Investigation of the function of Candida albicans Als3 by heterologous expression in Candida glabrata

Abstract

During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata, a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.

Fig 1

C. albicans Als3 is expressed on the surfaces of C. glabrata in a functional manner. (A) Flow cytometric analysis of C. glabrata expressing Als3 (Cg-Als3) or the wild-type control strain (Cg-control) after the organisms were stained with an anti-Als3 antibody. The histogram shows the results of analysis of 10,000 cells per strain. (B) Expression of Als3 in C. glabrata results in increased adherence to and endocytosis by endothelial cells in vitro. HUVECs were incubated with the indicated strains of C. glabrata for 90 min, after which the numbers of endocytosed and cell-associated organisms were determined using a differential fluorescence assay. Results are the means ± standard deviations of 3 experiments, each performed in triplicate. *, P < 0.001 versus Cg-control. HPF, high-power field. (C) Growth rate of the indicated strains of C. glabrata in minimal medium. Data are representative results of one of two independent experiments.

Fig 2

Mice infected with Cg-Als3 had increased brain fungal burdens. (A) Mice were infected intravenously with the indicated strains of C. glabrata and then sacrificed at the indicated times for brain fungal burden determination. Results are the medians ± interquartile ranges of 3 experiments, each consisting of 8 mice per strain of C. glabrata. *, P < 0.002 versus Cg-control. (B) Histopathology of the brains of mice infected for 7 days with the indicated strains of C. glabrata. Brain sections were stained with Gomori methenamine silver, and arrows indicate the organisms. Scale bar indicates 20 μm. (C) Expression of Als3 in vivo. Thin sections of the brains obtained after 7 days of infection were stained with an anti-Als3 antibody (red) and an anti-Candida antibody (green) and then imaged by confocal microscopy. Scale bar indicates 10 μm.

Fig 3

Infection with Cg-Als3 induced an increased inflammatory response in the brain. Mice were infected intravenously with the indicated strains of C. glabrata and then sacrificed at the indicated times. The levels of MPO (A), TNF (B), MCP-1 (C), and IFN-γ (D) in the brain homogenates were measured. Results are the medians ± interquartile ranges of 8 mice per strain of C. glabrata. *, P < 0.015 versus Cg-control.

Fig 4

Expression of Als3 in C. glabrata resulted in increased adherence to and endocytosis by brain endothelial cells in vitro. HBMECs were incubated with the indicated strains of C. glabrata for 180 min, after which the numbers of endocytosed and cell-associated organisms were determined using a differential fluorescence assay. Results are the means ± standard deviations of 3 experiments, each performed in triplicate. *, P < 0.0001 versus Cg-control.

Fig 5

Mice infected with Cg-Als3 had increased fungal persistence in the kidneys. (A) Mice were infected intravenously with the indicated strains of C. glabrata and then sacrificed at the indicated times for kidney fungal burden determination. Results are the medians ± interquartile ranges of 3 experiments, each consisting of 8 mice per strain of C. glabrata. *, P < 0.03 versus Cg-control. (B) Histopathology of the kidneys of mice infected with the indicated C. glabrata strains for 1 day. Kidney sections were stained with Gomori methenamine silver (top row) and periodic acid-Schiff (bottom row). Arrows indicate the organisms. Scale bar indicates 20 μm. (C) Expression of Als3 in vivo. Thin sections of the kidneys after 1 day of infection with the indicated strains were stained with an anti-Als3 antibody (red) and anti-Candida antibody (green) and then imaged by confocal microscopy. Scale bar indicates 10 μm.

Fig 6

Mice infected with Cg-Als3 and Cg-control had similar profiles of kidney inflammatory response. Mice were infected intravenously with the indicated strains of C. glabrata and then sacrificed at the indicated times. The levels of MPO (A), TNF (B), MCP-1 (C), and IFN-γ (D) in the kidney homogenates were measured. Results are the medians ± interquartile ranges of 8 mice per strain of C. glabrata. *, P < 0.03 versus Cg-control.

Fig 7

Expression of Als3 in C. glabrata had minimal effect on endothelial cell damage and stimulation of IL-8 release. HUVECs and HBMECs were incubated with Cg-control, Cg-Als3, or C. albicans for 16 h, after which the extent of endothelial cell damage (A) and secretion of IL-8 in to the medium (B) were determined. Results are the means ± standard deviations of 3 experiments, each performed in triplicate. *, P < 0.05 versus uninfected endothelial cells; †, P < 0.0001 versus Cg-Als3.