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Role of the Candida albicans MNN1 gene family in cell wall structure and virulence.

Bates S, Hall RA, Cheetham J, Netea MG, MacCallum DM, Brown AJ, Odds FC, Gow NA - BMC Res Notes (2013)

Bottom Line: 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 mnn14Δ 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life and Environmental Sciences, University of Exeter, Exeter, EX4 4QD, UK. s.bates@ex.ac.uk

ABSTRACT

Background: 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.

Findings: We generated single mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro 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 mnn14Δ 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 mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model.

Conclusion: 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.

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Related in: MedlinePlus

Glycosylation defects in C. albicans MNN1 family  mutants. The extent of N-glycosylation was determined by activity staining of Hex1 (β-N-acetylhexosaminidase) after protein samples were separated by non-denaturing gel electrophoresis (A). The continuous and dotted lines indicate the normal and decreased electrophoretic mobility of Hex1 respectively. The degree of N-glycosylation modification was also determined by western blot analysis of Hex1-V5-6× His tagged strains (B). Hex1-V5-6× His is apparent in both an unmodified (67 kDa) and a heavily glycosylated (~125 kDa) form. Acid-labile phosphomannan (C) and β-eliminated O-mannan (D) were released from [2-3H] mannose-labelled cells and separated by TLC. Samples are as follows: lane 1, wild type; lane 2, mnn1∆; lane 3, mnn12∆; lane 4, mnn13∆; lane 5, mnn14∆; lane 6, mnn15∆; lane 7, mnn16∆; lane 8, och1∆; lane 9, pmr1∆; lane 10, mnt1∆-mnt2∆.
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Figure 3: Glycosylation defects in C. albicans MNN1 family mutants. The extent of N-glycosylation was determined by activity staining of Hex1 (β-N-acetylhexosaminidase) after protein samples were separated by non-denaturing gel electrophoresis (A). The continuous and dotted lines indicate the normal and decreased electrophoretic mobility of Hex1 respectively. The degree of N-glycosylation modification was also determined by western blot analysis of Hex1-V5-6× His tagged strains (B). Hex1-V5-6× His is apparent in both an unmodified (67 kDa) and a heavily glycosylated (~125 kDa) form. Acid-labile phosphomannan (C) and β-eliminated O-mannan (D) were released from [2-3H] mannose-labelled cells and separated by TLC. Samples are as follows: lane 1, wild type; lane 2, mnn1∆; lane 3, mnn12∆; lane 4, mnn13∆; lane 5, mnn14∆; lane 6, mnn15∆; lane 7, mnn16∆; lane 8, och1∆; lane 9, pmr1∆; lane 10, mnt1∆-mnt2∆.

Mentions: The effect of disruption of the MNN1 family members on N-linked glycosylation was initially assessed through monitoring the electrophoretic mobility of secreted Hex1 (β-N-acetylhexosaminidase) by activity staining following native gel electrophoresis [7]. None of the MNN1 family mutants displayed the increased electrophoretic mobility of Hex1 that is normally associated with a deficiency in N-glycosylation, as demonstrated by och1Δ and pmr1Δ mutants [7,8], indicating no gross N-glycosylation defects in the MNN1 family mutants (Figure 3A). However, the mnn14Δ mutant instead demonstrated a noticeable decrease in Hex1 mobility following native gel electrophoresis (Figure 3A). This decrease in electrophoretic mobility could be suggestive of an increased level of glycosylation of Hex1 in this mutant. Alternatively, an alteration in the level, or extent of, phosphomannan modification could affect the surface charge of Hex1, which would impact on its mobility in a native gel.


Role of the Candida albicans MNN1 gene family in cell wall structure and virulence.

Bates S, Hall RA, Cheetham J, Netea MG, MacCallum DM, Brown AJ, Odds FC, Gow NA - BMC Res Notes (2013)

Glycosylation defects in C. albicans MNN1 family  mutants. The extent of N-glycosylation was determined by activity staining of Hex1 (β-N-acetylhexosaminidase) after protein samples were separated by non-denaturing gel electrophoresis (A). The continuous and dotted lines indicate the normal and decreased electrophoretic mobility of Hex1 respectively. The degree of N-glycosylation modification was also determined by western blot analysis of Hex1-V5-6× His tagged strains (B). Hex1-V5-6× His is apparent in both an unmodified (67 kDa) and a heavily glycosylated (~125 kDa) form. Acid-labile phosphomannan (C) and β-eliminated O-mannan (D) were released from [2-3H] mannose-labelled cells and separated by TLC. Samples are as follows: lane 1, wild type; lane 2, mnn1∆; lane 3, mnn12∆; lane 4, mnn13∆; lane 5, mnn14∆; lane 6, mnn15∆; lane 7, mnn16∆; lane 8, och1∆; lane 9, pmr1∆; lane 10, mnt1∆-mnt2∆.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Glycosylation defects in C. albicans MNN1 family mutants. The extent of N-glycosylation was determined by activity staining of Hex1 (β-N-acetylhexosaminidase) after protein samples were separated by non-denaturing gel electrophoresis (A). The continuous and dotted lines indicate the normal and decreased electrophoretic mobility of Hex1 respectively. The degree of N-glycosylation modification was also determined by western blot analysis of Hex1-V5-6× His tagged strains (B). Hex1-V5-6× His is apparent in both an unmodified (67 kDa) and a heavily glycosylated (~125 kDa) form. Acid-labile phosphomannan (C) and β-eliminated O-mannan (D) were released from [2-3H] mannose-labelled cells and separated by TLC. Samples are as follows: lane 1, wild type; lane 2, mnn1∆; lane 3, mnn12∆; lane 4, mnn13∆; lane 5, mnn14∆; lane 6, mnn15∆; lane 7, mnn16∆; lane 8, och1∆; lane 9, pmr1∆; lane 10, mnt1∆-mnt2∆.
Mentions: The effect of disruption of the MNN1 family members on N-linked glycosylation was initially assessed through monitoring the electrophoretic mobility of secreted Hex1 (β-N-acetylhexosaminidase) by activity staining following native gel electrophoresis [7]. None of the MNN1 family mutants displayed the increased electrophoretic mobility of Hex1 that is normally associated with a deficiency in N-glycosylation, as demonstrated by och1Δ and pmr1Δ mutants [7,8], indicating no gross N-glycosylation defects in the MNN1 family mutants (Figure 3A). However, the mnn14Δ mutant instead demonstrated a noticeable decrease in Hex1 mobility following native gel electrophoresis (Figure 3A). This decrease in electrophoretic mobility could be suggestive of an increased level of glycosylation of Hex1 in this mutant. Alternatively, an alteration in the level, or extent of, phosphomannan modification could affect the surface charge of Hex1, which would impact on its mobility in a native gel.

Bottom Line: 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 mnn14Δ 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life and Environmental Sciences, University of Exeter, Exeter, EX4 4QD, UK. s.bates@ex.ac.uk

ABSTRACT

Background: 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.

Findings: We generated single mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro 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 mnn14Δ 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 mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus