Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs

Abstract

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.

FIG. 1.

Histology of hypha- and proteinase-deficient C. albicans mutants. Four hours after i.p. infection, wild-type (wt) C. albicans yeast and hyphal cells adhered to and began to invade liver tissue (A), while efg1 mutant yeast cells (B) and sap4 to sap6 yeast and hyphal cells (C) did not invade the tissue after 4 h. At this time point and at later stages, sap4 to sap6 mutant cells appeared to be surrounded by larger amounts of inflammatory cells (granulocytes and macrophages) than the wild-type cells. These cells seemed to inhibit the growth and penetration of the fungal mutant cells more strongly than the wild-type cells. Twenty-four hours after infection, a large number of hyphal cells of the wild type (D) and the sap1 to sap3 mutant (E) deeply penetrated the liver. In contrast, hyphal cells of the sap4 to sap6 mutant poorly invaded the liver (F). No efg1 mutant cells were found close to the liver, and invasion of efg1 mutant cells into the pancreas was completely blocked as noted 24 h after infection (G), while the cph1 mutant (H) showed the same invasive phenotype as the wild type.

FIG. 2.

C. albicans cells invading host cells and ultralocalization of Sap antigen. Electron microscopy of single C. albicans wild-type cells invading parenchymal tissue. Proteinase antigens were detected by immunogold labeling using Sap1- to Sap3- and Sap4- to Sap6-specific antibodies. (A) Hyphal cell directly penetrating a hepatocyte with Sap1 to Sap3 antigens on the cellular surface. (B) Localization of Sap4 to Sap6 proteinases on an invading hyphal cell (liver, 24 h after i.p. infection). (C) Localization of Sap4 to Sap6 antigens on a hyphal cell penetrating a granulocyte (eosinophil) as identified by typical granulate material (arrows) and morphology (liver, 24 h after i.p. infection).

FIG. 2.

C. albicans cells invading host cells and ultralocalization of Sap antigen. Electron microscopy of single C. albicans wild-type cells invading parenchymal tissue. Proteinase antigens were detected by immunogold labeling using Sap1- to Sap3- and Sap4- to Sap6-specific antibodies. (A) Hyphal cell directly penetrating a hepatocyte with Sap1 to Sap3 antigens on the cellular surface. (B) Localization of Sap4 to Sap6 proteinases on an invading hyphal cell (liver, 24 h after i.p. infection). (C) Localization of Sap4 to Sap6 antigens on a hyphal cell penetrating a granulocyte (eosinophil) as identified by typical granulate material (arrows) and morphology (liver, 24 h after i.p. infection).

FIG. 3.

SAP4 to SAP6 expression profile pattern of wild-type (wt), cph1, and efg1 strains. Hyphal production in wild-type cells was induced by incubation in 5% serum (A) or by pH- and temperature-regulated transition (B). Cph1- and Efg1-deficient strains were incubated under the same conditions. Samples were collected 10, 30, 60, 90, and 120 min after induction, and total RNA was isolated. RT-PCR was used to amplify SAP4- to SAP6-specific transcripts as described in Materials and Methods. Only the results from 25 (A) and 30 (B) cycles are shown. EFB1 primers were used as an internal control and to show the absence of contaminating genomic DNA (30 cycles) (14, 35, 36). The notably reduced level of SAP4 to SAP6 expression by the efg1 mutant observed during serum induction (A) was also observed with 30 PCR cycles. Both cph1 and efg1 mutants had enhanced levels of SAP4 to SAP6 transcripts during pH- and temperature-regulated transition (B). Since the 30-min sample of the cph1 mutant in panel A showed only a weak signal for EFB1 expression due to technical reasons, the possibility that SAP4 to SAP6 were expressed at this time point cannot be excluded.

FIG.4.

In vivo expression of the SAP gene family in infected liver during i.p. infections. Mice were infected i.p. with 10⁸ cells/mouse, and organs were removed. Total RNA from each organ sample was used for RT-PCR detection of SAP transcripts as described in Materials and Methods. At least five organs were investigated at each time point. wt, wild type.

FIG. 5.

In vivo expression of SAP4 to SAP6 in hypha-deficient mutants. Mice were infected i.p. with 10⁸ cells/mouse, and livers were removed. Total RNA from each liver sample was used for RT-PCR detection of SAP transcripts. The expression patterns for the wild type (wt) and the cph1 and efg1 mutants 24 h after i.p. injection for SAP4 to SAP6 genes are shown. In comparison to wild-type and cph1 mutant cells, efg1 mutants had strongly reduced levels of SAP4 to SAP6 transcripts. After 35 PCR cycles, no signals for SAP5 or SAP6 and only one weak signal for SAP4 were detectable in three samples. After 40 PCR cycles, mRNA expression of all three genes was detectable but notably reduced in the efg1 mutant compared to the wild type.

FIG. 6.

Tissue damage cause by C. albicans proteinase-deficient mutants. ALT activity (liver) and AM activity (pancreas) in blood of mice infected with C. albicans wild-type and Sap-deficient mutant cells 24 h after intraperitoneal infection were measured as indicators of invasion and tissue damage of liver and pancreas. Four animals were tested for each strain. Both SC5314 and the URA3/ura3 heterozygote strain CAF2-1, derived from SC5314, were used as wild-type controls. CAF2-1 was not found to be less invasive than SC5314. ∗, significantly different from value for CAF2-1 (P ≤ 0.05) as determined by Student's t test, with corrections according to Bonferroni.

FIG. 7.

Invasiveness of the SAP6/sap6 heterozygote mutant, control strains, and a transformant carrying a SAP6-containing plasmid (pAF3). Tissue damage of liver and pancreas was investigated as described for Fig. 6 with the following strains: SC5314 (SAP6/SAP6), M1060 (CAI4 with pCIp10), DSY344 (SAP6/sap6), DSY346 (sap6/sap6), M1065 (sap6/sap6 with pCIp10), and M1067 (sap6/sap6 with pAF3). Four animals were tested for each strain except for M1060, M1065, and M1067 for AM (two animals each). ∗, significantly different from value for M1065 (P ≤ 0.05) as determined by Student's t test, with corrections according to Bonferroni.