Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype
Fig 3
Cryptococcus neoformans incubated for 8 days in hypoxia reactivated with pantothenic acid (PA) and culturable cells rewired in minimal medium (MM) dilutions.
Growth curves were determined on yeasts recovered as STAT or 8D-HYPOx and then refed in diluted MM. Latency and growth rate (slope) were then determined. Experiments were all repeated twice (independent experiments are identified as circle or triangle). Graph includes dot for the replicates, as well as median ± IQR (*p<0.01 compared to control in the same condition). A. The dilution of minimal medium from 100% to 10% (diluted MM) resulted in a progressive decrease of latency in 8D-HYPOx cells and to a lesser extend in STAT cells. (*p <0.01 compared to 100%MM). Two independent experiments were pooled. Each point represents the latency/well in two (STAT n = 5, 8D-HYPOx n = 15) B. 100-fold dilution of pantothenic acid (starting at 125 μM) were added to the suspension prior to growth curve determination. Pantothenic acid significantly decreased the latency at a concentration of 1.25x10-2 μM or more in 8D-HYPOx and to a lesser extend in STAT cells (* p<0.01). Two independent experiments were pooled. Each point represents the latency/well in two (STAT n = 9, 8D-HYPOx n = 6). C. Growth rate was significantly decreased in 20%MM and 10%MM in 8D-HYPOx D. A significantly increased growth rate was observed after addition of PA (1.25 μM or more) to 8D-HYPOx cells and at all concentrations for STAT. E. The addition of pantothenic acid at 125μM but not the dilution of MM increased the culturability of 8D-HYPOx based on the observation of the number of positive wells (100 cells per well) and the calculation of the probability of one cell to grow. Each dot represents the calculated culturability. Two independents experiments were pooled. F. Proposed model for the effect of hypoxia and nutrient depletion. From now on, 8D-HYPOx are called VBNC (Viable but not culturable cells). After 8 d of incubation in hypoxia and nutrient depletion, the decrease in latency observed upon addition of pantothenic acid on VBNC is explained by the reactivation of VBNC to culturable cells (increased culturability) and by the increased growth rate of culturable cells (increased slope). The decrease in latency observed upon dilutions of MM (decreased nutrient availability) is explained by a faster rewire of the culturable cells (decreased latency corresponding to decreased time to resume growth) without increased culturability or growth rate.