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. 1999 Feb;67(2):885-90.
doi: 10.1128/IAI.67.2.885-890.1999.

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages

S M Levitz  1 , S H Nong, K F Seetoo, T S Harrison, R A Speizer, E R Simons
Affiliations

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages

S M Levitz et al. Infect Immun. 1999 Feb.

Abstract

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 microM 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.

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Figures

FIG. 1
FIG. 1
Relationship between the ratio of excitation at 498 and 450 (Ex498/Ex450) and pH in MDM laden with C. neoformans labeled with OGITC and FITC. MDM were challenged for 3 h with C. neoformans labeled with OGITC or FITC and then permeabilized with nigericin-KCl in buffers at the pH indicated on the abscissa. Under such conditions, the pHs of the phagosome and the buffer are the same. Data are means ± SE of three independent experiments, each performed in duplicate.
FIG. 2
FIG. 2
Phagosomal pH of live and dead C. neoformans at various time points. MDM were challenged with OGITC-labeled live or dead fungi for 3 or 24 h, and then the pH of the intracellular fungi was determined as described in Materials and Methods. Data are means ± SE of four independent experiments, each performed in duplicate. P = 0.0004, comparing live fungi with dead fungi at 3 h; P = 0.0056, comparing live fungi at 3 and 24 h.
FIG. 3
FIG. 3
Effect of chloroquine on the phagosomal pH. MDM were challenged with OGITC- or FITC-labeled heat-killed C. neoformans for 3 h in the presence of the indicated concentration of chloroquine. The pH of the intracellular fungi was determined as described in Materials and Methods. Data are means ± SE of three independent experiments, each performed in duplicate.
FIG. 4
FIG. 4
Phagosome-lysosome fusion following MDM uptake of C. neoformans. MDM were challenged with live fungi for 3 h and then immunofluorescently stained for LAMP-1 with Alexa 488 and C. neoformans capsule with Alexa 494 as described in Materials and Methods. A single MDM that has phagocytosed four C. neoformans is shown. (a) LAMP-1 fluorescence (green) intensely distributed around the surface of the fungi (which faintly stains red). One fungus (arrows) appears to have been recently phagocytosed based on its appearance under phase-contrast microscopy (not shown) and has only a small rim of green fluorescence. LAMP-1 staining can also be appreciated in other parts of the cell except the nucleus. The same pattern of green fluorescence was seen in MDM stained for LAMP-1 with Alexa 488 but for which staining with the anticapsular antibody was not performed (not shown). (b) Capsule fluorescence (red) of the same cell. The C. neoformans capsule surrounding the four C. neoformans cells is readily apparent. Red fluorescence is also seen elsewhere in the MDM, mostly as a result of capsular polysaccharide secreted from the fungus. A similar pattern of fluorescence was seen in cells stained only for anticapsular antibody and not LAMP-1 (data not shown).
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