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

Abstract

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

Fig. 1. CaMNT1 and CaMNT2 region of chromosome 3 with intervening β-glucosidase gene (β-GLU) (top line) and the structure of the Camnt2Δ, Camnt1-Camnt2Δ and reconstituted heterozygous alleles with MNT1 or MNT2 integrated at the CIp10 locus

Hatched bars represent putative MNT promoter and terminator sequences. Restriction sites are: B, BglII; Bm, BamHI; E, EcoRI; H, HindIII, K, KpnI, and P, PstI.

Fig. 2

Morphology of colonies on Spider medium plates after 5 days at 30 °C showing reduction in filamentation in the Camnt1-Camnt2Δ mutant and complementation of this defect by addition of CaMNT1, but not CaMNT2.

Fig. 3. Thin-sections of wild type (A, CAF2-1) and the Camnt1-Camnt2Δ double mutant (B, C) showing extracellular matrix material between flocculating yeast cells (asterisks)

Scale bar, 3 μm, for all micrographs.

Fig. 4. Mannosyltransferase activity of mntΔ deletant and control strains

Membrane fractions were isolated from exponential phase C. albicans yeast cells and tested for the ability to transfer mannose from GDP-[³H]mannose to the acceptors methyl α-mannoside (solid bars) and methyl-α1,2-mannobiose (clear bars). Units are pmol of mannose transferred per mg of protein; error bars are S.D., n = 3. Bars marked with asterisks are statistically different from the control strain (CAI4 + CIp10) by Student’s t test (p ≤ 0.05). Samples are: 1 and 9, CAI-4 + CIp10; 2 and 10, Camnt1Δ+ CIp10; 3 and 11, Camnt1Δ + CIp10-MNT1; 4 and 12, Camnt2Δ + CIp10; 5 and 13, Camnt2Δ+ CIp10-MNT2; 6 and 14, Camnt1-Camnt2Δ + CIp10; 7 and 15, Camnt1-Camnt2Δ + CIp10-MNT1; 8 and 16, Camnt1-Camnt2Δ + CIp10-CaMNT2.

Fig. 5. TLC analysis of β-eliminated O-glycans of mntΔ mutant strains

A, cell walls were labeled by growing whole exponentially growing yeast cells in d-[2-³H]mannose and removing O-glycans by β-elimination then separating them by TLC. Labeled products then visualized by autofluorography were from: lane a, wild-type C. albicans CAF2-1; lane b, Camnt1Δ; lane c, Camnt2Δ; lane d, Camnt1-Camnt2Δ; and lane e, wild-type S. cerevisiae serving as a labeled Man₁-Man₅ control. B, control strains with: lane f, the parent CAI4+CIp10; lane g, Camnt1-Camnt2Δ + CIp10-MNT1; and lane h, Camnt1-Camnt2Δ + CIp10-MNT2. C, confirmation of α-1,2 linkages in O-glycans by mannosidase digestion. Standards (lane i), and untreated (lane j) purified cell walls of CAF-2 were used and were extracted by reductive β-elimination. Cell walls were then digested with Jack Bean mannosidase (lane k) and A. satoi mannosidase (lane l). Positions of Man-OH, Man₁, Man₂, and Man₃ are shown. Or, origin.

Fig. 6. Dionex HPAEC glycan profile of the wild type (A) and the mutant (B) strain

Separation of the glycan was performed on a CarboPac PA-100 column eluted with 200 mm sodium acetate as described under “Experimental Procedures.” The small peak labeled (x) with a retention time slightly lower than Man₂ was found not to be a glycan.

Fig. 7. Sensitivity of wild-type, Camnt1Δ, and Camnt2Δ, mutant strains and re-integrant strains to cell wall disruptive agents

YPD agar plates with and without Calcofluor White (100 μg ml⁻¹) or SDS (0.025% (w/v)) are shown after incubation of 5000, 500, and 50 cells at 30 °C for 24 h.

Fig. 8. Relative virulence of control strains and Mnt-deficient mutants, in a systemic mouse model of infection

Six mice were used per strain, with mice being infected intravenously with 2.6 ×10⁴ cfu/g body weight. The control parental strain (CAI-4 + CIp10) is shown as closed circles, the Camnt1-Camnt2Δ double mutant as open circles, Camnt1-Camnt2Δ + CIp10-MNT1 as closed squares, and Camnt1-Camnt2Δ + CIp10-CaMNT2 as closed triangles. The single mutant Camnt1Δ + CIp10 is shown as open squares and Camnt2Δ + CIp10 as open triangles.

Fig. 9. Structure of O-linked mannan and the enzymes involved in O-glycosylation in C. albicans as described in the text

Arrows indicate the linkage between each residue. The major enzyme involved in each step is shown in bold.