KRE genes are required for beta-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

Abstract

The polysaccharide beta-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in beta-1,6-glucan synthesis. The H99 deletion mutants kre5Delta and kre6Deltaskn1Delta contained less cell wall beta-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Delta and kre6Deltaskn1Delta. Our results indicate that KRE5, KRE6 and SKN1 are involved in beta-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.

Fig. 1

Phylogenetic tree of the Kre6/Skn1 proteins. The tree was created using the neighbor-joining algorithm in Phylip from a multiple alignment of the Kre6 and Skn1 protein sequences from S. cerevisiae, C.neoformans, and C. albicans, with the C. neoformans Kre5 protein used as an out group.

Fig. 2

Domain organization of C. neoformans and S. cerevisiae Kre6/Skn1 homologs. Domains were identified using InterProScan.

Fig. 3

Northern blot mRNA expression analysis of KRE5 and the KRE6/SKN1 homologs in C. neoformans. H99 was grown in liquid culture for 24 h at the indicated temperatures. Relative gene expression was determined utilizing ImageQuant (version 5.1) and was normalized to the level of actin. Values are the average of two independent biological replicates.

Fig. 4

β-1,6-glucan dot blot assay. (A) Polysaccharide competition analysis. Alkali soluble cell wall material from H99 was spotted onto membrane. The primary anti-β-1,6-glucan antiserum was preincubated with the indicated purified polysaccharides for 30 min before probing the membrane. (B) Alkali-soluble cell wall β-1,6-glucan analysis of H99 and deletion strains. Cells were grown at 30°C to mid-log phase and alkali-soluble polysaccharides were isolated and spotted in 1:2 serial dilutions onto nitrocellulose membrane. Membranes were probed with polyclonal anti-β-1,6-glucan antiserum. (C) Alkali-insoluble, chitinase-released cell wall β-1,6-glucan analysis of H99 and deletion strains.

Fig. 5

Morphology of C. neoformans H99, kre5Δ and kre6Δskn1Δ. Cells were grown on YPD medium at 30°C for 72h and stained with Pontamine FastScarlet. All images are at 1000X magnification. Scale bar = 25µm.

Fig. 6

Sensitivity of mutants to temperature (A) and cell wall inhibitors (B). Strains were grown overnight in YPD medium, then diluted to an OD₆₅₀ of 1.0. Tenfold serial dilutions were made in PBS and 5 µl of each was spotted on medium containing the indicated inhibitors. Plates were incubated at the indicated temperatures for 2 days.

Fig. 7

Capsule analysis (A) India ink staining of capsule of H99 and deletion strains. Strains grown for 3 days on DME medium at 30°C with 5% CO2 to induce capsule production were stained with India ink and visualized by light microscope. Arrows point to the rough appearance of the polysaccharide sloughing from capsular edge. Scale bars in the left and right panels are 25 and 10 µm, respectively. (B) Penetration of the cryptococcal capsule by TMR-dextrans. Top panel shows representative images of wild type cells stained with India ink or incubated with 70 kDa TMR-dextran and illustrates the measurements taken and the equations used to calculate the penetration zone. CD = capsule diameter; ED = exclusion diameter; CR = capsule radius. Strains were grown as in (A) and incubated 60 min with TMR-dextrans with average molecular masses of 10, 40 and 70 kDa, and the radius of penetration was determined by examination of digital images. Results shown are mean ± SD for at least 24 cells in two independent biological replicates. * denotes p≤ 0.02 compared to wild type H99 with 70 kDa dextran.

Fig. 8

Melanin production. Strains were grown overnight in YPD medium, diluted to an OD₆₅₀ of 1.0. and 5 µl of each tenfold serial dilution (in PBS) and was plated on medium containing 1 mM L-DOPA. Plates were incubated at 30°C for 7 days.

Fig. 9

Virulence analysis. Mice (10 per strain) were inoculated with 1×10⁶ cryptococcal cells, dripped into the nares. Mice were weighed before infection and sacrificed once they reached 80% of their original body weight.

Fig. 10

Cellular distribution of PLB activity (A) and the Plb1 protein (B). (A) Subcellular fractions were prepared from concentrated cell suspensions after an overnight incubation period and harvesting of the secreted proteins. PLB activity in the crude membrane fraction was negligible. * denotes p<0.005 (unpaired 2-tail t-test) compared to the corresponding fraction from H99. Similar results for all three strains were obtained in a second independent experiment (not shown). (B) Plb1 protein in the secreted and cell wall fractions were detected by western blotting with an anti-Plb1 peptide antibody. Top and bottom arrows indicate the position of full-length, fully glycosylated Plb1 and a breakdown product (most prominent in kre5Δ secretions), respectively.

Fig. 11

Eosin Y staining of H99 and kre5Δ and kre6Δskn1Δ. Strains were incubated at 30 °C for 72 hours and stained with eosin Y. Images were captured at 1000X magnification. Bright cells in the eosin Y panels of the mutants are presumably dead cells that have taken up dye, as seen previously (Gerik et al. 2008). Scale bar = 25µm.