Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans

Abstract

Quorum sensing (QS) is a cell density-dependent mechanism of communication between microorganisms, characterized by the release of signaling molecules that affect microbial metabolism and gene expression in a synchronized way. In this study, we investigated cell density-dependent behaviors mediated by conditioned medium (CM) in the pathogenic encapsulated fungus Cryptococcus neoformans. CM produced dose-dependent increases in the growth of planktonic and biofilm cells, glucuronoxylomannan release, and melanin synthesis, important virulence attributes of this organism. Mass spectrometry revealed the presence of pantothenic acid (PA) in our samples, and commercial PA was able to increase growth and melanization, although not to the same extent as CM. Additionally, we found four mutants that were either unable to produce active CM or failed to respond with increased growth in the presence of wild-type CM, providing genetic evidence for the existence of intercellular communication in C. neoformans. C. neoformans CM also increased the growth of Cryptococcus albidus, Candida albicans, and Saccharomyces cerevisiae. Conversely, CM from Cryptococcus albidus, C. albicans, S. cerevisiae, and Sporothrix schenckii increased C. neoformans growth. In summary, we report the existence of a new QS system regulating the growth and virulence factor expression of C. neoformans in vitro and, possibly, also able to regulate growth in other fungi.

Importance: Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium--culture medium in which C. neoformans has previously grown--to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis.

FIG 1

CM accelerates the growth of C. neoformans strain 24067 and decreases the time for growth resumption after culture dilution. C. neoformans 24067 cells were grown overnight and diluted to new cultures at 10⁴ cells/ml. The cells were grown in MM or MM complemented with increasing concentrations of CM (5 to 100%) at 30°C. The results shown are the averages of three measurements. (A) To determine cell number, the cultures were followed for 36 h and aliquots collected every 12 h for cell counting in a hemocytometer. (B) To determine fold change in growth, the number of cells in each system containing CM was normalized by the number of cells in the control system (MM not supplemented with CM). (C) To evaluate cell density, the cultures were incubated in an automated microbiology growth curve analysis system, and their absorbance was read every 30 min for 84 h. OD, optical density. (D) To determine fold change in growth, the absorbance of each system containing CM was normalized to the absorbance of the cells in the control system (MM not supplemented with CM).

FIG 2

CM affects GXM release in culture. C. neoformans strain 24067 cells were grown overnight and diluted to new cultures at 10⁴ cells/ml in minimal medium alone or in the presence of increasing concentrations of CM (5 to 100%). (A) The supernatants of the cultures were collected at three different time points and used in a capture ELISA assay to measure GXM released to the medium during cell growth. (B) The concentration of GXM (µg/ml) at each time point was divided by the cell number of each culture at each time interval. Error bars show standard deviations.

FIG 3

CM effects on C. neoformans melanization. (A) C. neoformans strain H99 cells were grown overnight and washed 3 times, and 10⁵ cells were spotted on solid minimal medium supplemented with L-DOPA and with increasing concentrations of CM. The colonies were followed visually for melanin production for 40 h. We can observe that cultures in the presence of CM already showed melanization on a dose-response basis at 24 h, while the control in MM started to show the pigment only at 32 h. (B) We followed colonies growing in MM or in the presence of 10% CM and observed that small satellite colonies in the wells containing CM melanized much earlier than the satellite colonies in the wells containing MM alone, even though the main colony was already melanized 48 h earlier. Similar results were obtained with C. neoformans strain 24067 cells. This experiment is representative of several replicates done over 5 years.

FIG 4

CM affects the growth of C. neoformans cells in biofilms. Cells of C. neoformans strain B3501 were grown overnight, washed 3 times with PBS, and inoculated at different initial cell densities (10⁴, 10⁵, 10⁶, or 10⁷ cells/ml) in 96-well polystyrene plates with increasing concentrations of CM that was added to the plates at different time points. (A) 0h, C. neoformans cells and CM were added at the same time; 4h, C. neoformans cells were added to the plate 4 h before the addition of CM; 24h, C. neoformans cells were added to the plate 24 h before the addition of CM. After CM addition, the plates were incubated for 24 h at 37°C and the biofilm formation was evaluated with the XTT reduction assay. Error bars represent standard deviations. The results were analyzed with linear regression to generate trend lines and evaluate whether the slopes of these lines were significantly different from zero (*, P < 0.05). (B) B3501 biofilms were prepared with initial cell density of 10⁴ cells/ml in coverslips placed in 6-well culture plates. As described above, after 0, 4, and 24 h, CM (100% final concentration) or MM was added and the coverslips incubated for another 24 h. After vigorous washing, attached cells were stained with the cell wall stain Calcofluor white (blue) and the GXM antibody 18B7 (green) and imaged by confocal microscopy. The scale bar measures 50 µm.

FIG 5

Comparison of the activities of pantothenic acid and CM on C. neoformans cells. Cells of the H99 strain of C. neoformans were grown overnight, washed three times, and inoculated at a density of 10⁴ cells/ml in different concentrations of pantothenic acid (A) or CM (B). The cultures were incubated at 30°C for 24 h, and their growth was measured by their absorbance at 600 nm. The fold increase represents the ratio between the growth in the cultures containing PA or CM and the growth of the control culture in MM. (C) HPLC analysis to evaluate the presence of PA in H99 CM. CM from H99 cells was analyzed in a C₁₈ column. Commercial PA was used as a standard, and its elution profile was compared to the elution profile of CM derived from H99 cells. mAU, milli-absorbance unit.

FIG 6

Effects of CNI27702 deletion on CM growth effects. C. neoformans H99 cells were grown for 24 h and diluted to begin new cultures at 10⁴ cells/ml. The cells were grown in MM or MM complemented with increasing concentrations of H99 CM or CNI27702Δ CM at 30°C. (A and B) Fold change values (growth in CM/growth in MM) for H99 wild-type cells grown in the presence of their own CM (gray) or with CNI27702Δ CM (black) at 24 h or 48 h. (C and D) Fold change values of CNI27702Δ cells incubated with H99 CM after 24 h or 48 h of incubation. At 24 h, we observed a dose-dependent increase in growth of the H99 cells in the presence of H99 CM, while the cells growing in CNI27702Δ CM were not different from the MM control. At 48 h, the effects of the H99 CM began to decrease and approached the control levels, while the cells grown in the presence of CNI27702Δ CM finally began to show some increase in the fold change. The results shown in panels C and D demonstrate that CNI27702Δ cells respond similarly to H99 cells with faster growth when exposed to wild-type CM.

FIG 7

Comparison of the effects of CNI27702Δ CM and wild-type (WT) CM on melanization. C. neoformans H99 cells were grown for 24 h and washed 3 times, and 10⁵ cells were spotted in solid MM supplemented with L-DOPA and with increasing concentrations of H99 CM or CNI27702Δ CM. The colonies were visually followed for melanin production for 40 h. At 24 and 32 h, it was possible to visualize the dose-dependent melanization of cells at higher concentrations of wild-type CM. Only at 32 h was the melanization of cells grown in CNI27702Δ CM noticeable; however, the melanization levels are similar to those of the control cells grown in MM alone.

FIG 8

Growth effects of wild-type CM on wild-type, opi3Δ, and pka1Δ cells. Each strain was grown for 24 h and diluted to new cultures at 10⁴ cells/ml. The cells were then grown in MM or MM supplemented with increasing concentrations of H99 CM at 30°C in an automated microbiology growth curve analysis system where their absorbance was read every 30 min for 72 h. Left side plots show the absorbance of each culture at 600 nm, while the right plots show fold change (growth in CM/growth in MM). The values shown represent the means of three wells for each condition.

FIG 9

Comparison of the effects of wild-type CM on melanization of wild-type, pka1Δ, and opi3Δ cells. Each strain was grown for 24 h and washed 3 times, and 10⁵ cells were spotted in agar MM supplemented with L-DOPA and with increasing concentrations of CM from wild-type cells (H99). The colonies were followed visually for melanin production for 32 h. At 24 h, it was possible to discriminate visually the dose-dependent melanization of wild-type cells. The opi3Δ colonies only started to melanize at 32 h, whereas the pka1Δ colonies did not melanize at all.

FIG 10

Comparison of the growth effects of wild-type CM on wild-type and lhc1Δ cells. Both strains were grown for 24 h and diluted to seed new cultures starting at 10⁴ cells/ml. The cells were grown in either MM or MM supplemented with increasing quantities of H99 CM at 30°C. The plots on the left show the absorbance at 600 nm, whereas the plots on the right show the fold change values (growth in CM/growth in MM) during the growth curve. The values represent the means of three wells.

FIG 11

Comparison of the effects of CM on melanization of wild-type and lhc1Δ cells. Cells of the two strains were grown for 24 h and washed 3 times, and 10⁵ cells were spotted in solid MM supplemented with L-DOPA and with increasing concentrations of H99 CM or lhc1Δ CM. The colonies were visually followed for melanin production for 32 h. At 24 h, it was possible to visualize the dose-dependent melanization of H99 cells but not yet of lhc1Δ cells, which began to show melanization only by 32 h of incubation.