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 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.
Table 1.
Relative proportions of carbohydrates in the cell wall extracted from the MNN2 gene family mutants.
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 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 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 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 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 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.
Table 2.
Deletion of MNN2 gene family members attenuates virulence in the 3-day murine infection model.
Figure 9.
Hypothetical model for the actions of the six Mnn2 mannosyltransferases.
See text for details.