Soil amoebae help to unravel fungal pathogenesis

Cryptococcus neoformans is a facultative intracellular fungal pathogen that can cause life-threatening meningitis in immunocompromised patients. Its ecological niche is soil and the normal route of infection is inhalation of infectious particles. C. neoformans cells are encapsulated by a cell wall composed of polysaccharide, which protects the fungus from intracellular destruction by phagocytic cells. Legionella pneumophila was the first bacterial pathogen shown to multiply and persist in macrophages and amoebae. Amoebae and macrophages phagocytose particles into vacuoles and secrete lysosomal enzymes that digest the particles; the intracellular events after ingestion of L. pneumophila by amoebae and macrophages are similar.Amoebae of the genus Acanthamoeba feed on bacteria and fungi. Acanthamoeba is a freshwater and soil protozoan that was originally isolated from cultures of C. neoformans, and has been used to study bacteria–amoeba interactions. A previous study using fungal cells demonstrated that Acanthamoeba cells can phagocytose and kill 78%–99% of cells using several C. neoformans isolates. Streenbergen et al. [1xCryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Steenbergen, J.N. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15245–15250CrossRef | PubMed | Scopus (230)See all References][1] have now tested the hypothesis that C. neoformans is capable of intracellular growth in amoebae and that the mechanism of subversion of macrophage function is selected to facilitate survival of interactions with environmental phagocytic cells.To examine the interaction between amoebae and fungal cells, several wild-type, capsular strains were compared with several mutant, acapsular strains (including a pseudohyphal mutant) in a multitude of cellular assays. The addition of fungal cells to amoebae resulted in phagocytosis of the fungal cells. The efficiency of phagocytosis was determined to be between 17%–23% for capsular strains and 34%–65% for mutants defective in capsule or pseudohyphal production. This strongly suggested that fungal cells lacking capsules were phagocytosed at a significantly higher rate than capsular cells, suggesting a role for this cellular structure in avoiding ingestion. The number of ‘buds’ formed on the surface of the amoebae after infection, an indicator of intracellular replication, was also determined. In wild-type, capsular strains, the number of buds increased over time. Mutant strains of C. neoformans, however, showed no significant levels of budding, suggesting that they were probably being destroyed intracellularly. Trypan-blue exclusion of amoebae revealed that mutant C. neoformans cells were not able to kill the amoebae, whereas wild-type strains readily lysed them. Scanning electron micrographs of infected amoebae indicated signs of intracellular development of wild-type strains, and lack of development of mutant strains. An amoeba-killing assay was also used to quantify the levels of killing of amoebae by fungal cells. Again, those cells with wild-type capsules had a greater number of colony-forming units than those lacking the polysaccharide coat. A striking contrast between this study and that of L. pneumophila–amoebae interactions should be noted here: fungal cells destroy amoebae, probably to prevent intracellular destruction, rather than using them as hosts for replication, as is the case with the bacterial pathogen.This elegant study details for the first time the cellular interaction between C. neoformans and amoebae, cells which fungal particles often encounter in nature. Amoebae are also similar to macrophages found in the human host, and therefore provide insight into pathogenic mechanisms in the human host. This study also develops a useful tool to test the role of potential fungal virulence factors. One can also extrapolate from this biological example and begin to address questions regarding the evolutionary adaptation of pathogens to their vertebrate hosts.