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Infection and Immunity, February 1999, p. 885-890, Vol. 67, No. 2
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cryptococcus neoformans Resides in an Acidic Phagolysosome of Human Macrophages

Stuart M. Levitz,1,* Shu-Hua Nong,1 Kurt F. Seetoo,2 Thomas S. Harrison,3 Robert A. Speizer,1 and Elizabeth R. Simons2

Evans Memorial Department of Clinical Research and Department of Medicine1 and Department of Biochemistry,2 Boston University Medical Center, Boston, Massachusetts, and Department of Infectious Diseases, St. George's Hospital Medical School, London, England3

Received 2 October 1998/Returned for modification 5 November 1998/Accepted 17 November 1998

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus 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. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. 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. neoformans does 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 of C. neoformans and 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. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

* Corresponding author. Mailing address: Room E336, Boston Medical Center, 88 E. Newton St., Boston, MA 02118. Phone: (617) 638-7904. Fax: (617) 638-8070. E-mail: slevitz{at}bu.edu.

Infection and Immunity, February 1999, p. 885-890, Vol. 67, No. 2
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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