Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

Abstract

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.

Fig. 1

C. neoformans cells differing in capsule size manifest different susceptibility to H₂O₂. C. neoformans cells with small and large capsule (induced in diluted Sabouraud in MOPS buffer) were incubated with 0.5 mM H₂O₂ at 37°C. Parallel samples not exposed to H₂O₂ were carried out in parallel. At the indicated times, 100 μl aliquots were plated in Sabouraud, and viability was calculated at each time point as described in Experimental procedures. Solid line, small capsule; dotted line, cells with large capsule. At each time, the mean value and standard deviation of the viabilities from four different experiments are plotted. Asterisk indicates statistical differences at the time point indicated (P < 0.05).

Fig. 2

Susceptibility of C. neoformans cells isolated from mice to H₂O₂. Cells with enlarged capsule were obtained from the lungs of infected mice as described in Experimental procedures. The cells were exposed to 0.5 mM (A) or 2 mM (B) H₂O₂, and survival was monitored as in (A) and as described in Experimental procedures. Open bars, cells grown in Sabouraud; closed bars, cells obtained from mice. Asterisks indicate statistical differences between cells grown in Sabouraud or isolated from infected mice, and the corresponding P-value is shown.

Fig. 3

Purified capsular polysaccharide protects C. neoformans and S. cerevisiae. Purified capsular polysaccharide was obtained from cells with enlarged capsule by γ-irradiation as described in Experimental procedures. This polysaccharide was lyophilized and dissolved at 10 mg ml⁻¹ (A and B) in distilled water. Cells from C. neoformans (A) or S. cerevisiae (B–D) were grown in Sabouraud, washed, and suspended in distilled water or capsular polysaccharide. A and B. H₂O₂ 0.5 mM was added to both samples (closed bars), and parallel samples to which no H₂O₂ was added were carried out in parallel (open bars). Viability was measured after 2 h of incubation at 37°C. Asterisk denotes statistical differences (P < 0.01). C. Dose–response protection, using S. cerevisiae. Serial dilutions of the capsular polysaccharide were prepared (from 10 to 0.08 mg ml⁻¹), and S. cerevisiae cells were added to each suspension. H₂O₂ (0.5 mM) was added to these samples. As controls, we included H₂O₂-treated and non-treated samples suspended in distilled water (left and right bars). Viability was measured after 2 h of incubation at 37°C. The experiment was performed in triplicates. In all the panels, the survival mean value and the standard deviation are plotted. Asterisk denotes statistical differences with the control where the cells were treated with H₂O₂ suspended in water (right bar). D. Cells were treated as in (B), but plated after 24 h of incubation and pictures of the plates of the different samples were taken. E. Cells from the wild-type strain (B3501) and the acapsular mutant (C536, cap59 disruptant) were placed in triplicates in wells from microdilution plates (10⁶ cells per well, approximately) in the presence or absence of 0.5 mM H₂O₂. After 3 h incubation, the wells were washed, and XTT/menadione mixture was added to each well. Viability was calculated as Experimental procedures. Closed bars, controls without H₂O₂; open bars, 2 mM H₂O₂.

Fig. 4

Capsular polysaccharide affects NBT reduction in the NADH/PMS electron transfer system. NBT reduction induced by NADH and PMS was monitored by measuring absorbance at 540 nm in the absence or presence of different concentrations of capsular polysaccharide. A. Optical density (O.D.) increase during 30 min. B. Inhibition percentage of NBT reduction in the presence of different polysaccharide concentrations. The experiment was performed in duplicates, obtaining almost identical results (P < 0.05).

Fig. 5

Reactive oxygen species affect the size of the polysaccharide molecules. The distribution of the molecular size of polysaccharide samples was measured after treatment with 0.5 mM H₂O₂ during 2 h or overnight at 37°C as described in Experimental procedures.

Fig. 6. C. neoformans cells manifest different susceptibility to antimicrobial agents depending on their capsule size

A. Cells with small and large capsule were exposed to different defensin concentrations, and after 30 min, viability was estimated by plating 100 μl on Sabouraud plates. The mean value and standard deviation from three different experiments are plotted. Solid line, cells with small capsule; dotted line, cells with large capsule. Asterisk denotes statistical differences between cells with small and large capsule at the defensin concentration indicated. B. Cells with small and large capsule were exposed to different Amphotericin B concentrations during 3 h at 37°C, and viability was estimated by cfu enumeration as described in Experimental procedures. The average and the standard deviation of four different experiments are plotted. Solid line, cells with small capsule; dotted line, cells with large capsule. Asterisk denotes statistical differences between cells with small and large capsule at the Amphotericin B concentration indicated (P = 0.05).

Fig. 7. C. neoformans cells with different capsule size manifest differences in susceptibility of phagocytosis by A. castellanii

A. C. neoformans cells were incubated overnight in PBS, and then transferred to Sabouraud or PBS for 1 or 2 h. Afterwards the cells were washed and used in phagocytosis experiments in the presence of A. castellanii as described in Experimental procedures. The mean value and standard deviation of three different experiments are shown. Statistical differences between samples are highlighted with arrows, and the P-value of the comparison is shown. B. C. neoformans cells were incubated overnight in PBS or in PBS + 10% FCS or in Sabouraud, and then transferred to Sabouraud, PBS or PBS + 10% FCS for 2 h. Each bar is labelled first with the condition in which the cells were placed overnight and then with the condition in which they were placed for 2 h. The experiment was performed in triplicate, and the mean value and standard deviation are shown. Statistical differences between samples are highlighted with arrows, and the P-value of the comparison is shown.