Cell wall chitosan is necessary for virulence in the opportunistic pathogen Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.

Fig. 1.

Chitosan and chitin levels from KN99 recovered from mouse lungs 16 days p.i. Chitosan and chitin levels from mouse lungs were compared to levels measured for KN99 grown in YPD at 30°C for 1 day and 3 days from a starting optical density at 600 nm of 0.10. Error bars indicate standard deviations for averaged measurements from four mice, and five 1-day YPD and four 3-day YPD cultures.

Fig. 2.

Growth curves of strains in liquid YNB at 30°C (A) or 37°C (B). The inset of panel B shows csr2Δ, chs3Δ, cda1Δ cda2Δ cda3Δ, and trx1Δ strains on an expanded y axis. Strains are listed on the right side of panel A. The x axis is in hours. Data consist of three bioreplicates, each with two technical replicates, with standard errors shown.

Fig. 3.

Virulence of chitosan-deficient strains. Strains are listed. Data represent results for two independent isolates per deletion strain.

Fig. 4.

Fungal burden over time for selected strains. Data are from four mice per strain. Strains are listed to the right of each graph. nd, not determined; a dash indicates no live cells were recovered. (A) *, significant difference between deletion strains and wild type at 2 h (chs3Δ, P = 0.009; csr2Δ, P = 0.010; cda1Δ cda2Δ cda3Δ, P = 0.003); P < 0.001 for the 8-h and 24-h time points based on one-way analysis of variance with Dunnett's post hoc test. Data represent one of two independent isolates for each chitosan-deficient strain after the 24-h time point. Standard errors are shown. (B) Fungal burden of the complemented strain. Wild-type and cda2Δ cda3Δ controls were included.

Fig. 5.

Average cryptoccocal cell numbers in longitudinal lung sections. Cryptoccocal cells in three stepwise lung sections from each mouse were stained with Grocott's methenamine silver (GMS). All cells in lung sections were counted by viewing with a light microscope. The number of cells for time points 8 to 48 h of each strain were calculated as a percentage of cells counted for each respective 2-h time point. Error bars indicate standard deviations between the four mice (12 lung sections) in each group.