An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

Abstract

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans

“…We also investigated PLC␥ by pretreating YT cells with the PLC␥ inhibitor, U-73122, at concentrations found to inhibit tumor killing (34). The anti-cryptococcal activity was examined, and we found that the addition of YT cells significantly reduced the number of cryptococcal cfu, which is consistent with our previous observations of killing (1,27,35). Despite using concentrations of U-73122 in excess to inhibit PLC␥, there was no impact on YT anti-cryptococcal killing, suggesting that PLC␥ was not involved in NK cell-mediated cryptococcal killing (Fig.…”

Cryptococcus gattii Infections

“…They exert a direct cytotoxic effect on cryptococcal cells via exocytosis of lytic granules containing the effector compound perforin (164). It was recently reported that perforin degranulation is increased in an acidic environment, at pH levels reported previously to be generated within cryptococcomas (163); this effect was observed with both C. gattii and C. neoformans (165).…”

Fungal colonization of the brain: anatomopathological aspects of neurological cryptococcosis

“…Space occupying lesions can be observed in infected individuals with the formation of cysts, abscesses or granulomas during neurological cryptococcosis (Watabe et al 1984, Lee et al 1996b, Kamezawa et al 2000. Granulomas originated from C. neoformans infections are commonly called cryptococcomas (Chen et al 2000, Islam et al 2013.…”