Primaquine, an antimalarial drug that controls the growth of cryptococcal cells.

INTRODUCTION: The treatment of Cryptococcus neoformans with fluconazole and amphotericin B is, at times, characterised by clinical failure. Therefore, this study sought to re-purpose primaquine (PQ) as an anti-Cryptococcus compound. METHOD: The susceptibility profile of some cryptococcal strains towards PQ was determined using EUCAST guidelines, and PQ's mode of action was examined. In the end, the ability of PQ to enhance in vitro macrophage phagocytosis was also assessed. RESULTS: We show that PQ had a significant inhibitory effect on the metabolic activity of all tested cryptococcal strains, with 60 µM, defined as MIC50 in this preliminary study, as it reduced the metabolic activity by more than 50%. Moreover, at this concentration, the drug was able to affect mitochondrial function adversely, as treated cells displayed significant (p < 0.05) loss of mitochondrial membrane potential, cytochrome c (cyt c) leakage and overproduction of reactive oxygen species (ROS) when compared to non-treated cells. It is our reasoned summation that the produced ROS targeted the cell walls and cell membranes, inducing observable ultrastructural changes and a significant (p < 0.05) increase in membrane permeability when compared to non-treated cells. Concerning the PQ effect on macrophages, it was noted that it significantly (p < 0.05) enhanced macrophage phagocytic efficiency compared to non-treated macrophages. CONCLUSION: This preliminary study highlights the potential of PQ to inhibit the in vitro growth of cryptococcal cells. Moreover, PQ could control the proliferation of cryptococcal cells inside macrophages, which they often manipulate in a Trojan horse-like manner.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Elucidation of the Role of 3-Hydroxy Fatty Acids in Cryptococcus-amoeba Interactions.

We previously reported that 3-hydroxy fatty acids promoted the survival of cryptococcal cells when acted upon by amoebae. To expand on this, the current study sought to explain how these molecules may protect cells. Our data suggest that 3-hydroxy fatty acids may subvert the internalization of cryptococcal cells via suppression of the levels of a fetuin A-like amoebal protein, which may be important for enhancing phagocytosis. Additionally, we show that an acapsular strain (that is devoid of 3-hydroxy fatty acids) was protected against the effects of hydrogen peroxide when exogenous 3-hydroxy fatty acids were present, but not in the absence of 3-hydroxy fatty acids. A similar response profile was noted when a strain with a capsule was challenged with hydrogen peroxide. We also show that cryptococcal cells that naturally produce 3-hydroxy fatty acids were more resistant to the effects of amoebapore (an amoeba-specific hydrolytic enzyme), compared to cells that do not produce these molecules. Taken together, our findings suggest that 3-hydroxy fatty acids possess an anti-phagocytic activity that may be expressed when cells interact with macrophages. This may allow the yeast cells to evade immuno-processing.

Cryptococcal 3-Hydroxy Fatty Acids Protect Cells Against Amoebal Phagocytosis.

We previously reported on a 3-hydroxy fatty acid that is secreted via cryptococcal capsular protuberances - possibly to promote pathogenesis and survival. Thus, we investigated the role of this molecule in mediating the fate of Cryptococcus (C.) neoformans and the related species C. gattii when predated upon by amoebae. We show that this molecule protects cells against the phagocytic effects of amoebae. C. neoformans UOFS Y-1378 (which produces 3-hydroxy fatty acids) was less sensitive toward amoebae compared to C. neoformans LMPE 046 and C. gattii R265 (both do not produce 3-hydroxy fatty acids) and addition of 3-hydroxy fatty acids to C. neoformans LMPE 046 and C. gattii R265 culture media, causes these strains to become more resistant to amoebal predation. Conversely, addition of aspirin (a 3-hydroxy fatty acid inhibitor) to C. neoformans UOFS Y-1378 culture media made cells more susceptible to amoebae. Our data suggest that this molecule is secreted at a high enough concentration to effect intracellular signaling within amoeba, which in turn, promotes fungal survival.