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. 2011 Oct;7(10):e1002305.
doi: 10.1371/journal.ppat.1002305. Epub 2011 Oct 6.

Mechanisms of Candida albicans trafficking to the brain

Yaoping Liu  1 , Rahul Mittal, Norma V Solis, Nemani V Prasadarao, Scott G Filler
Affiliations

Mechanisms of Candida albicans trafficking to the brain

Yaoping Liu et al. PLoS Pathog. 2011 Oct.

Abstract

During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.

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Conflict of interest statement

SGF is one of the founders of NovaDigm Therapeutics and serves on its scientific advisory board.

Figures

Figure 1
Figure 1. A vps51Δ/Δ mutant has attenuated overall virulence, but increased brain tropism during hematogenously disseminated candidiasis.
(A and B) Survival of mice after intravenous inoculation with 5×105 (A) or 3×106 (B) yeast phase cells of the indicated strains of C. albicans (n = 10 mice per strain). (C – E) Fungal burden of the kidneys (C), liver (D), and brain (E) of mice at the indicated times after inoculation with 5×105 cells of the various strains. Results from days 1, 4, 7, 14, and 21 are median ± interquartile range of a single experiment with 7 mice per strain at each time point. Data from day 3 are the combined results from two experiments, each with 6–7 mice per strain. Only mice infected with the vps51Δ/Δ mutant were analyzed at the 7, 14, and 21 day time points because all of the mice infected with the other C. albicans strains had died. At these later time points, the absence of a bar indicates that the fungal burden was below the limits of detection. *p<0.01 compared to the wild-type (WT) and vps51Δ/Δ+pVPS51 complemented strains by the Wilcoxon Rank Sum Test. (F). Histopathology of the hypothalamus of mice after 3 days of infection with the indicated strains. Sections were stained with Gomori methenamine silver. Scale bar 10 µm. Arrows indicate the organisms.
Figure 2
Figure 2. Deletion of VPS53 causes reduced mortality and increased brain fungal burden during hematogenously disseminated candidiasis.
(A) Survival of mice after intravenous inoculation of with 3×106 yeast phase cells of the indicated strains. 10 mice were infected with each strain. (B) Increased brain fungal burden of mice 3 days after intravenous inoculation of 5×105 cells per strain. Results are the median ± interquartile ranges of 7 mice per strain. *p<0.01 compared to the wild-type and vps53Δ/Δ+pVPS53 complemented strains.
Figure 3
Figure 3. The vps51Δ/Δ mutant interacts differently with human umbilical vein endothelial cells (HUVECs) versus human brain microvascular endothelial cells (HBMECs).
(A and B) Adherence of germ tubes of the indicated C. albicans strains to HUVECs (A) and HBMECs (B). (C and D) Endocytosis of hyphae of the indicated strains by HUVECs (C) and HBMECs (D). The results are expressed as a percentage of the wild-type strain and are the mean ± SD of 3 experiments, each performed in triplicate. The mean adherence of the wild-type strain to HUVECs and HBMECs was 47% and 28%, respectively. The mean number of wild-type cells endocytosed by HUVECs and HBMECs was 109 and 35 organisms per 10 high-powered fields (HPF), respectively. *p<0.01 compared to both the wild-type strain and the vps51Δ/Δ+pVPS51 complemented strain by Analysis of Variance.
Figure 4
Figure 4. C. albicans endocytosis by HBMECs is mediated by gp96.
(A) Reduced HBMEC endocytosis of wild-type and vps51Δ/Δ mutant strains of C. albicans by an anti-gp96 antibody. (B and C) Knockdown of gp96 by siRNA inhibits the endocytosis of C. albicans by HBMECs (B), but not by HUVECs (C). (D) Gp96 knockdown has no effect on transferrin endocytosis by HBMECs. (E and F) Overexpression of human gp96 in HBMECs (E) and Chinese Hamster Ovary (CHO) cells (F) results in increased endocytosis of C. albicans. The results are expressed as a percentage of the wild-type strain and are the mean ± SD of 3 experiments, each performed in triplicate. The mean number of wild-type cells endocytosed by control HBMECs, HUVECs, and CHO cells was 41, 49, and 48 organisms per 10 HPF, respectively. *p<0.01 compared to control strains. Images to the right of the graphs are of representative immunoblots showing the effects of the interventions on total gp96 and β-actin protein levels in the cells.
Figure 5
Figure 5. Deletion of SSA1 or ALS3 reduces HBMEC endocytosis of C. albicans.
(A and B) Endocytosis of the indicated strains of C. albicans by HBMECs. The results are expressed as a percentage of the wild-type strain and are the mean ± SD of 3 experiments, each performed in triplicate. A mean 65 of wild-type cells per HPF was endocytosed by the HBMECs. *p<0.01 compared to the wild-type strain; †p<0.01 compared to the vps51Δ/Δ mutant.
Figure 6
Figure 6. Both Als3 and Ssa1 induce HBMEC endocytosis, but Als3 has a greater effect than Ssa1.
(A and B) Endocytosis by HUVECs (A) and HBMECs (B) of S. cerevisiae expressing C. albicans SSA1, or the backbone vector (control). (C and D) Endocytosis by HUVECs (C) and HBMECs (D) of S. cerevisiae expressing C. albicans Als3 or the backbone vector (control). (E) Effect of siRNA knockdown of gp96 on HBMEC endocytosis of S. cerevisiae expressing C. albicans ALS3. Image on the right is of a representative immunoblot of total HBMEC lysates probed for gp96 and β-actin. The endocytosis data are the mean ± SD of 3 experiments, each performed in triplicate. The results in (A–D) are expressed as a percentage of the control strain containing the backbone vector and the results in (E) are expressed as a percentage of the endocytosis of the ALS3 expressing strain by HBMEC transfected with control siRNA. The mean number of control cells endocytosed by the HUVECs and HBMECs in (A–D) was 3.2 and 1.2 organisms per 10 HPF, respectively. A mean of 49 cells of the Als3 expressing strain per HPF was endocytosed by HBMEC transfected with the control siRNA in (E). *p<0.01 compared to control strains.
Figure 7
Figure 7. Effects of deletion of VPS51, SSA1, and ALS3 on binding to gp96 and fungal surface expression of Als3.
(A and B) Immunoblots of HBMEC membrane proteins that were bound by the indicated strains of C. albicans (A) or S. cerevisiae (B). Both blots were probed with an anti-gp96 antibody. (C) Flow cytometric analysis of Als3 exposure on the surface of hyphae of the wild-type, vps51Δ/Δ mutant, and vps51Δ/Δ+pVPS51 complemented strains.
Figure 8
Figure 8. Deletion of SSA1 and ALS3 have different effects on brain fungal burden.
(A and B) Brain fungal burden of mice after 3 days of infection with the indicated strains of C. albicans. Results are median ± interquartile ranges of 7 mice per strain. *p<0.02 compared to mice infected with the wild-type strain; † p<0.001 compared to mice infected with the vps51Δ/Δ mutant.
Figure 9
Figure 9. Model of the receptor-ligand interactions that mediate the endocytosis of C. albicans by HBMECs.
C. albicans Als3 binds to gp96 on the surface of HBMECs and induces endocytosis. C. albicans Ssa1 binds to an HBMEC surface protein other than gp96, which also induces endocytosis. PM, plasma membrane.
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