American Society for Microbiology, Eukaryotic Cell, 5(12), p. 761-774, 2013
DOI: 10.1128/ec.00073-13
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Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evo-lutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellu-larly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter pro-tein 1), is involved in the transport of 5-and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capac-ity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better under-standing of how C. neoformans adapts to the mammalian host. C ryptococcus neoformans is an opportunistic pathogen often found in soils contaminated with bird excreta (1). Infection by C. neoformans, which occurs through inhalation of propagules from the environment by the host, seems to be accidental since C. neoformans is a saprophytic fungus that does not require an ani-mal host for replication and survival (2). In the case of human exposure to C. neoformans, the establishment of infection and subsequent development of cryptococcosis depend both on the host's immune response and the virulence of the fungus (3) One of the first lines of defense in the lung are alveolar macrophages, which are able to phagocytose C. neoformans efficiently (4). Phagocytosis is followed by phagosome acidification and fusion of lysosomes (5). However, this process does not always result in the death of yeast cells and C. neoformans can survive and replicate within macrophages in vivo, as shown by Feldmesser et al. (6). The ability of C. neoformans to survive in the intracellular en-vironment of phagocytes might have a critical role in disease pro-gression and probably contributes to the propensity of the fungus to cause chronic and latent infections (3, 7, 8). However, it is known that the intracellular microenvironment of phagocytes is extremely inhospitable to internalized microorganisms due to nu-tritional and oxidative stress and exposure to antimicrobial pep-tides and hydrolytic enzymes (9, 10, 11). To survive the harsh environment of the phagosome, C. neoformans is able to repro-gram its gene expression profile. Twenty-four hours after phago-cytosis by murine macrophages, C. neoformans responds to car-bon starvation by upregulating genes that encode sugar transporters and proteins involved in the utilization of alternative carbon sources, including enzymes of the glyoxylate cycle and fatty acid metabolism. Genes related to the oxidative stress re-sponse were also induced (4). Since C. neoformans is a free-living fungus, its mammalian in-tracellular lifestyle is particularly curious, given that this organism has no obvious requirement for animal virulence in its life cycle. In this sense, the evolutionary origin and the maintenance of viru-lence strategies that allow for survival of C. neoformans within macrophages have been an issue of interest, and the investigation of the C. neoformans ecological niche may be informative in this regard. Steenbergen et al. (12) suggested that the competence of C. neoformans to proliferate within mammalian phagocytic cells was initially selected to confer an advantage against environmental predators. This idea was supported by the fact that interaction of C. neoformans with the amoeba Acanthamoeba castellanii results in the ingestion of the fungus followed by its intracellular replica-tion and the accumulation of vesicles containing polysaccharide in the cytoplasm of amoeboid cells (12). This result is similar to those previously observed in the interaction of C. neoformans with macrophages. Furthermore, mutant strains defective in capsule