Divalent Metal Cations Potentiate the Predatory Capacity of Amoeba for Cryptococcus neoformans

Abstract

Among the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that of Acanthamoeba castellanii and Cryptococcus neoformans The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg²⁺ and Ca²⁺ in phosphate-buffered saline (PBS), C. neoformans was consistently killed when incubated with A. castellaniiA. castellanii survived better in the presence of Mg²⁺ and Ca²⁺, even when incubated with C. neoformans In the absence of Mg²⁺ and Ca²⁺, C. neoformans survived when incubated with A. castellanii, and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg²⁺ and Ca²⁺ can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba. Of the two metals, Mg²⁺ had a stronger effect than Ca²⁺ The cations enhanced A. castellanii activity against C. neoformans via enhanced phagocytosis, which is the major mechanism by which amoebae kill fungal cells. We found no evidence that amoebae use extracellular killing mechanisms in their interactions with C. neoformans In summary, the presence of Mg²⁺ and Ca²⁺ enhanced the cell adhesion on the surfaces and the motility of the amoeba, thus increasing the chance for contact with C. neoformans and the frequency of phagocytosis. Our findings imply that the divalent cation concentration in soils could be an important variable for whether amoebae can control C. neoformans in the environment.IMPORTANCE The grazing of soil organisms by phagocytic predators such as amoebae is thought to select for traits that enable some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoebae and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the interaction between an amoeba and the pathogenic fungus C. neoformans is influenced by the presence in the assay of magnesium and calcium, which potentiate amoebae. The results may also have practical applications, since enriching soils with divalent cations may reduce C. neoformans numbers in contaminated soils.

Keywords: Cryptococcus neoformans; amoeba; cations; fungi; predation.

FIG 1

Presence of magnesium and calcium affects the outcome of the A. castellanii-C. neoformans interaction. (A) The presence of magnesium and calcium decreases the survival of C. neoformans during the incubation with A. castellanii (AC). The survival of C. neoformans was determined by CFU after incubation with A. castellanii for 0, 24, and 48 h. The CFU counts at 24 h and 48 h were normalized to the initial CFU at time zero. Data represent the means from four biological samples. Error bars are SDs. ****, P < 0.0001 by Student's t test. (B) Phase-contrast and fluorescence images of Uvitex 2B-stained C. neoformans cells coincubated with or without A. castellanii in DPBS with or without magnesium and calcium. In the phase-contrast images, disrupted C. neoformans cells are dark (red arrow), while intact cells are refractile (white arrows). More disrupted C. neoformans cells appeared under the condition with A. castellanii incubation in DPBS containing magnesium and calcium. Scale bars are 10 μm. (C) A. castellanii cells survived better in the presence of Mg²⁺ and Ca²⁺ during the incubation with C. neoformans (CN). The viability of A. castellanii cells was determined by a trypan blue exclusion assay. The percentages of dead A. castellanii cells were determined by counting the numbers of blue stained cells per the total cell number counted. Four independent biological experiments were performed. Error bars represent 95% confidence intervals of the means. ***, P = 0.0008; ****, P < 0.0001 by Fisher's exact tests.

FIG 2

Magnesium had a stronger effect than calcium on reducing the survival of C. neoformans when incubated with A. castellanii. The survival of C. neoformans (CN) was determined by CFU after incubation with A. castellanii (AC) for 0, 24, and 48 h in DBPS (A), DPBS with calcium (B), DPBS with magnesium (C), and DPBS with calcium and magnesium (D). The CFU counts were normalized to the initial CFU at time zero. Data represent the means from three biological samples. Error bars are SDs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student's t tests. The viabilities of A. castellanii cells were also determined by trypan blue exclusion assays after incubation with C. neoformans in DBPS (E), DPBS with calcium (F), DPBS with magnesium (G), and DPBS with calcium and magnesium (H). The percentages of dead A. castellanii cells were determined by counting the numbers of blue stained cells per total cell number counted. Three independent biological experiments were performed. Error bars represent 95% confidence intervals of the mean. ****, P < 0.0001 by Fisher's exact tests.

FIG 3

Presence of calcium and magnesium enhances the adherence of A. castellanii to the surface and its motility. (A) Phase-contrast images of A. castellanii cells on glass surfaces in DPBS with or without Ca²⁺ and Mg²⁺. Scale bars are 50 μm. (B) To quantify cell spreading, the areas of A. castellanii cells in DPBS with or without Ca²⁺ and Mg²⁺ were measured. At least 100 cells were analyzed for each condition. Error bars indicate SDs. *, P = 0.01572; ****, P < 0.0001 by Student's t test. (C) Images of A. castellanii cell trajectories on glass surfaces in DPBS with or without Ca²⁺ and Mg²⁺. Ten A. castellanii cells were randomly selected for manually centroid tracked for a total duration of 2 h under each condition. The interval of each track is 30 s. Color lines indicate manually generated tracks. Two distinct morphologies of amoeboid cells, round and spread, in DPBS were tracked separately. Scale bars are 100 μm. (D) The total distance, the mean velocity, and the frequency of contact with C. neoformans cells were quantified. The total distance was defined as the sum of the distances the amoeboid cells travelled from the starting point to the endpoint of the cell trajectory. The mean velocity was calculated as the mean from all velocity measurements from amoeboid cells moving in each 30-s interval. Error bars indicate SDs. ****, P < 0.0001 by Student's t tests. (E) The presence of magnesium and calcium induces the phagocytosis of C. neoformans by A. castellanii. A. castellanii and Uvitex 2B-labeled C. neoformans were incubated at a 1:1 ratio in DPBS with or without magnesium and calcium for 2 h to allow phagocytosis. The phagocytic index was determined by the number of internalized C. neoformans cells per 100 A. castellanii cells. Data were obtained from four biologically independent experiments. Error bars represent 95% confidence intervals of the means. ****, P < 0.0001 by Fisher's exact test.

FIG 4

Phagocytosis is the major mechanism for A. castellanii to kill C. neoformans. (A) Scheme of the Transwell assay. A. castellanii and C. neoformans were separated in DPBS with Ca²⁺ and Mg²⁺ by porous membrane (pore size, 0.4 μm). The upper compartment contained C. neoformans while the lower compartment contained A. castellanii. As a positive control, C. neoformans was incubated with A. castellanii in the lower compartment. As a negative control, C. neoformans was incubated without A. castellanii in the upper compartment. (B) The survival of C. neoformans was determined by CFU after the Transwell assay for 0, 24, and 48 h. The CFU counts were normalized to the initial CFU at time zero. Data represent the means from four biological samples. Error bars are SDs. ***, P = 0.0004; ****, P < 0.0001 by Student's t tests. (C) Schematic for the production of conditioned media from coincubation of C. neoformans (CN) and A. castellanii (AC) and exposure of the fungal cells. After exposure of A. castellanii to C. neoformans in DPBS with Ca²⁺ and Mg²⁺, conditioned media were collected, filtered, and added to fresh cultures of C. neoformans. As a negative control, A. castellanii-conditioned medium was collected and added to C. neoformans. As a positive control, A. castellanii-conditioned medium was collected and added to the coincubation of C. neoformans and A. castellanii. (D) The survival of C. neoformans (CN) was determined by CFU after exposure to conditioned medium (CM) for 0, 24, and 48 h. The CFU counts were normalized to the initial CFU at time zero. Data represent the means from three biological samples. Error bars are SDs. ***, P = 0.00223; ****, P < 0.0001 by Student's t tests. (E) Time-lapse phase-contrast images of coincubation of C. neoformans and A. castellanii in DPBS with Ca²⁺ and Mg²⁺. Top images show the phagocytosis of C. neoformans (green arrows) by A. castellanii (white arrows). Bottom images show the exocytosis of disrupted C. neoformans (appears dark; red arrows) from A. castellanii (white arrows). Scale bar is 25 μm.