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Cited by 128Fungal and yeast genetics researchGlycosylation and Glycoproteins ResearchAntifungal resistance and susceptibility

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The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans

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Hall, Rebecca A., Bates, Steven, Lenardon, Megan D., MacCallum, Donna M., Wagener, Jeanette, Lowman, Douglas W., Kruppa, Michael D., Williams, David L., Odds, Frank C., Brown, Alistair J. P., Gow, Neil A. R.·2013·PLoS Pathogens
DOI 10.1371/journal.ppat.1003276PMC PMC3636026
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Abstract

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.

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Figure 1 The MNN2 gene family provides the mannan scaffold to which the phosphomannan is attached. Mutants were incubated in 30 µg/ml Alcian Blue for 10 min and the amount of dye bound to the cell wall estimated by absorbance. Data represent the mean amount of dye bound per cell ± SD from 6 independent experiments, *p<0.05, **p<0.01.
Figure 1 The MNN2 gene family provides the mannan scaffold to which the phosphomannan is attached. Mutants were incubated in 30 µg/ml Alcian Blue for 10 min and the amount of dye bound to the cell wall estimated by absorbance. Data represent the mean amount of dye bound per cell ± SD from 6 independent experiments, *p<0.05, **p<0.01.CC BY · PubMed Central
Figure 2 The MNN2 gene family is required for cell wall integrity. (A) Stationary phase cultures were diluted to OD600 0.5 in YPD and 3 µl from 1∶10 serial dilutions spotted onto YPD agar plates containing either 100 µg/ml CFW, 0.03% SDS, 100 µg/ml Congo Red (CR) and incubated at 30°C for 48 h, or spotted on YPD agar and incubated at 42°C. (B) Activation of the cell wall salvage pathway was determined by western blot on crude protein extracts prepared from exponentially growing cells. The PhosphoPlus P44/42 antibody detects phosphorylated (activated) Mkc1. As a positive control for Mkc1 activation wild type (CAI-4+CIp10) cells were exposed to 0.0032 µg/ml caspofungin for 10 min. Lane 1 wild type +CIp10, lane 2 wild type+caspofungin, lane 3 mnn2Δ, lane 4 mnn2Δ+MNN2, lane 5 mnn26Δ, lane 6 mnn26Δ+MNN26, lane 7 mnn2Δ/mnn26Δ, lane 8 mnn2Δ/mnn26Δ+MNN2/MNN26, lane 9 mnn23Δ/mnn26Δ, lane 10 mnn24Δ/mnn26Δ, lane 11 triple mutant, lane 12 quintuple mutant, lane 13 sextuple mutant, lane 14 sextuple mutant+MNN2/MNN26.
Figure 2 The MNN2 gene family is required for cell wall integrity. (A) Stationary phase cultures were diluted to OD600 0.5 in YPD and 3 µl from 1∶10 serial dilutions spotted onto YPD agar plates containing either 100 µg/ml CFW, 0.03% SDS, 100 µg/ml Congo Red (CR) and incubated at 30°C for 48 h, or spotted on YPD agar and incubated at 42°C. (B) Activation of the cell wall salvage pathway was determined by western blot on crude protein extracts prepared from exponentially growing cells. The PhosphoPlus P44/42 antibody detects phosphorylated (activated) Mkc1. As a positive control for Mkc1 activation wild type (CAI-4+CIp10) cells were exposed to 0.0032 µg/ml caspofungin for 10 min. Lane 1 wild type +CIp10, lane 2 wild type+caspofungin, lane 3 mnn2Δ, lane 4 mnn2Δ+MNN2, lane 5 mnn26Δ, lane 6 mnn26Δ+MNN26, lane 7 mnn2Δ/mnn26Δ, lane 8 mnn2Δ/mnn26Δ+MNN2/MNN26, lane 9 mnn23Δ/mnn26Δ, lane 10 mnn24Δ/mnn26Δ, lane 11 triple mutant, lane 12 quintuple mutant, lane 13 sextuple mutant, lane 14 sextuple mutant+MNN2/MNN26.CC BY · PubMed Central
Figure 3 Deletion of MNN2 gene family members increases chitin content and β-glucan exposure. Exponentially growing cells were stained with A) WGA-FITC to visualise chitin B) Fc Dectin-1 to visualise β-glucan and C) ConA-TR to visualise mannan. Scale bar represents 10 µm.
Figure 3 Deletion of MNN2 gene family members increases chitin content and β-glucan exposure. Exponentially growing cells were stained with A) WGA-FITC to visualise chitin B) Fc Dectin-1 to visualise β-glucan and C) ConA-TR to visualise mannan. Scale bar represents 10 µm.CC BY · PubMed Central
Figure 4 The Mnn2 family of mannosyltransferases regulates mannan fibril length. Electron micrographs showing the ultrastructure of the cell walls of the (A) wild type (CAI-4+CIp10), (B) mnn2Δ/mnn26Δ, (C) sextuple mutant, (D) sextuple mutant+MNN2/MNN26 strains. The scale bar represents 500 nm. (E) Mannan fibril length was measured in 8 randomly selected cells. Each cell was measured 10 times in different locations. Data represent the means ± SD, **p<0.01.
Figure 4 The Mnn2 family of mannosyltransferases regulates mannan fibril length. Electron micrographs showing the ultrastructure of the cell walls of the (A) wild type (CAI-4+CIp10), (B) mnn2Δ/mnn26Δ, (C) sextuple mutant, (D) sextuple mutant+MNN2/MNN26 strains. The scale bar represents 500 nm. (E) Mannan fibril length was measured in 8 randomly selected cells. Each cell was measured 10 times in different locations. Data represent the means ± SD, **p<0.01.CC BY · PubMed Central
Figure 5 Deletion of MNN2 gene family members results in a lower molecular weight and less complex N-mannan. (A) C. albicans N-mannans were extracted from cells and analysed by GPC. Solid line represents wild type (CAI-4+CIp10), dotted line represents mnn26Δ, dashed line represents mnn2Δ/mnn26 and the dashed and dotted line represents mnn24Δ/mnn26Δ. The numbers designate the peaks discussed in the text. (B) Representative proton NMR spectra for the N-mannan extracted from wild type, mnn2Δ, mnn26Δ, mnn2Δ/mnn26Δ, sextuple mutant and the reconstituted sextuple mutant.
Figure 5 Deletion of MNN2 gene family members results in a lower molecular weight and less complex N-mannan. (A) C. albicans N-mannans were extracted from cells and analysed by GPC. Solid line represents wild type (CAI-4+CIp10), dotted line represents mnn26Δ, dashed line represents mnn2Δ/mnn26 and the dashed and dotted line represents mnn24Δ/mnn26Δ. The numbers designate the peaks discussed in the text. (B) Representative proton NMR spectra for the N-mannan extracted from wild type, mnn2Δ, mnn26Δ, mnn2Δ/mnn26Δ, sextuple mutant and the reconstituted sextuple mutant.CC BY · PubMed Central
Figure 6 Predicted structures of the C. albicans N-mannan in the respective mutants as identified by NMR. The N-mannan structure is based on the work by Shibata and colleagues [26], [43], [45], [48].
Figure 6 Predicted structures of the C. albicans N-mannan in the respective mutants as identified by NMR. The N-mannan structure is based on the work by Shibata and colleagues [26], [43], [45], [48].CC BY · PubMed Central
Figure 7 Deletion of MNN2 gene family members reduces immune recognition. PBMCs were co-incubated with heat-killed C. albicans cells at an MOI of 0.4 for 24 h. The concentration of secreted cytokines was measured by ELISA. Data represent the means ± SEM from 8 independent experiments, *p<0.05, **p<0.01.
Figure 7 Deletion of MNN2 gene family members reduces immune recognition. PBMCs were co-incubated with heat-killed C. albicans cells at an MOI of 0.4 for 24 h. The concentration of secreted cytokines was measured by ELISA. Data represent the means ± SEM from 8 independent experiments, *p<0.05, **p<0.01.CC BY · PubMed Central
Figure 8 Mnn2 members are required for virulence of C. albicans. (A) Mutants were screened for virulence defects in the G. mellonella infection model. Larvae were infected with 2.5×105 C. albicans cells and incubated at 37°C. Data represent the mean larvae survival times ± SD from 3 independent experiments, *p<0.05, **p<0.01. (B) wild type (CAI-4+CIp10), mnn2Δ/mnn26Δ, mnn2Δ/mnn26Δ+MNN2/MNN26 and the sextuple mutant were tested for their ability to cause infection in the 28 day mouse model of systemic infection.
Figure 8 Mnn2 members are required for virulence of C. albicans. (A) Mutants were screened for virulence defects in the G. mellonella infection model. Larvae were infected with 2.5×105 C. albicans cells and incubated at 37°C. Data represent the mean larvae survival times ± SD from 3 independent experiments, *p<0.05, **p<0.01. (B) wild type (CAI-4+CIp10), mnn2Δ/mnn26Δ, mnn2Δ/mnn26Δ+MNN2/MNN26 and the sextuple mutant were tested for their ability to cause infection in the 28 day mouse model of systemic infection.CC BY · PubMed Central
Figure 9 Hypothetical model for the actions of the six Mnn2 mannosyltransferases. See text for details.
Figure 9 Hypothetical model for the actions of the six Mnn2 mannosyltransferases. See text for details.CC BY · PubMed Central

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Metadata

Authors
Hall, R. A., Bates, S., Lenardon, M. D., MacCallum, D. M., Wagener, J., Lowman, D. W., Kruppa, M. D., Williams, D. L., Odds, F. C., Brown, A. J. P., & Gow, N. A. R.
Affiliations
  • University of Aberdeen (GB)
  • University of Exeter (GB)
  • East Tennessee State University (US)
Corresponding author
Gow, Neil A. R.
ORCID
Venue
PLoS Pathogens
Publisher
Public Library of Science (PLoS)
Published
2013-04-25
Volume
9
Issue
4
Pages
e1003276
ISSN
  • 1553-7366 (ISSN-L)
  • 1553-7374
DOI
10.1371/journal.ppat.1003276
PMC
PMC3636026
OpenAlex
W2086193922
Cited by
128
References
60
Citations by year
2026: 8 · 2025: 9 · 2024: 13 · 2023: 4 · 2022: 12 · 2021: 6 · 2020: 9 · 2019: 10 · 2018: 16 · 2017: 9 · 2016: 11 · 2015: 9 · 2014: 6 · 2013: 6
FWCI
4.78
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Pathogens (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Kent Academic Repository (University of Kent) (acceptedVersion)
  • Aberdeen University Research Archive (Aberdeen University) (submittedVersion)
  • Digital Commons - East Tennessee State University (East Tennessee State University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Open Research Exeter (University of Exeter) (submittedVersion)
  • University of Birmingham Research Portal (University of Birmingham) (submittedVersion, cc-by)
  • University of Birmingham Research Portal (University of Birmingham) (submittedVersion, other-oa)
Funders
  • Wellcome Trust (099215, 086827, 080088, 075470)
  • European Commission (260338)
  • Medical Research Council (MR/J008230/1)
  • Biotechnology and Biological Sciences Research Council (BB/F00513X/1)
  • National Institute of General Medical Sciences (R01GM053522)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
Keywords
Candida albicans, fungal cell wall, glycosyltransferases, pathogen-associated molecular patterns, innate immune recognition, inflammatory cytokines, fungal virulence, GPI-anchored proteins, N-glycans
MeSH terms
Animals, Candida albicans, Candidiasis, Cell Wall, Female, Fungal Proteins, Humans, Mannans, Mannose, Mannosyltransferases, Membrane Glycoproteins, Mice, Inbred BALB C, Polysaccharides, Sequence Alignment, Sequence Deletion, Receptors, Pattern Recognition, Mice

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