans BWP17 were the parental strains for construction of all mutants.

Table 1.

Yeast strains used in this study.

Name Relevant genotype Source
Candida albicans
 BWP17 SC5314 and ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG
arg4::hisG/arg4::hisG 		Wilson et al. (1999)
 HNY31 BWP17 and leu2Δ/leu2Δ eno1Δ:: CaCas9 Ng and Dean (2017)
 NDY203 BWP17 and + PADH1-SS-yEmRFP-N-HA This study
 NDY250 BWP17 and PADH1- SS-yEmRFP-X-HA-URA3 This study
 NDY265 BWP17 and PADH1- yEmRFP-X-HA-URA3 This study
 NGY205 CAI4 and och1Δ::hisG/och1Δ::hisG Bates et al. (2006)
 RJY56 NGY205 (och1Δ/Δ) and PADH1-SS-yEmRFP-N-HA-URA3 This study
 NDY254 NGY205 (och1Δ/Δ) and PADH1- SS-yEmRFP-X-HA-URA3 This study
 Camnn9Δ CAI4 and mnn9Δ::hisG/mnn9Δ::hisG Southard et al. (1999)
 NDY207 Camnn9Δ and PADH1- SS-yEmRFP-N-HA-URA3 This study
 SKY68 BWP17 and mnn10Δ::HIS1/mnn10Δ::ARG4 This study
 RJY30 BWP17 and MNN11/mnn11Δ:: SATR This study
 RJY31 BWP17 and MNN11/mnn11Δ:: ARG4 This study
 RJY32 BWP17 and mnn11Δ::ARG4/mnn11Δ:: SATR This study
 RJY42 BWP17 and mnn10Δ::HIS1 mnn10Δ::ARG4 mnn11Δ::URA3dpl200/mnn11Δ:: SATR This study
 RJY45 BWP17 and MNN11/mnn11Δ::URA3dpl200 This study
 RJY52 BWP17 and mnn10Δ::HIS1/mnn10Δ::ARG4 + PADH1-SS-yEmRFP-N-HA-URA3 This study
 RJY53 BWP17 and mnn10Δ::HIS1/mnn10Δ::ARG4 mnn11Δ::URA3dpl200/mnn11Δ:: SATR + PADH1-SS-yEmRFP-N-HA-URA3 This study
 RJY55 BWP17 and mnn11Δ::ARG4/mnn11Δ:: SATR + PADH1-SS-yEmRFP-N-HA-URA3 This study
 SKY43 BWP17 and + PADH1-GFP-URA3 Keppler-Ross et al. (2010)
 RJY66 BWP17 and mnn10Δ::HIS1/mnn10Δ::ARG4 mnn11Δ::URA3dpl200/mnn11Δ:: SATR + pADH-GFP This study
 RJY67 BWP17 and mnn10Δ::HIS1/mnn10Δ::ARG4 + PADH1-GFP-URA3 This study
 RJY68 BWP17 and mnn11Δ::ARG4/mnn11Δ:: SATR + PADH1-GFP-URA3 This study
 RJY69 BWP17 and mnn11Δ::ARG4/mnn11Δ:: SATR + PADH1yEmRFP-URA3 This study
 SKY38 BWP17 and PADH1yEmRFP-URA3 Keppler-Ross et al. (2010)
 RJY64 NGY205 and PADH1-GFP-URA3 This study
 GCY1 HNY31 and anp1Δ486-848/anp1Δ486-848 This study
 GCY2 GCY1 (anp1Δ/Δ) and PADH1-SS-yEmRFP-N-HA-URA3 This study
 JDY1-1 HNY31 and van1Δ21-570/van1Δ21-570 This study
 JDY3-1 JDY1-1 (van1Δ/Δ) and PADH1-SS-yEmRFP-N-HA-URA3 This study
S. cerevisiae
 SEY6210 MAT alpha ura3-52 his3-Δ200 trp1-Δ901 lys2-801 leu2-3 112 suc2-Δ9 Robinson et al. (1988)
 RJY47 SEY6210 + PADH1SS-yEmRFP-N-HA3-URA3 This study
 NDY238 SEY6210 + PADH1SS-yEmRFP-X-HA3-URA3 This study
 RJY70 SEY6210 + PADH1 yEmGFP This study
 RJY71 SEY6210 + PADH1yEmRFP This study
 RJY3 SEY6210 and mnn10Δ::Sp his5+ This study
 RJY48 SEY6210 and mnn10Δ:: Sp his5++ PADH1SS-yEmRFP-N-HA3-URA3 This study
 RJY6 SEY6210 and mnn10Δ:: Sp his5++ PADH1CaMNN10 This study
 RJY72 SEY6210 and mnn10Δ:: Sp his5 + PADH1yEmGFP This study
 RJY73 SEY6210 and mnn10Δ:: Sp his5 + YEp PGAPyEmRFP This study
 RJY4 SEY6210 and mnn11Δ::Sp his5+ This study
 RJY49 SEY6210 and mnn11Δ:: Sp his5 + PADH1SS-yEmRFP-N-HA3-URA3 This study
 RJY5 SEY6210 and mnn11Δ:: Sp his5++ pAdh CaMNN11 This study
 RJY74 SEY6210 and mnn11Δ::HIS3 + YEp PGAPyEmRFP This study
 RJY9 SEY6210 and mnn10Δ:: Sp his5+mnn11Δ::TRP1 This study
 RJY50 SEY6210 and mnn10Δ:: Sp his5 mnn11Δ::TRP1 + PADH1-SS-yEmRFP-N-HA3-URA3 This study
 RJY75 SEY6210 and mnn10Δ:: Sp his5 mnn11Δ::TRP1 + YEp PGAPyEmRFP This study
 NDY269 SEY6210 and mnn9Δ:: Sp his5 This study
 NDY202 SEY6210 and mnn9Δ:: Sp his5 PADH1SS-yEmRFP-N-HA This study
 GCY4 SEY6210 and anp1Δ:: Sp his5 This study
 GCY3 SEY6210 and anp1Δ:: Sp his5 PADH1SS-yEmRFP-N-HA This study
 BZY1 SEY6210 and van1Δ:: Sp his5 This study
 NDY270 SEY6210 and van1Δ PADH1SS-yEmRFP-N-HA This study
 NDY21 SEY6210 and och1Δ::LEU2 Nakayama et al. (1992)
 NDY271 och1Δ + PADH1SS-yEmRFP-N-HA3-URA3 This study
 NDY260 och1Δ + PADH1SS-yEmRFP-X-HA3-URA3 This study
Open in a new tab

Saccharomyces cerevisiae mnn9Δ, van1Δ, anp1Δ, mnn10Δ, and mnn11Δ strains (NDY269, BZY1, GCY4, RJY3, RJY4, respectively) were constructed by PCR-mediated recombination using the standard pFA6a-HIS3 series of plasmids as templates (Longtine et al. 1998). The ORF of each gene (including the ATG and stop codon) was replaced with the S. pombe his3 ortholog. The S. cerevisiae mnn10Δ::HIS3 mnn11Δ::TRP1 double mutant (RJY9) was constructed by replacing MNN11 with TRP1 in the mnn10Δ::HIS3 strain.

Homozygous and heterozygous C. albicans mnn10 mutants were made by replacing MNN10 with mnn10Δ::HIS1 or mnn10Δ::ARG4 cassettes (described below). The mnn10Δ/Δ mnn11Δ/Δ double mutant (RJY42) was made by sequentially replacing MNN11 alleles with mnn11Δ::SAT1 and mnn11Δ::ura3-dpl200 cassettes in the mnn10Δ::ARG4 mnn10Δ::HIS1 strain (SKY68). The ura3-dpl200 allele contains URA3 flanked by a direct 200 bp repeat (Wilson et al. 2000). Candida albicans mnn10Δ/Δ mnn11Δ/Δ uracil auxotrophs were isolated by plating ura3-dpl200 strains on media containing 5-flouro-orotic acid (FOA) to select for loss of ura3-dpl200 by homologous recombination between the flanking direct repeats. Deletion strains were verified by PCR of genomic DNA, using primer pairs where 1 primer hybridized within the site of integration and 1 outside (Supplementary Fig. 1). For complementation analyses, CaMNN10 and CaMNN11 were introduced into S. cerevisiae mnn10Δ or mnn11Δ by transformation with pADH-CaMNN10 or pADH-CaMNN11 plasmids (see below).

Candida albicans homozygous deletions of ANP1 and VAN1 were made by CRISPR/Cas-mediated deletion as described (Ng and Dean 2017; Dean and Ng 2018). Briefly, 23 bp oligonucleotide duplexes, encoding guide RNAs that target PAM sites in each of these genes were hybridized and cloned in the SapI site of the ura3-dpl200-marked gRNA expression plasmid pND501 (Dean and Ng 2018). Targeted PAM sites (relative to +1 of the ORF) were at positions 810 of ANP1 and 299 of VAN1. These plasmids were introduced in the CAS9-expressing strain, HNY31 (Dean and Ng 2018), along with donor repair fragments whose recombination in the chromosome resulted in deletions of each respective ORF. Deletions, relative to the +1 initiating ATG were as follows: ANP1 was deleted for 362 bp from nucleotide 486–848 of the 1,287 bp ORF. VAN1 was deleted for 459 bp from nucleotide 21 to 570 of the 1,555 bp ORF. Strains were screened by PCR analysis of genomic DNA (Supplementary Fig. 1). Homozygous deletions generated by CRISPR/Cas9 were obtained at a frequency of 60–90%. To “pop-out” the ura3-dpl200Δ gRNA-expressing plasmid, uracil auxotrophs were isolated by FOA selection as described above.

Candida albicans strains auxotrophic for uracil, histidine, and arginine were made prototrophic by targeting IRO1-URA3 to the ura3Δ::imm434 locus using the IRO1-URA3 plasmid pBSK-URA3 linearized by digestion with Not1/Pst1 (Park et al. 2005), by targeting HIS1 to the his1::hisG locus using the integrative pGEM-HIS plasmid (Wilson et al. 1999) linearized with Nru1 or by targeting ARG4 to the RP10 locus using the ARG4 integrative plasmid, Clp-ARG4 linearized with Stu1.

Plasmid constructions

Plasmids used in this study and their relevant features are listed in Table 2. Details of plasmid construction are described in Supplementary Material 2. Plasmid sequences were verified by DNA sequence analysis. PCR primer sequences that were used for DNA amplification are available upon request.

Table 2.

Plasmids used in this study.

Name Alias Relevant features Reference
pBSK-URA3 NA IRO1-URA3 Park et al. (2005)
pGEM-HIS NA CaHIS1 Wilson et al. (1999)
CIp10 pND294 CaURA3 integrative plasmid Murad et al. (2000)
CIp-HIS1 pND500 CaHIS1 integrative plasmid Keppler-Ross et al. (2010)
CIp-ARG4 pND383 CaARG4 integrative plasmid This study
CIp-SAT pND347 CaSAT integrative plasmid This study
pADH-yEGFP pND133 PADH1-GFP in CaURA3/ARS plasmid Keppler-Ross et al. (2010)
pADH-yEmRFP pND292 PADH1-RFP in CaURA3/ARS plasmid Keppler-Ross et al. (2010)
pADH-ssRFP-HDEL pND336 PADH1-ssRFP-HDEL in CaURA3/ARS/2 µ plasmid This study
pADH-ssRFP-NDT-HA pND462 PADH1-ssRFP-N -HA in CaURA3/ARS plasmid This study
pADH-ssRFP-X-HA pND517 PADH1-ssRFP-X-HA in CaURA3/ARS plasmid This study
pADH-RFP-X-HA pND537 PADH1-RFP-X-HA in CaURA3/ARS plasmid This study
pADH-CaMNN10 pND393 PADH1-CaMNN10 in CaURA3/ARS plasmid This study
pADH-CaMNN11 pND415 PADH1-CaMNN11 in CaURA3/ARS plasmid This study
CIp-ADH-MNN10 pND394 PADH1-CaMNN10 in CaURA3 integrative plasmid This study
pDDB57 pND418 CaURA “mini” blaster (ura3-dpl200) Wilson et al. (2000)
pCamnn10Δ::HIS1 pND389 Contains Camnn10Δ::HIS1 deletion allele This study
pCamnn10Δ::ARG4 pND390 Contains Camnn10Δ::ARG4 deletion allele This study
pCamnn11Δ::ura3-dpl200 pND457 Contains Camnn11Δ:: ura3-dpl200 deletion allele This study
pCamnn11Δ::ΔSAT pND 420 Contains Camnn11Δ::SATR deletion allele This study
pADHtAgSapHDV pND501 C. albicans gRNA expression plasmid Dean and Ng (2018)
P501gAnp1-1 pND487 Contains gANP1-1 This study
p501gVan1-1 pND515 Contains gVAN1-1 This study
Open in a new tab

Protein glycosylation assays

Protein extracts were prepared in one of the 2 ways. Extracts containing both secreted and intracellular proteins were obtained by precipitation of protein in 0.8 ml of overnight culture (∼8 OD600 units of C. albicans or 0.8 OD600 units of S. cerevisiae) by the NaOH, β-mercaptoethanol/trichloroacetic acid method (Chi et al. 1996). Extracts enriched for secreted proteins were isolated by acetone precipitation of supernatants from overnight cultures. One milliliter culture from S. cerevisiae strains (OD600 ∼ 1) or C. albicans strains (OD600∼10) were harvested by centrifugation (5’ at 14,000 g). Supernatants (300 µl) were transferred to fresh tubes and precipitated by the addition of 4 volumes of ice-cold acetone. After 20 min at −20°C, samples were centrifuged 5’ at 14,000 g and the supernatant aspirated. An additional 300 µl of supernatant was added to the precipitate along with 1.2 ml acetone. These were kept at −20°C for 20 min, centrifuged, and the acetone supernatant aspirated. These steps were repeated in order to pool secreted proteins. The precipitated protein pellets were resuspended in 100 µl Laemmli’s sample buffer (0.001% bromophenol blue, 1% β-mercaptoethanol, 1% PMSF, 4% SDS, 30% glycerol, and 120 mM Tris–HCl, pH 6.8) or phosphate buffered saline (PBS) containing 0.1% Triton X-100, 1% β-mercaptoethanol, and 1 mM PMSF. The concentration of solubilized protein samples was determined using the Bradford colorimetric assay (Bio-Rad) with bovine serum albumin as a standard. Proteins were analyzed directly by 10% SDS-PAGE and western blotting, or stored at 20°C for up to 1 week. Before electrophoresis, samples were denatured by heating at 95°C for 2 min and centrifuged for 30 s. To compensate for much higher levels of plasmid-borne RFP protein expression in S. cerevisiae vs C. albicans, different amounts of protein were loaded per well. Generally, 3 µl of S. cerevisiae samples (∼0.45 µg protein) precipitated from culture supernatant or 15 µl (∼2.25 µg protein) of C. albicans samples were loaded per well.

After transfer to PVDF and blocking with milk, PVDF membranes were incubated with anti-HA antibody (1:1,000) followed by mouse IgG lambda binding protein-HRP (1:10,000; Santa Cruz Biotechnology). Proteins on blots were detected by chemiluminescence (WesternBright Quantum, Advansta for C. albicans blots or WesternBright-ECL for S. cerevisiae blots) using the ChemiDoc system (BioRad) or film.

Hygromycin B sensitivity assay

Hygromycin B sensitivity was determined by serial dilution and growth of yeast on solid media (Dean 1995). Overnight cultures were adjusted to 107 cells/ml, serially diluted and 3 µl of each dilution (105, 104, 103, 102, 10 cells/ml) were spotted onto YPAD plates containing various concentrations of hygromycin B (from 50 to 150 µg/ml). Plates were incubated at 30°C for 2–3 days.

Measurement of cell wall chitin content

Cell wall chitin was measured by a modification of the Morgan–Elson method (Leloir and Cardini 1953). Briefly, overnight cultures grown in YPAD +Uri were harvested, boiled in 1% SDS, washed 3 times with H2O, pelleted, and weighed to produce a cell wall pellet of 50 mg of (wet) cells. Cell wall polysaccharides were hydrolyzed in 1 ml 6 M HCl at 100°C overnight. After lyophilization, acid-resistant amino sugars were resuspended in 1 ml H2O. Samples (0.1 ml) were mixed with 0.1 ml 1.5 M Na2CO3 in 4% acetylacetone and incubated at 100°C for 20 min to form a chromogenic product. After cooling to room temperature, 700 µl 95% ethanol was added. The penta-acetyl glucosamine derivative in samples gives a red color after incubation for an additional 1 h with 0.1 ml of Erlich reagent (2.6% p-dimethylaminobenzaldehyde in 1:1 95% ethanol/concentrated HCl). Derivatized glucosamine concentration was measured by absorbance at 520 nm. Absolute cell wall glucosamine was calculated by comparison to a standard curve generated using 0–50 µg glucosamine taken through the same reactions.

Cell wall chitin content was indirectly assayed as a function of bound Calcofluor white, measured by the mean intensity of fluorescence using microscopy. One hundred microliters of overnight cultures grown in YPAD + Uri were washed, resuspended in 100 µl PBS containing 1 µg/ml Calcofluor white, and observed by microscopy using a DAPI filter. Relative calcofluor white fluorescence levels in different strains were determined using Scion Image software (Zeiss), measuring the differences in exposure time (ms) required to auto balance each sample.

Macrophage phagocytosis competition assay

Phagocytosis of fluorescent yeast cells by the murine BALB/C macrophage-like J774 cell line was assayed as described (Keppler-Ross et al. 2010). It should be noted that all yeast strains used in these phagocytosis experiments were prototrophic for all nutritional markers. Competing yeast strains, expressing either yEGFP (Cormack et al. 1997) or yEmRFP (Keppler-Ross et al. 2008) were grown overnight in SD-Ura media, harvested, washed in PBS, and resuspended to 0.1 ODA600/ml in PBS. An equal number of competing red and green yeast cells were mixed and added at an MOI of 5 to J744 macrophages grown on coverslips in 12 well plates. After a 90-min incubation, Calcofluor white was added (1 µg/ml final concentration). Cover slips were removed, placed upside down on glass slides, sealed, and viewed by fluorescence microscopy. The % internalization was calculated as described (Keppler-Ross et al. 2010), by quantitating the number of red and green yeast that were inside (phagocytosed) or outside of macrophages (blue). Each experiment was repeated 3 times and ∼300 yeast cell counted per experiment.

Results

Phenotypic analyses of Camnn10Δ and Camnn11Δ

Unlike other yeast species deleted for MNN10, C. albicans mnn10Δ/Δ strains do not display obvious cell wall phenotypes [Zhang et al. (2016) and see Fig. 1]. One explanation for these observations is that Mnn10-mediated protein glycosylation in C. albicans can also be catalyzed by another redundant α1,6 MTase in C. albicans. CaMnn10 shares significant peptide sequence similarity to Mnn11 (E = ∼7e10), raising the possibility that these 2 proteins have overlapping functions. To test this, heterozygous and homozygous single and double C. albicans mnn10 and mnn11 deletion strains were constructed and analyzed for various phenotypes (see Materials and methods; Fig. 1). Candida albicans strains deleted for either MNN10, MNN11 or both genes were indistinguishable from wild-type. By microscopy, the shape and size of single Camnn10Δ/Δ and mnn11Δ/Δ and double mnn10Δ/Δ mnn11Δ/Δ strains were morphologically identical to their wild-type parents. In contrast, both S. cerevisiae mnn10Δ and mnn11Δ appeared clumped and misshapen (Fig. 1a). Candida albicans single and double mutants also grew at rates indistinguishable from the parent while Scmnn10Δ and mnn11Δ grew at markedly reduced rates (∼3- and 1.5-fold, respectively) compared with wild-type (Fig. 1b). Finally, C. albicans single and double mnn10Δ/Δ mnn11Δ/Δ mutants were resistant to 60 µg/ml hygromycin B (Fig. 1c), a drug to which sensitivity is commonly associated with cell wall or glycosylation defects (Dean 1995). This concentration completely inhibited the growth of S. cerevisiae Scmnn10Δ and to a lesser extent, Scmnn11Δ, as well as Caoch1Δ/Δ, which lacks the N-linked outer chain (Fig. 1c). As a test for functionality, the C. albicans MNN10 and MNN11 genes were cloned and plasmid-borne copies were introduced into the corresponding S. cerevisiae mutants. Each gene complemented the hygromycin B sensitivity of the corresponding S. cerevisiae mutants (Fig. 1d). In summary, these experiments demonstrated that cell morphology, growth rate, and drug sensitivity of the Camnn10Δ/Δ mnn11Δ/Δ single and double mutants were virtually indistinguishable from the wild-type parent, and also ruled out the idea that these genes are redundant. These results also demonstrated that unlike their S. cerevisiae counterparts, neither Mnn10 nor Mnn11 is required for normal growth in C. albicans.

Fig. 1.

Fig. 1.

Open in a new tab

Phenotypic analysis of C. albicans and S. cerevisiae single and double mnn10 and mnn11 deletion mutants. a) Light microscopy (40×) of wild-type, mnn10Δ/Δ, mnn11Δ/Δ, mnn10Δ/Δ/mnn11Δ/Δ, and och1Δ/Δstrains. Saccharomyces cerevisiae strains are in the upper panel and corresponding C. albicans Δ/Δ mutants are in lower panel. b) Wild-type or mnn10Δ/Δ/mnn11Δ/Δ strains were diluted to 0.20 OD600 units/ml in liquid YPAD. Growth at 30°C was monitored over time (up till 27 h) by taking aliquots and measuring the absorbance at OD600. Triplicate measurements were collected for each time point. Means and standard deviations were plotted using GraphPad Prism. c) Hygromycin sensitivity of wild-type or C. albicans mnn10Δ/Δ mnn11Δ/Δ strains. Yeast were grown to early logarithmic stage, adjusted to 1 OD600 units/ml and 10-fold serially diluted. Three microliters of each dilution (104, 103, 102, 101, and 10°) were plated on YPAD containing hygromycin B at the indicated concentrations. Note that concentrations of hygromycin B above 100 µg/ml inhibit the growth of the wild-type parental C. albicans strain. d) Complementation of the hygromycin B-sensitive phenotype of S. cerevisiae mnn10Δ and mnn11Δ mutants by CaMNN10 and CaMNN11. Saccharomyces cerevisiae WT (SEY6210), mnn10Δ, and mnn11Δ harboring a vector, or plasmid-borne CaMNN10 or CaMNN11 were serially diluted as described in c), and plated on YPAD plus or minus 50 µg/ml hygromycin B. Plates were incubated at 30°C for 3 days.

Single and double Camnn10Δ/Δ mnn11Δ/Δ mutants were further analyzed for other phenotypes indicative of virulence attributes, including efficiency of hyphal induction and phagocytosis by macrophage. Hyphal formation was measured in response to the presence of serum when growth temperature was raised from 30°C to 37°C. As shown in Fig. 2, both the rate and extent of hyphae formed in C. albicans lacking MNN10, MNN11, or both were the same as the parental wild-type cells. This is in marked contrast to the severe filamentation defect displayed by Caoch1Δ/Δ, a phenotype reported previously (Bates et al. 2006). To analyze phagocytosis of C. albicans by murine macrophage, we used previously established competition assays that measure preferential uptake of red or green fluorescently tagged fungi of differing genotypes by macrophage (Keppler-Ross et al. 2010). By this and other assays, the Och1-dependent N-linked outer chain is strictly required for efficient phagocytosis (Bulik et al. 2003; Bates et al. 2006; Keppler-Ross et al. 2010; Lewis et al. 2012). To determine if fungal recognition by macrophage was similarly reduced by loss of MNN10 and/or MNN11, wild-type and mutant strains expressing either GFP or RFP were mixed prior to phagocytosis by macrophage (Fig. 3). The relative rate of fungal uptake by macrophage for wild-type vs mutants was measured by quantitating the ratio of green vs red internalized fungal cells. As shown previously, both S. cerevisiae and C. albicans wild-type cells were phagocytosed 5- to 10-fold more efficiently than och1Δ (Fig. 3, a and b). However, phagocytosis of single or double Camnn10Δ/Δ mnn11Δ/Δ mutants occurred with the same efficiency as wild-type C. albicans (Fig. 3a). This was different than phagocytosis of S. cerevisiae, where the wild-type was phagocytosed ∼3-fold more efficiently than Scmnn10Δ mnn11Δ (Fig. 3b). These results demonstrated that MNN10 and/or MNN11 are not required for hyphal formation or recognition of C. albicans by macrophage in vitro.

Fig. 2.

Fig. 2.

Open in a new tab

MNN10 and MNN11 are not required for hyphal formation Hyphal formation was examined in wild-type parental (BWP17) or isogenic mnn10Δ/Δ, mnn11Δ/Δ, mnn10Δ/Δ mnn11Δ/Δ, or och1Δ/Δ strains. Cultures were grown to early logarithmic stage (0.5 OD600 units/ml) and induced to form hyphae by the addition of 20% calf serum and a temperature shift from 30°C to 37°C. Aliquots were removed at the indicated times and viewed by light microscopy (a) or incubated for 1 min with calcofluor white and viewed by fluorescence microscopy (×40 magnification) (b).

Fig. 3.

Fig. 3.

Open in a new tab

Mnn10 is required for macrophage uptake of S. cerevisiae but not C. albicans. Phagocytosis of yeast by macrophage was quantitated by competition assays as described in Materials and Methods. RFP or GFP-expressing yeast strains of differing genotypes were mixed in equal numbers and added to J774 macrophages at an MOI of 5. After 90 min, calcofluor white was added, and red and green yeast were scored by fluorescence as being inside (calcofluor white negative) or outside (calcofluor white positive) of macrophages. Top and bottom panels show competition assays between C. albicans or between S. cerevisiae strains, respectively.

MNN10 and MNN11 are required for N-linked backbone elongation

CaMnn10 reportedly has α1,6 mannosyltransferase activity in vitro (Zhang et al. 2016). However, the absence of any obvious cell wall-associated phenotypes raised the question of whether or to what extent in vivo N-linked mannosylation is affected by loss of CaMNN10 and/or MNN11. To address this question, we developed a protein reporter for the N-linked glycosylation pathway. This reporter is based on a yeast codon-optimized RFP (Keppler-Ross et al. 2008) with an N-terminal signal sequence to direct ER translocation, a single recognition motif (N/D/T) for N-glycan attachment, and a C-terminal HA tag for detection by western blotting. This reporter, SS-yEmRFP-N-HA (which we will hereafter refer to as ssRFP-N-HA) shown schematically in Fig. 4a, was cloned in a plasmid that could be expressed in S. cerevisiae or C. albicans (see Materials and methods).

Fig. 4.

Fig. 4.

Open in a new tab

MNN10 and MNN11 are required for N-glycosylation. a) Schematic diagram of N-linked glycosylation RFP reporter. ssRFP-N-HA encodes yEmRFP tagged with an N-terminal signal sequence, a C-terminal HA tag and a single N/D/T N-glycan attachment recognition motif. ssRFP-X-HA is identical except it lacks the N/D/T recognition motif and therefore is not an acceptor for the N-linked glycan. RFP-X-HA lacks both the N/D/T and N-terminal signal sequences and therefore cannot enter the secretory pathway. b) Localization of ssRFP-N-HA in C. albicans. Yeast cells expressing ssRFP-N-HA, or ssRFP-HDEL that carries a C-terminal HDEL ER retention sequence instead of the N/D/T-HA, were grown to mid-log phase and viewed by fluorescence microscopy (using 100× magnification). Arrow denotes perinuclear ER staining. c) Western blot analysis of total (T) and secreted (s) ssRFP-N-HA or RFP-X-HA protein isolated from C. albicans wild-type or mnn10Δ/Δ. Cells were grown to mid-log phase and proteins were extracted by precipitation of proteins in an aliquot of the culture (T) or from culture supernatants (S), as described in Materials and Methods. Equal cell equivalents of protein extracts were analyzed by 10% SDS-PAGE and detected by chemiluminescence with anti-HA antibody. d) Western blot analysis of ss-RFP-N-HA and ss-RFP-X-HA in wild-type and och1 mutants of C. albicans and S. cerevisiae. C. albicans or S. cerevisiae wild-type and och1 mutants expressing secreted RFP (ssRFP) with (N) or without (X) a N-glycosylation site are indicated below each lane. Secreted proteins in culture supernatants were acetone precipitated as described in Materials and Methods. Protein extracts (2.25 µg/lane for C. albicans samples and 0.45 µg/lane for S. cerevisiae samples) were separated by 10% SDS-PAGE and detected by western blotting with anti-HA. e) N-glycosylation defects in C. albicans and corresponding S. cerevisiae α1,6 MTase mutant strains. Secreted ssRFP-N-HA was isolated from the culture supernatants of S. cerevisiae or C. albicans wild-type, mnn10Δ/Δ, mnn11Δ/Δ, mnn10Δ/Δ mnn11Δ/Δ, and och1Δ/Δ strains by acetone precipitation as described in Materials and Methods. Protein extracts (2.25 µg/lane for C. albicans samples and 0.45 µg/lane for S. cerevisiae samples) were separated by 10% SDS-PAGE and detected by western blotting with anti-HA antibody as in (d).

We reasoned that if ssRFP-N-HA undergoes N-linked glycosylation in the ER and Golgi, then mutations that affect outer chain synthesis will lead to its decreased molecular weight (MW) and an increased electrophoretic mobility shift on SDS-polyacrylamide gels. In cells expressing this reporter and analyzed by fluorescence microscopy, most of the RFP fluorescence was evenly distributed at the outermost cell surface, as predicted if ssRFP-N-HA were secreted. Some intracellular staining was also observed in a pattern characteristic of the ER (Fig. 4b). An ER marker (ssRFP-HDEL) showed a similar ER localization but was absent from the cell surface (Fig. 4b). These results suggested that most of ssRFP-N-HA was secreted but that a portion also accumulated intracellularly. To test this and determine if secreted ssRFP-N-HA undergoes N-glycosylation, protein extracts prepared from C. albicans wild-type and mnn10Δ/Δ cells expressing ssRFP-N-HA were analyzed by SDS-PAGE and western blotted with anti-HA antibodies. As a control, extracts were also prepared from strains expressing an RFP-HA variant that lacks both the signal sequence and N/D/T motif (“RFP-X”; see Fig. 4a). After growth in liquid media, aliquots containing both cells and growth media were removed for protein extraction (labeled “T” for total protein). To enrich for secreted proteins (“S”), proteins from equivalent amounts of culture supernatants were acetone precipitated and analyzed in parallel (see Materials and Methods). The results, shown in Fig. 4c, led to several conclusions. First, secretion of this RFP reporter was dependent on the signal sequence since RFP-HA lacking the signal sequence was detected in whole cell extracts but not in the culture supernatant (Fig. 4c). Second, ssRFP-N-HA isolated from wild-type C. albicans culture supernatants migrated as a large heterogeneous “smear,” whose average MW was between 95 and 170 kDa. RFP-HA that lacks the secretion signal peptide and the NDT recognition motif migrated with an MW of about 32 kDa. This difference in MW was consistent with the N-linked glycosylation of ssRFP-N-HA. Third, the MW of ssRFP-N-HA from Camnn10Δ/Δ cells was reduced significantly compared with its expression in the wild-type. In contrast, the migration of nonsecreted, nonglycosylated RFP-HA (∼32 kDa) was the same regardless of strain genotype.

To demonstrate that the increased MW shift of ss-RFP-N-HA is due to an N-linked glycan, the mobility of ss-RFP-N-HA was compared with ss-RFP-X-HA, which contains a signal sequence but lacks an N-glycosylation recognition motif (Fig. 4a). Secreted proteins in culture supernatants from C. albicans and S. cerevisiae wild-type or och1 mutants expressing these reporters were analyzed by western blotting. Since ss-RFP-X-HA lacks a N-glycan recognition site, we expected that it would migrate according to its MW of ∼35 kDa in both wild-type and in the Golgi N-glycosylation defective och1 mutant. This prediction was borne out in S. cerevisiae, where the mobility of ssRFP-X-HA was identical in wild-type and Scoch1Δ (Fig. 4d). In Scoch1Δ, ssRFP-N-HA migrated with a slightly larger MW than ssRFP-X-HA as expected since och1Δ affects Golgi but not ER glycosylation; this slight increased MW of ssRFP-N-HA is due to the presence of core ER N-glycan (∼1.6 kDa) that ssRFP-X-HA lacks. Unexpectedly, ssRFP-X-HA in C. albicans ran much larger than in S. cerevisiae. It appeared as heterogenous smear with an average MW of ∼60 kDa. Although larger, as in S. cerevisiae, the size of ssRFP-X-HA was the same in both C. albicans wild-type and Caoch1Δ/Δ. These results demonstrated that ss-RFP-X-HA is post-translationally modified in the secretory pathway of C. albicans but not S. cerevisiae and that this modification is independent of N-glycosylation. This modification(s) may include β1,2 mannosylation or another as yet unidentified modification (Shibata et al. 1995). Nevertheless, ss-RFP-N-HA was heavily glycosylated in wild-type strains but ran with a decreased MW when expressed in och1 mutants of both species (Fig. 4d), establishing its utility as a N-glycosylation reporter.

To compare the effect of mnn10Δ and mnn11Δ mutations on the extent of N-glycan extension, ssRFP-N-HA secreted from wild-type and orthologous mnn10 mnn11, mnn10 mnn11, and och1 strains of C. albicans and S. cerevisiae was analyzed by western blot (Fig. 4e). As described above, ssRFP-N-HA migrated with a significantly larger MW when expressed in C. albicans than in S. cerevisiae. This was the case when comparing ssRFP-N-HA mobility between C. albicans and S. cerevisiae wild-type strains or between any of the orthologous mutants, including och1Δ. We also observed that ssRFP-N-HA mobility in the mnn10Δ/Δ mnn11Δ/Δ double mutant resembled that of mnn10Δ/Δ, in both C. albicans and S. cerevisiae, suggesting MNN10 is epistatic to MNN11 in both species. Finally, the reduction in MW of ssRFP-N-HA from Camnn10Δ/Δ approached that seen in och1Δ/Δ (and see Fig. 5b), suggesting that loss of MNN10 affected Golgi glycosylation in C. albicans almost as severely as loss of OCH1. Therefore, despite the lack of obvious cell wall phenotypes, deletion of MNN10 and to a lesser extent, MNN11, leads to severely truncated N-glycans. Together, these results provide additional evidence that C. albicans, unlike S. cerevisiae, does not require the Mnn10-dependent highly mannosylated outer chain for normal growth.

Fig. 5.

Fig. 5.

Open in a new tab

Colony morphology and glycosylation phenotypes of C. albicans Golgi α1,6 MTase mutants. a) Wild-type or homozygous C. albicans mutants defective in each of the Golgi α1,6 MTase genes (mnn11Δ/Δ, mnn10Δ/Δ, anp1Δ/Δ, van1Δ/Δ, mnn9Δ/Δ, and och1Δ/Δ) were spotted on YPAD solid media. Individual colonies were incubated for 3 days at 30°C before photography. b) Western blot analyses of secreted proteins from C. albicans (left panel) and S. cerevisiae (right panel) MTase mutants expressing ss-RFP-N-HA. Proteins from culture supernatants were acetone precipitated as described in Materials and Methods. Protein extracts (2.25 µg/lane for C. albicans samples and 0.45 µg/lane for S. cerevisiae samples) were separated by 10% SDS-PAGE and detected by western blotting with anti-HA antibody. Note that gels with C. albicans samples were run longer than S. cerevisiae.

Candida albicans and S. cerevisiae diverge phenotypically at the Van1-dependent step of N-glycosylation

The contrast in wall phenotypes between S. cerevisiae and C. albicans regarding outer chain backbone dependency was unexpected. While complete loss of the backbone, as in Caoch1Δ/Δ, cannot be tolerated, hyper-mannosylation appears dispensable for normal growth of C. albicans but not for S. cerevisiae. To pinpoint where along the Golgi N-glycosylation biochemical pathway C. albicans and S. cerevisiae have diverged in their outer chain backbone dependency, we undertook a phenotypic comparison of C. albicans and S. cerevisiae strains deleted for each of the Golgi α1,6 MTases that build the backbone, between Och1 and Mnn10 (see Fig. 7 for schematic diagram of this pathway). These deletion strains, including och1Δ/Δ, mnn9Δ/Δ, van1Δ/Δ, anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ (Table 1) were analyzed for a variety of cell wall phenotypes (Figs. 5 and 6). From these experiments, the first indication that typical cell wall phenotypes in C. albicans are Van1-dependent came from their easily visualized colony morphology after growth on solid media (Fig. 5a). Candida albicans homozygous och1Δ/Δ, mnn9Δ/Δ and van1Δ/Δ colonies displayed irregular borders and very wrinkled colony surface. In contrast, mnn11Δ/Δ, mnn10Δ/Δ, and anp1Δ/Δ colonies were smooth and even edged like the wild-type.

Fig. 7.

Fig. 7.

Open in a new tab

Schematic diagram of N-linked backbone extension in Golgi of S. cerevisiae and C. albicans. After core synthesis and transfer to proteins in ER, the N-linked glycan is extended by MTases in the Golgi. In S. cerevisiae, loss of α1,6 MTases Och1, Mnn, Van1, Mnn10, or Anp1 leads to increases in cell wall chitin. In C. albicans, loss of Och1, Mnn9, or Van 1 also stimulates cell wall chitin deposition but backbone truncations beyond Van1 do not trigger the cell wall integrity pathway.

Fig. 6.

Fig. 6.

Open in a new tab

Analysis of cell wall chitin C. albicans and S. cerevisiae α1,6 MTase mutants. a) Candida albicans strains were stained with calcofluor white and imaged by fluorescence microscopy as described in Materials and Methods; ×40 images were taken with the same exposure settings. b) Saccharomyces cerevisiae strains were stained with calcofluor white and imaged as in (a). c) Calcofluor white fluorescence intensity of cells [as described in (a) and (b)] was measured by microscopy, as described in Materials and Methods. d) Cell wall chitin was measured by the Morgan–Elson assay (Leloir and Cardini 1953). After purifying cell wall, absolute levels of glucosamine were measured spectrophotometrically by absorbance at 520 nm (see Materials and methods).

To assay protein glycosylation, ss-RFP-N-HA was introduced into each of these C. albicans and S. cerevisiae strains, and secreted ss-RFP-N-HA from culture supernatants was analyzed by western blotting. This experiment demonstrated the expected hierarchy of N-glycan extensions in these strains, ordered as follows from the longest to the shortest glycan: wild-type → mnn11 → mnn10 = anp1 → van1= mnn9 = och1. In both C. albicans and in S. cerevisiae, the electrophoretic migration of ssRFP-N-HA was similar in anp1Δ and mnn10Δ strains, and in mnn9Δ, van9Δ, and och1Δ (Fig. 5b). While both species displayed the same hierarchical order of glycan extension, when comparing mutants of each particular ortholog, the MW of ssRFP-N-HA was always larger in C. albicans than in S. cerevisiae, presumably due to the N-independent modification we described above (Fig. 4d) that is absent in S. cerevisiae.

The Anp1/Mnn10-dependent backbone is not required for increased wall chitin deposition in C. albicans

Defects in the cell wall activate well characterized signaling pathways that allow yeast to compensate for a weakened wall, by upregulating expression of cell wall biosynthetic genes. In S. cerevisiae, decreased mannan leads to increased deposition of chitin cell wall as reinforcement (Bulik et al. 2003; Lesage and Bussey 2006; Levin 2011). Candida albicans och1Δ/Δ and mnn9Δ/Δ behave like S. cerevisiae as both mutations lead to increased cell wall chitin [Southard et al. 1999; Bates et al. 2006 and see Fig. 6]. The lack of any wall phenotypes in C. albicans mnn10Δ/Δ, mnn11Δ/Δ, and anp1Δ/Δ suggested that the signaling pathway that induces cell wall chitin deposition was not activated in these mutants despite their mannosylation defects. To test this idea, cell wall chitin levels were analyzed in these strains. Wall chitin was first measured indirectly, by assaying fluorescence intensity of cells stained with the chitin-binding fluorescent dye, calcoflour white (see Materials and methods). By this microscopic assay, increased calcoflour white fluorescence was evident in Cavan1Δ/Δ, Camnn9Δ/Δ, Caoch1Δ/Δ but not in Camnn10Δ/Δ, mnn11Δ/Δ, or anp1Δ/Δ (Fig. 6a). As expected, all S. cerevisiae α1,6 MTase mutants had increased levels calcofluor white fluorescence (Fig. 6b). These experiments also underscored the correlation of increased cell wall chitin with irregular cell size and the tendency to form clumps (Fig. 6a). The relative fluorescence level in these strains was quantitated by measuring fluorescence intensity (see Material and methods). By this assay, there was ∼5- to 10-fold increased fluorescence in all the S. cerevisiae MTase mutants compared with the parental strain (Fig. 6c). In C. albicans, similar differences were observed between the parent and van1Δ/Δ, mnn9Δ/Δ, and och1Δ/Δ. However, calcofluor white fluorescence levels of Camnn10Δ/Δ, mnn11Δ/Δ, and anp1Δ/Δ were indistinguishable from the parent (Fig. 6c).

To verify these results using an independent assay, cell wall chitin content was determined directly using the Morgan–Elson assay, in which enriched cell wall fractions were hydrolyzed, and the GlcNAc concentration measured spectroscopically (Fig. 6d;Leloir and Cardini 1953). These results mirrored those obtained by calcofluor white staining; i.e. cell wall chitin levels in C. albicans mnn10Δ/Δ, mnn11Δ/Δ, and anp1Δ/Δ were similar to those of the wild-type parent while van1Δ/Δ, mnn9Δ/Δ, and och1Δ/Δ levels were increased. These different assays yielded different levels of cell wall chitin reduction (5- vs ∼2-fold), a difference that has been previously noted (Lenardon et al. 2010) and likely represents the different sensitivities of the methods. Nevertheless, taken together, these results demonstrated that the mannan reduction due to loss of ANP1, MNN10, or MNN11 does not induce cell wall integrity pathways that occur in the corresponding S. cerevisiae mannan mutants.

Discussion

The outer chain of N-glycans S. cerevisiae and C. albicans are similar in structure (Ballou 1990; Shibata et al. 2007). Thus it has been assumed that they are also similar in function (for review Martinez-Duncker et al. 2014). Our comparative phenotypic analyses of α1,6 Golgi MTase mutants established that this assumption is incorrect. Despite the apparent similarity of their backbone structure, the dependence on these glycans has diverged considerably between S. cerevisiae and C. albicans. While both species mount a robust cell wall stress response when defective in the earliest steps of backbone synthesis, S. cerevisiae cannot tolerate any defects in backbone extension while C. albicans appears to be relatively unaffected by much of its absence.

This study was initially motivated by our finding that the C. albicans mnn10Δ/Δ strain lacked any expected cell wall phenotypes (Figs. 1–3). Candida albicans is notorious for its expansion of gene families whose members have overlapping functions in Golgi mannosylation, for instance the 5-membered Mnt1/Kre2 (Mora-Montes et al. 2010), the 6-membered Mnn1 (Bates et al. 2013), the 6-membered Mnn2 (Hall et al. 2013), and the 9-membered Bmt family (Mille et al. 2008). Presumably, this redundancy has been selected during fungal evolution because it confers fitness. Given these precedents, a redundant gene whose activity masked loss of MNN10 seemed a plausible explanation for the lack of mnn10Δ/Δ phenotypes. As MNN10 and MNN11 are significantly similar in sequence, MNN11 seemed like a good candidate for such a gene. However, this idea was incorrect since neither the single nor the double Camnn10Δ/Δ mnn11Δ/Δ were impaired in any aspect of growth, hyphal formation, or even phagocytosis by macrophage.

We demonstrated that the step in α1,6 backbone biosynthesis where S. cerevisiae and C. albicans diverge with regards to their cell wall phenotypes and stress response is Van1-dependent (Fig. 7). Loss of VAN1 or mutants that acts upstream, including mnn9Δ/Δ and och1Δ/Δ, resemble van1Δ/Δ in phenotypic severity while mutants downstream VAN1 (i.e. anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ) have no significant impact on cell division or hyphal formation. Importantly, this Van1-dependent step also defined the point at which activation of cell wall stress response occurred, as measured by induction of cell wall chitin deposition (Figs. 6 and 7).

These data raise the important question of how C. albicans compensates for the apparent large-scale reduction of cell wall N-linked mannose in anp1/mnn10/mnn11 mutants. Several possibilities, not mutually exclusive, can be envisaged. One is that O-linked glycosylation plays a more prominent role in bolstering the C. albicans cell wall than S. cerevisiae. However, evidence from past studies suggests otherwise. Complete loss of C. albicans Pmt MTases that initiate O-linked mannose addition in the ER leads to inviability but this phenotype is likely related to protein misfolding in the ER rather than defects in bulk cell wall mannan (Prill et al. 2005). Furthermore, loss of MNT1 and/or MNT2, which participate in Golgi O-glycosylation, also fail to induce the cell wall integrity pathway (Munro et al. 2005). A second possibility is that C. albicans cell wall glycoproteins are modified in additional ways that compensate for the reduction of glycan length resulting from Anp1/Mnn10/Mnn11 reduced activity. In this regard, our observation that ssRFP-N-HA migrated with an increased MW in C. albicans compared with S. cerevisiae, irrespective of genetic background, may be relevant. The additional MW of ssRFP-N-HA in C. albicans is due to post-translational modifications that are dependent on its entry into the secretory pathway since RFP lacking the signal sequence accumulates with the same size in both S. cerevisiae and C. albicans. Moreover, this C. albicans-specific modification is independent of N-glycosylation since it is seen on ssRFP-X-HA, which is otherwise identical to ssRFP-N-HA except for the absence of the critical asparagine (Fig. 4, c–e). The additional modifications we observed on ssRFP-N-HA is probably not an artifact of this reporter, as a comparison of bulk glycoproteins from C. albicans and S. cerevisiae by lectin blots shows a similar phenomenon (our unpublished data). Although the nature of these modification(s) is currently unknown, they do not occur in S. cerevisiae. It is therefore tempting to speculate that these additional modifications may serve to bolster C. albicans cell wall strength in a manner that can compensate for the absence of Anp1-dependent N-glycan.

A third possibility is that the “readout” for cell wall stress response in response to mannan defects, i.e. increased chitin deposition at the wall, may not benefit C. albicans in the way it does other fungal and yeast species. The experiments performed in this study did not address the roles of these MTase genes in the environment normally habituated by C. albicans, i.e. the human gut. With regard to balancing loss or gain of fitness as a commensal, it is possible that cell wall chitin, glucan, or other molecules that are induced by mannan reduction in nonpathogenic yeast may not benefit C. albicans as a gut-evolved commensal.

In either case, the experiments in this study demonstrate that C. albicans has a different “mannan” threshold for what constitutes a weakened wall compared with S. cerevisiae. Our data suggest a model in which the signal for activation of the cell wall salvage pathway is limited by the length of Van1-dependent backbone (Fig. 7). Only when levels of N-linked mannan fall below this threshold will C. albicans attempt to reinforce wall strength by upregulating chitin synthesis and thereby preventing lysis. These results suggest C. albicans has developed an alternate compensatory mechanism to maintain the cell wall’s integrity when N-glycans are truncated. Future experiments that investigate how C. albicans can tolerate the loss of N-linked mannan will be necessary to better define how C. albicans compensates its loss.

Finally, it is important to note that multiple studies in the current literature cite the importance of N-linked glycans during host fungal interactions but many employ och1Δ/Δ or mnn9Δ/Δ mutants as surrogates for all N-linked backbone outer chain mutants. The results described here demonstrate that it is no longer appropriate to extrapolate cell wall integrity pathways from S. cerevisiae to C. albicans. The availability of additional α1,6-MTase mutants described here, with incremental loss of the N-linked outer chain backbone, opens new avenues to study the roles of these important carbohydrates in C. albicans pathogenesis.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author. All strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material is available at GENETICS online.

Supplementary Material

iyac048_Supplemental_Material_Figure_S1
Click here for additional data file. (4.2MB, pdf)
iyac048_Supplemental_Material
Click here for additional data file. (17.5KB, docx)

Acknowledgments

The authors wish to thank Sabine Keppler-Ross and Binghao Zhang for strain construction (SKY68 and BZY1). This work is dedicated to the memories of Clint Ballou and Bill Lennarz.

Conflicts of interest

None declared.

Literature cited

  1. Ballou CE. Isolation, characterization and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 1990;185:440–470. [DOI] [PubMed] [Google Scholar]
  2. Bates S, Hall RA, Cheetham J, Netea MG, MacCallum DM, Brown AJP, Odds FC, Gow NAR.. Role of the Candida albicans MNN1 gene family in cell wall structure and virulence. BMC Res Notes. 2013;6(294):294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates S, Hughes HB, Munro CA, Thomas WPH, MacCallum DM, Bertram G, Atrih A, Ferguson MAJ, Brown AJP, Odds FC, et al. Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J Biol Chem. 2006;281(1):90–98. [DOI] [PubMed] [Google Scholar]
  4. Bulik DA, Olczak M, Lucero HA, Osmond BC, Robbins PW, Specht CA.. Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell. 2003;2(5):886–900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chapman RE, Munro S.. The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway. Eur J Mol Biol. 1994;13:4896–4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chi JH, Roos J, Dean N.. The OST4 gene of Saccharomyces cerevisiae encodes an unusually small protein required for normal levels of oligosaccharyltransferase activity. J Biol Chem. 1996;271(6):3132–3140. [DOI] [PubMed] [Google Scholar]
  7. Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ.. Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology. 1997;143:303–311. [DOI] [PubMed] [Google Scholar]
  8. Dean N. Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci U S A. 1995;92(5):1287–1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dean N. Asparagine-linked glycosylation in the yeast Golgi. Biochim Biophys Acta. 1999;1426(2):309–322. [DOI] [PubMed] [Google Scholar]
  10. Dean N, Ng H.. Method for CRISPR/Cas9 Mutagenesis in Candida albicans. Bio Protoc. 2018;8(8):e2814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dean N, Poster J.. Molecular and phenotypic analysis of the S. cerevisiae MNN10 gene identifies a family of related glycosyltransferases. Glycobiology. 1996;6:73–81. [DOI] [PubMed] [Google Scholar]
  12. Gemmill TR, Trimble RB.. Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta. 1999;1426:227–237. [DOI] [PubMed] [Google Scholar]
  13. Gietz RD, Schiestl RH.. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2(1):31–34. [DOI] [PubMed] [Google Scholar]
  14. Gow NA, van de Veerdonk FL, Brown AJ, Netea MG.. Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nat Rev Microbiol. 2011;10(2):112–122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gow NAR, Latge JP, Munro CA.. The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr. 2017;5(3) [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hall RA, Bates S, Lenardon MD, Maccallum DM, Wagener J, Lowman DW, Kruppa MD, Williams DL, Odds FC, Brown AJP, et al. The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans. PLoS Pathog. 2013;9(4):e1003276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hall RA, Gow NA.. Mannosylation in Candida albicans: role in cell wall function and immune recognition. Mol Microbiol. 2013;90(6):1147–1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hashimoto H, Yoda K.. Novel membrane protein complexes for protein glycosylation in the yeast Golgi apparatus. Biochem Biophys Res Commun. 1997;241(3):682–686. [DOI] [PubMed] [Google Scholar]
  19. Jigami Y, Odani T.. Mannosylphosphate transfer to yeast mannan. Biochim Biophys Acta. 1999;1426(2):335–345. [DOI] [PubMed] [Google Scholar]
  20. Jungmann J, Munro S.. Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with a-1,6-mannosyltransferase activity. EMBO J. 1998;17:423–434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jungmann J, Rayner JC, Munro S.. The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. J Biol Chem. 1999;274(10):6579–6585. [DOI] [PubMed] [Google Scholar]
  22. Keppler-Ross S, Douglas L, Konopka JB, Dean N.. Recognition of yeast by murine macrophages requires mannan but not glucan. Eukaryot Cell. 2010;9:1776–1787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keppler-Ross S, Noffz C, Dean N.. A new purple fluorescent color marker for genetic studies in Saccharomyces cerevisiae and Candida albicans. Genetics. 2008;179(1):705–710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leloir LF, Cardini CE.. The biosynthesis of glucosamine. Biochim Biophys Acta. 1953;12(1–2):15–22. [DOI] [PubMed] [Google Scholar]
  25. Lenardon MD, Munro CA, Gow NA.. Chitin synthesis and fungal pathogenesis. Curr Opin Microbiol. 2010;13(4):416–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lesage G, Bussey H.. Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006;70(2):317–343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Levin DE. Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics. 2011;189(4):1145–1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lewis LE, Bain JM, Lowes C, Gillespie C, Rudkin FM, Gow NAR, Erwig L-P.. Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants. PLoS Pathog. 2012;8(3):e1002578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Longtine MS, McKenzie A III, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR.. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998;14:953–961. [DOI] [PubMed] [Google Scholar]
  30. Lussier M, Sdicu AM, Bussereau F, Jacquet M, Bussey H.. The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J Biol Chem. 1997;272(24):15527–15531. [DOI] [PubMed] [Google Scholar]
  31. Lussier M, Sdicu AM, Bussey H.. The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim Biophys Acta. 1999;1426(2):323–334. [DOI] [PubMed] [Google Scholar]
  32. Martinez-Duncker I, Diaz-Jimenez DF, Mora-Montes HM.. Comparative analysis of protein glycosylation pathways in humans and the fungal pathogen Candida albicans. Int J Microbiol. 2014;2014:267497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mille C, Bobrowicz P, Trinel P-A, Li H, Maes E, Guerardel Y, Fradin C, Martínez-Esparza M, Davidson RC, Janbon G, et al. Identification of a new family of genes involved in beta-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans. J Biol Chem. 2008;283(15):9724–9736. [DOI] [PubMed] [Google Scholar]
  34. Mora-Montes HM, Bates S, Netea MG, Castillo L, Brand A, Buurman ET, Díaz-Jiménez DF, Jan Kullberg B, Brown AJP, Odds FC, et al. A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions. J Biol Chem. 2010;285(16):12087–12095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mora-Montes HM, Ponce-Noyola P, Villagómez-Castro JC, Gow NA, Flores-Carreón A, López-Romero E.. Protein glycosylation in Candida. Future Microbiol. 2009;4(9):1167–1183. [DOI] [PubMed] [Google Scholar]
  36. Munro CA, Bates S, Buurman ET, Hughes HB, Maccallum DM, Bertram G, Atrih A, Ferguson MAJ, Bain JM, Brand A, et al. Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence. J Biol Chem. 2005;280(2):1051–1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murad A, Lee PID, Broadbent I, Barelle C, Brown A.. CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast. 2000;16:325–327. [DOI] [PubMed] [Google Scholar]
  38. Nakanishi-Shindo Y, Nakayama K, Tanaka A, Toda Y, Jigami Y.. Structure of the N-linked oligosaccharides that show the complete loss of a1,6 polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J Biol Chem. 1993;268:26338–26345. [PubMed] [Google Scholar]
  39. Nakayama K, Nagasu T, Shimma Y, Kuromitsu J, Jigami Y.. OCH1 encodes a novel membrane-bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharidesa. EMBO J. 1992;11(7):2511–2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ng H, Dean N.. Dramatic improvement of CRISPR/Cas9 editing in Candida albicans by increased single guide RNA expression. mSphere. 20172(2):e00385–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Park H, Myers CL, Sheppard DC, Phan QT, Sanchez AA, E Edwards J, Filler SG.. Role of the fungal Ras-protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis. Cell Microbiol. 2005;7(4):499–510. [DOI] [PubMed] [Google Scholar]
  42. Prill SK-H, Klinkert B, Timpel C, Gale CA, Schröppel K, Ernst JF.. PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol Microbiol. 2005;55(2):546–560. [DOI] [PubMed] [Google Scholar]
  43. Rayner JC, Munro S.. Identification of the MNN2 and MNN5 mannosyltransferases required for forming and extending the mannose branches of the outer chain mannans of Saccharomyces cerevisiae. J Biol Chem. 1998;273(41):26836–26843. [DOI] [PubMed] [Google Scholar]
  44. Robinson JS, Klionsky DJ, Banta LM, Emr SD.. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988;8(11):4936–4948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rodionov D, Romero PA, Berghuis AM, Herscovics A.. Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with alpha-1,6 mannosylpolymerase activity. Protein Expr Purif. 2009;66(1):1–6. [DOI] [PubMed] [Google Scholar]
  46. Shibata N, Arai M, Haga E, Kikuchi T, Najima M, Satoh T, Kobayashi H, Suzuki S.. Structural identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B-792 (serotype B) and J-1012 (serotype A) as beta-1,2-linked oligomannosyl residues. Infect Immun. 1992;60(10):4100–4110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shibata N, Ikuta K, Imai T, Satoh Y, Satoh R, Suzuki A, Kojima C, Kobayashi H, Hisamichi K, Suzuki S, et al. Existence of branched side chains in the cell wall mannan of pathogenic yeast, Candida albicans. Structure-antigenicity relationship between the cell wall mannans of Candida albicans and Candida parapsilosis. J Biol Chem. 1995;270(3):1113–1122., [DOI] [PubMed] [Google Scholar]
  48. Shibata N, Suzuki A, Kobayashi H, Okawa Y.. Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms. Biochem J. 2007;404(3):365–372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Southard SB, Specht CA, Mishra C, Chen-Weiner J, Robbins PW.. Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins. J Bacteriol. 1999;181(24):7439–7448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stolz J, Munro S.. The components of the Saccharomyces cerevisiae mannosyltransferase complex M-Pol I have distinct functions in mannan synthesis. J Biol Chem. 2002;277(47):44801–44808. [DOI] [PubMed] [Google Scholar]
  51. Wilson RB, Davis D, Enloe BM, Mitchell AP.. A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast. 2000;16:65–70. [DOI] [PubMed] [Google Scholar]
  52. Wilson RB, Davis D, Mitchell AP.. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol. 1999;181(6):1868–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yadav B, Mora-Montes HM, Wagener J, Cunningham I, West L, Haynes K, Brown AJP, Gow NAR.. Differences in fungal immune recognition by monocytes and macrophages: N-mannan can be a shield or activator of immune recognition. Cell Surf. 2020;6:100042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang SQ, Zou Z, Shen H, Shen SS, Miao Q, Huang X, Liu W, Li LP, Chen SM, Yan L, et al. Mnn10 maintains pathogenicity in Candida albicans by extending alpha-1,6-mannose backbone to evade host Dectin-1 mediated antifungal immunity. PLoS Pathog. 2016;12(5):e1005617. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

iyac048_Supplemental_Material_Figure_S1
Click here for additional data file. (4.2MB, pdf)
iyac048_Supplemental_Material
Click here for additional data file. (17.5KB, docx)

Data Availability Statement

The data underlying this article will be shared on reasonable request to the corresponding author. All strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material is available at GENETICS online.

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES