Cryptococcus neoformansResides in an Acidic Phagolysosome of Human Macrophages

Autor: Levitz, Stuart M., Nong, Shu-Hua, Seetoo, Kurt F., Harrison, Thomas S., Speizer, Robert A., Simons, Elizabeth R.
Zdroj: Infection and Immunity; February 1999, Vol. 67 Issue: 2 p885-890, 6p
Abstrakt: ABSTRACTRecently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogenCryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformansin cell-free media and pH. These data suggested that C. neoformanswas well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containingC. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformansdoes not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization ofC. neoformansand the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformansdoes not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.
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