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Role of the Candida albicans MNN1 gene family in cell wall structure and virulence
© Bates et al.; licensee BioMed Central Ltd. 2013
Received: 30 April 2013
Accepted: 24 July 2013
Published: 26 July 2013
The Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation.
We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitr o and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14 Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14 Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model.
Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence.
Candida albicans is the most common opportunistic fungal pathogen of humans causing superficial infections of the mucosa, and life threatening systemic infections in immunocompromised and severely ill patients [1–3]. The fungal cell wall is a dynamic structure required for maintaining cell shape and providing protection from changes in the extracellular environment. In addition, the cell wall acts as the first point of contact with the host and plays an essential role in the host-fungal interaction. The cell wall is composed of an inner skeletal layer of β-glucans and chitin, decorated with an outer layer enriched in mannoproteins that are heavily post-translationally modified through the addition of N- and O-linked mannans [4–6]. These mannans have been shown to play a vital role in cell wall integrity, adhesion and virulence, and constitute one of the main C. albicans pathogen associated molecular patterns (PAMPs) recognised by the host innate immune system [7–16].
In C. albicans structural studies of O-mannan have shown that it typically consists of one to five α1,2-linked mannose residues attached to serine or threonine, and that these are required for full virulence [11, 12]. In addition studies using anti-β-mannan specific antibodies have demonstrated that O-mannan may also contain β1,2-linked mannose residues , presumably added by members of the BMT family . This is different to Saccharomyces cerevisiae where O-mannan consists of one to two α1,2-linked mannose residues capped with α1,3-linked mannose residues transferred by members of the Mnn1 family. N-glycosylation is initiated in the endoplasmic reticulum (ER) with the transfer of the N-mannan precursor (Glc3Man9GlcNAc2) to specific asparagine residues in the protein. The precursor is then processed by ER resident glycosidases to form the mature Man8GlcNAc2 core conserved across eukaryotes . In C. albicans as well as other fungi, the core is then extensively modified through outer chain elaboration as the protein passes through the Golgi [7, 14, 16, 17]. Outer chain elaboration is initiated through the addition of a single α1,6-linked mannose residue to the core by Och1 , and the subsequent extension of the α1,6-backbone by the sequential action of the mannan polymerase I and II enzyme complexes [17, 18]. The α1,6-backbone is then further elaborated with side chains, which in C. albicans consists of α1,2-, α1,3-, and β1,2-linked mannose residues [14, 16, 19]. In addition both N-mannan and O-mannan structures can be further modified through the addition of phosphomannan, which in C. albicans consists of a chain of β1,2-linked mannose residues attached through a phosphodiester linkage [10, 20, 21].
Previous work has clearly identified outer chain N-glycosylation as an important factor in the host-fungal interaction and virulence [7, 8, 10, 14–16]. However, most work to date has focused on the early stages of outer chain elaboration where the mutants display gross defects in glycosylation. In S. cerevisiae the MNN1/2 family contains genes involved in the elaboration of O- and N-mannan; in particular the MNN1 sub-family encodes α1,3-mannosyltranferases important for the addition of the terminal mannose residues in O- and N-mannan [22, 23]. In C. albicans we have identified six MNN1 family members and here we report the first analysis of this gene family in C. albicans. Null mutants in most of its members displayed no alteration of phenotype, suggesting possible functional redundancy. However, the mnn14 Δ mutant, whilst displaying only subtle cell wall changes, was severely attenuated in virulence, demonstrating that specific glycans are important in the host-fungal interaction.
Results and discussion
Analysis and disruption of C. albicans MNN1 family members
Homology of MNN1 gene family products
% Identity (% similarity)a
Glycosylation defects in the MNN1 family mutants
To screen for more subtle glycosylation defects, and to discount the impact of altered surface charge on electrophoretic mobility, we epitope tagged Hex1 with V5-6xHis in the null mutants and conducted Western blot analysis of Hex1-V5-6xHis following standard denaturing gel electrophoresis. Following Western blot analysis Hex1-V5-6xHis was apparent in both an unmodified (67 kDa) and a heavily glycosylated (~125 kDa) form in soluble protein extracts. In the pmr1 Δ mutant, which is known to display glycosylation defects , the glycosylated form of Hex1-V5-6xHis demonstrated a clear increase in electrophoretic mobility characteristic of this mutant’s gross defect in glycosylation. However, no change in electrophoretic mobility of the glycosylated form of Hex1-V5-6xHis was observed for any of the MNN1 family mutants (Figure 3B), demonstrating that none of these mutants displayed a gross defect in N-glycosylation. In addition, this would suggest that the decreased electrophoretic mobility of Hex1 seen in the mnn14 Δ null mutant following native gel electrophoresis and activity staining is likely to be due to differences in the proteins surface charge and not the overall level of glycosylation.
Alcian blue binding
Alcian blue binding ± SD (μg bound/OD6001.0)
183.1 ± 8.7
148.2 ± 21.6
143.3 ± 37.4
175.3 ± 7.4
181.9 ± 3.9
184.1 ± 2.6
191.1 ± 6.7
25.2 ± 2.2
The structure of O-linked mannans was also assessed by TLC and no significant differences were observed in any of the MNN1 family mutants (Figure 3D). This is consistent with the previous finding that Mnt1 and Mnt2 are the principal enzymes involved in the extension of O-glycans, and that C. albicans lacks α1,3-linked residues in its O-mannan .
Cell wall associated defects
Host-pathogen interaction and virulence
As cell wall mannan has been shown to play an important role in the stimulation of the host response  we tested the ability of the null mutants to induce cytokine production by human PBMCs. All the MNN1 family mutants were as potent at stimulating TNFα and IL-6 production as wild type C. albicans (data not shown). Virulence of the single mutants was tested in a Galleria mellonella larvae model of infection. In this model all larvae infected with the wild type strain succumbed to infection by day 2, whereas 75% of larvae infected with the pmr1 Δ mutant, which displays a gross defect in glycosylation, survived to the end of the experiment. However, none of the MNN1 family mutants displayed a virulence defect, demonstrating that none of the individual family members are required for virulence in this model. Because the mnn14 Δ mutant displayed an altered phenotype in vitro we tested its virulence in a murine model of disseminated infection; in addition the mnn1 Δ and mnn12 Δ mutants were also tested. The mnn1 Δ and mnn12 Δ null mutants were unaltered in virulence. However, the mnn14 Δ null mutant was clearly attenuated in virulence (log-rank test; p < 0.001) with all mice surviving to the end of the experiment at 28 days compared to a median survival time of 9 days for the wild type control. Mice infected with the mnn14 Δ null mutant also displayed a clear reduction in tissue burdens at 28 days compared to that seen post-mortem following infection with the wild type strain, with a >2 log reduction in kidney burdens (log10 CFU/g 4.0 ± 1.3 cf. 6.5 ± 0.4) and a 1 log reduction in the brain (log10 CFU/g 3.3 ± 0.9 cf. 4.3 ± 0.4). Hence the mnn14 Δ null mutant was significantly attenuated in virulence in the murine model of systemic candidiasis.
The C. albicans cell wall is the immediate point of contact between the invading fungus and the host, and previous work has clearly identified both O- and N- mannan structures as important in the host-pathogen interaction and virulence. In this study, we present the first analysis of the C. albicans MNN1 gene family, which is predicted to encode capping enzymes involved in the terminal stages of glycosylation. We generated single null mutants in the six members of the C. albicans MNN1 gene family. The majority of the mutants generated, with the exception of the mnn14 Δ null mutant, demonstrated no discernible change in phenotype potentially due to functional redundancy as seen with some other C. albicans mannosyltransferase gene families [9, 11, 16]. Disruption of MNN14 however led to both in vitro and in vivo defects. The mnn14 Δ null mutant displayed subtle morphogenesis defects and hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting altered cell wall structure or permeability. However, no gross changes in cell wall composition or N-glycan extension were identified, although an extension of phosphomannan chains was apparent. This extension of phosphomannan in the mnn14 Δ null mutant could be a compensatory mechanism for its absence, through an increase in the expression, or activity, of other mannosyltransferases. Alternatively Mnn14 could potentially act as a capping enzyme to terminate extension similar to the proposed role of MNN1 in S. cerevisiae. Although the mnn14 Δ null mutant only displayed subtle cell wall defects this mutant did display a severe defect in virulence in a murine infection model, apparent both in terms of overall survival and associated tissue burdens. This virulence defect was not associated with differences in the induction of inflammatory responses, as no defect was seen in the TNFα and IL-6 host cytokine production in vitro, which suggests that the virulence defect may not be due to an alteration of the host response. Overall, therefore, the MNN1 family appears to display redundancy, with the exception of MNN14, which may therefore play a distinct role either in the synthesis of specific epitopes or in the modification of a discrete protein(s) required for virulence and the host-pathogen interaction.
Strains, media and culture conditions
C. albicans strains
ura3Δ ::imm434/ura3 Δ::imm434
ura3Δ::imm434/ura3Δ::imm434, leu2Δ/leu2Δ, his1Δ/his1Δ
As CAI-4 but RPS1/rps1Δ::CIp10
As CAI-4 but MNN1/mnn1Δ::hisG-URA3-hisG
As CAI-4 but MNN1/mnn1Δ::hisG
As CAI-4 but mnn1Δ::hisG/mnn1Δ::hisG-URA3-hisG
As CAI-4 but MNN12/mnn12Δ::hisG-URA3-hisG
As CAI-4 but MNN12/mnn12Δ::hisG
As CAI-4 but mnn12Δ::hisG/mnn12Δ::hisG-URA3-hisG
As CAI-4 but mnn1Δ::hisG/mnn1Δ::hisG
As CAI-4 but mnn12Δ::hisG/mnn12Δ::hisG
As CAI-4 but mnn1Δ::hisG/mnn1Δ::hisG, RPS1/rps1Δ::CIp10
As CAI-4 but mnn12Δ::hisG/mnn12Δ::hisG, RPS1/rps1Δ::CIp10
As SN78 but MNN13/mnn13Δ::CmLeu2
As SN78 but MNN14/mnn14Δ::CmLeu2
As SN78 but MNN15/mnn15Δ::CmLeu2
As SN78 but MNN16/mnn16Δ::CmLeu2
As SN78 but mnn13Δ::CmLeu2/mnn13Δ ::CdHIS1
As SN78 but mnn14Δ::CmLeu2/mnn14Δ ::CdHIS1
As SN78 but mnn15Δ::CmLeu2/mnn15Δ ::CdHIS1
As SN78 but mnn16Δ::CmLeu2/mnn16Δ ::CdHIS1
As SN78 but mnn13Δ::CmLeu2/mnn13Δ ::CdHIS1, RPS1/ rps1Δ::CIp10
As SN78 but mnn14Δ::CmLeu2/mnn14Δ ::CdHIS1, RPS1/ rps1Δ::CIp10
As SN78 but mnn15Δ::CmLeu2/mnn15Δ ::CdHIS1, RPS1/ rps1Δ::CIp10
As SN78 but mnn16Δ::CmLeu2/mnn16Δ ::CdHIS1, RPS1/ rps1Δ::CIp10
As SN78 but mnn13Δ::CmLeu2/mnn13Δ ::CdHIS1, RPS1/ rps1Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As SN78 but mnn14Δ::CmLeu2/mnn14Δ ::CdHIS1, RPS1/ rps1Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As SN78 but mnn15Δ::CmLeu2/mnn15Δ ::CdHIS1, RPS1/ rps1Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As SN78 but mnn16Δ::CmLeu2/mnn16Δ ::CdHIS1, RPS1/ rps1Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As CAI-4 but mnn1 Δ::hisG/mnn1 Δ::hisG, RPS1/rps1 Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As CAI-4 but mnn12 Δ::hisG/mnn12 Δ::hisG, RPS1/rps1 Δ::CIp10, HEX1/HEX1-V5-6xHis-NAT1
As NGY152 but HEX1/HEX1-V5-6xHis-NAT1
pmr1 Δ::hisG/pmr1 Δ::hisG HEX1/HEX1-V5-6xHis-URA3
As CAI-4 but pmr1 Δ::hisG/pmr1 Δ::hisG, RPS1/rps1 Δ::CIp10
As CAI-4 but och1 Δ::hisG/och1 Δ::hisG, RPS1/rps1 Δ::CIp10
As CAI-4 but mnt1-mnt2 Δ::hisG/mnt1-mnt2 Δ::hisG, RPS1/rps1 Δ
Oligonucleotides used for strain construction
Cell wall and glycosylation analysis
Null mutants were screened for sensitivity to cell wall stressing agents by the microdilution method as previously reported . Briefly, standardised inocula (A600 = 0.01) of strains in YEPD medium were incubated with cell wall stressing agents, across a range of doubling dilutions, for 16 h at 30°C and the A600 was determined. The agents tested (maximum concentrations in parentheses) included Calcofluor White (500 μg/ml), Congo Red (500 μg/ml), SDS (0.1%), hygromycin B (500 μg/ml), NaCl (2 M) and tunicamycin (50 μg/ml). Phosphomannan content and N-glycosylation status were determined by Alcian Blue binding assays and β-N-acetylhexosaminidase (Hex1) native PAGE zymograms as reported elsewhere . In addition the N-glycosylation status was assessed by Western blot analysis of mutant strains carrying the V5-6xHis tagged Hex1 protein as a glycosylation reporter as described previously . Cell wall carbohydrate composition was determined by acid hydrolysis of the cell wall carbohydrate polymers and quantification of constituent monosaccharides following high performance anion exchange chromatography . Finally, for the analysis of phosphomannan and O-linked mannan, strains were labelled with D-[2-3H] mannose, mannans released through mild acid treatment and β-elimination respectively and separated through thin layer chromatography (TLC) .
Isolation of human peripheral blood mononuclear cells (PMBCs) and cytokine stimulation assays were carried out as described previously . For the wax moth larvae infection model groups of 8 larvae were infected with 3×105 CFU of C. albicans through the last left proleg and survival monitored over 7 days at 37°C. To test virulence in a murine infection model groups of 6 female BALB/c mice were intravenously challenged with 3.6×104 CFU/g (body weight) C. albicans and monitored over 28 days. Animals showing signs of illness or disease were humanely terminated and recorded as dying the following day, and those surviving the course of the experiment were terminated on day 28. The kidneys and brain were aseptically removed post-mortem, homogenised, and tissue burdens determined by viable counting. Experiments were conducted under the terms of a UK Home Office licence and were approved by the University of Aberdeen Ethical Review Committee.
This work was supported by a Wellcome Trust Programme grant (080088) to NG, FO and AB and by a FP7-2007-2013 grant agreement (HEALTH-F2- 2010-260338–ALLFUN) and BBSRC SABR (CRISP) award. MGN was supported by a Vici grant of the Netherlands Organization for Scientific Research. JC was supported by a UK Biotechnology and Biological Sciences Research Council project grant (BB/F009232/1) to SB.
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