Limited Role of Secreted Aspartyl Proteinases Sap1 to Sap6 in Candida albicans Virulence and Host Immune Response in Murine Hematogenously Disseminated Candidiasis

Mice.Male BALB/c mice, 8 to 10 weeks old, were purchased from Charles River (Barcelona, Spain) and kept under specific-pathogen-free conditions at the Animal Facility of the Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal. All procedures involving mice were performed according to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123), the 86/609/EEC directive, and Portuguese rules (DL 129/92).

Candida albicans and culture conditions.The C. albicans strains used in this study are listed in Table 1. All strains were maintained as frozen stocks in 30% glycerol at −80°C. Yeast growth was tested in synthetic glucose minimal (2% glucose, 0.67% Bacto yeast nitrogen base without amino acids) and complex yeast-peptone-dextrose (YPD) (2% glucose, 1% yeast extract, 2% Bacto peptone) liquid media at 30°C and 37°C in a shaking incubator for 24 h. Growth was measured at 60-min intervals, and the generation times were calculated. Hypha formation was induced by the addition of calf serum (10%) (Invitrogen, Carlsbad, CA) or N-acetylglucosamine (2.5 mM) (Sigma, St. Louis, MO) at 37°C. To prepare the inocula for infection, C. albicans strains were grown in a shaking incubator for 14 h at 30°C in Winge medium (0.2% glucose, 0.3% yeast extract). Yeast cells were harvested, washed twice with sterile, nonpyrogenic phosphate-buffered saline (PBS), counted in a hemocytometer, and resuspended at the appropriate concentrations. Inocula were confirmed by CFU counts on Sabouraud dextrose agar (Difco, Detroit, MI) for up to 48 h at 37°C.

C. albicans hematogenously disseminated infections.Mice were injected intravenously (i.v.) in the lateral tail vein with 1 × 10⁵ or 5 × 10⁵C. albicans yeast cells in 0.2 ml PBS. To evaluate the progress of hematogenously disseminated candidiasis, mice were weighed and monitored twice daily. Moribund mice were humanely terminated, and their deaths were recorded as occurring on the following day.

Other groups of mice were infected with 5 × 10⁴ yeast cells and sacrificed 3 and 7 days postinfection to determine organ fungal burden and/or immunological parameters. Control mice were injected i.v. with PBS. Kidneys were aseptically removed, weighed, homogenized, and quantitatively cultured on Sabouraud dextrose agar (Difco) at 37°C. Values are expressed as log CFU per gram of tissue. Alternatively, kidneys, liver, lungs, and brain were fixed in 10% phosphate-buffered formaldehyde, followed by periodic acid-Schiff (PAS) reagent staining and counterstaining of the paraffin-embedded tissues with hematoxylin in order to evaluate both fungal morphology and the composition and distribution of inflammatory infiltrates.

Quantitative real-time PCR (qRT-PCR).BALB/c mice were i.v. infected with 1 × 10⁵C. albicans SC5314, SAP123MS4C, and SAP456MS4A yeast cells. Total RNA was isolated from the kidneys of three mice per group 3 and 7 days after infection. Briefly, the kidneys were removed, homogenized in PBS, and centrifuged at 1,500 × g at 4°C for 10 min. Pellets were washed twice with ice-cold RNase-free water and frozen in liquid nitrogen until RNA extraction, which was performed by using the hot acidic phenol method (4). Total RNA was incubated with DNase I, amplification grade (Invitrogen), for 15 min at room temperature to eliminate genomic DNA contamination. DNase I was inactivated according to the manufacturer's instructions.

The Superscript III Platinum two-step qRT-PCR kit with SYBR green (Invitrogen) was used to generate first-strand cDNA from each DNase I-treated RNA sample, as follows: 10 min at 65°C, 60 min at 37°C, and 10 min at 65°C. Quantitative PCR (qPCR) was performed with Platinum SYBR green qPCR SuperMix-UDG (Invitrogen). Three microliters of each cDNA sample was added to a 25-μl PCR mixture containing 12.5 μl of Platinum SYBR green qPCR SuperMix-UDG, 0.5 μl of 10 μM specific forward and reverse primers (Table 2), and 8.5 μl of RNase-free water. Each reaction was performed with a Corbett Rotor-Gene 6000 instrument (Qiagen). Thermocycling conditions for SAP and ACT1 quantification were 2 min at 50°C (UDG incubation) and 5 min at 95°C, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The specificity of each primer pair was verified by the presence of a single melting-temperature peak. The calibration and efficiency of SAP and ACT1 primers were assessed in titration experiments using C. albicans SC5314 genomic DNA (500 ng to 5 pg) in serial dilutions. A negative (water) control and a four-point curve of SC5314 genomic DNA were included in each run. DNase I-treated RNA was used to exclude genomic DNA contamination. SAP1 to SAP10 gene expression was normalized to the housekeeping gene ACT1 and analyzed by using both the standard curve and the comparative threshold cycle (C_T) (ΔΔC_T) methods. Data are presented as the fold difference in expression relative to wild-type (WT) gene expression from infected mice. Each experimental condition was performed in triplicate, and reactions were done in duplicate on different days for reproducibility purposes.

Flow cytometric analysis.The assessment of cell surface and cytoplasmic lineage or activation markers on different splenic leukocyte populations was performed by flow cytometric analysis (fluorescence-activated cell sorter [FACS] analysis). Spleens were aseptically removed and homogenized to single-cell suspensions in Hanks’ balanced salt solution (HBSS) (Sigma). A total number of 1 × 10⁶ leukocytes were stained per sample.

The following monoclonal antibodies (MAbs), along with the respective isotype controls, were used (at previously determined optimal dilutions) for immunofluorescence cytometric analysis with a FACScan apparatus (Becton Dickinson, San Jose, CA) using CELLQUEST software (Becton Dickinson): phycoerythrin (PE) anti-mouse CD40 (1C10) and biotin anti-mouse major histocompatibility complex (MHC) class II (NIMR-4) (Southern Biotechnology Associates, Birmingham, AL); fluorescein isothiocyanate (FITC) anti-mouse/rat Foxp3 (FJK-16S), PE-Cy5 anti-mouse CD4 (L3T4) (RM4-5), and PE anti-mouse F4/80 antigen (BM8) (eBioscience, San Diego, CA); and FITC anti-mouse CD11c (HL3), FITC anti-mouse Ly-6G and Ly-6C (Gr-1) (RB6-8C5), PE anti-mouse CD25 (PC61), FITC anti-mouse CD45R/B220 (RA3-6B2), PE anti-mouse CD80 (B7-1) (16-10A1), PE anti-mouse CD86 (B7-2) (GL1), PE rat anti-mouse interleukin-4 (IL-4) (BVD4-1D11), FITC anti-mouse gamma interferon (IFN-γ) (XMG1.2), and PE anti-mouse IL-10 (JES5-2A5) (BD Pharmingen, San Diego, CA). Biotin-conjugated MAbs were revealed with streptavidin-PE-Cy5 (BD Pharmingen). Cells were preincubated for 15 min with anti-FcγR (a kind gift of Jocelyne Demengeot, Gulbenkian Institute of Science, Oeiras, Portugal) before CD11c and Foxp3 staining. The Foxp3 Staining Buffer set (eBioscience) was used for the fixation and permeabilization of splenocytes previously surface stained with CD4 and CD25 MAbs.

The intracellular expression of the cytokines IFN-γ, IL-4, and IL-10 was detected in splenic CD4⁺ T lymphocytes. The intracellular expression of the cytokines IFN-γ and IL-4 was also detected in renal CD4⁺ T lymphocytes. Splenocytes were obtained as described above. Red blood cell lysis was performed by incubation with 0.15 M ammonium chloride. Cells were washed and resuspended in complete RPMI medium (Sigma) (RPMI 1640 medium supplemented with 50 U of penicillin/ml, 50 μg of streptomycin/ml, 1% HEPES buffer [Sigma], 10% fetal calf serum [FCS] [Invitrogen], and 5 μM 2-mercaptoethanol). The kidneys were minced with a razor blade and incubated for 30 min at 37°C in RPMI 1640 complete medium containing collagenase D (Sigma-Aldrich) at 2 μg/ml. Cells were homogenized to single-cell suspensions, washed, and resuspended in RPMI 1640 complete medium. Mononuclear cells were separated from the above-described suspensions by layering 5 ml onto 2.5 ml of a polysucrose-sodium ditrizoate solution (Histopaque 1083; Sigma) and centrifuged at 800 × g for 20 min at room temperature. Mononuclear cells collected from the medium-Histopaque interface were washed and resuspended in RPMI 1640 complete medium. Spleen and renal cells (1 × 10⁶ cells) were transferred into 96-well tissue culture plates (Nunc, Roskilde, Denmark) and stimulated for 4.5 h with 20 ng/ml phorbol myristate acetate (Sigma) and 200 ng/ml ionomycin (Sigma) in the presence of 10 μg/ml of brefeldin A (Roche, Penzberg, Germany).

The staining of the cell surface marker CD4 was performed as described above, after a preincubation step of 15 min with anti-FcγR, followed by fixation with 2% formaldehyde. Cells were permeabilized with 0.5% saponin in flow cytometric buffer (PBS containing 1% bovine serum albumin [BSA] and 10 mM sodium azide), and subsequently, cells were incubated for 15 min with anti-FcγR and stained for 30 min at room temperature with the appropriate antibody. Intracellular staining with the isotypic controls was performed to confirm the specificity of antibody binding.

Th1 cells were defined as being CD4⁺ IFN-γ⁺ IL-4⁻, and Th2 cells were defined as being CD4⁺IFN-γ⁻ IL-4⁺. Ratios of Th1/Th2 cells were generated to determine the presence of a polarized immune response. The immune response in noninfected mice was defined as being unpolarized.

Serum IFN-γ, IL-4, and IL-10 measurements.The concentration of IL-4 in the sera of C. albicans-infected mice and noninfected controls was quantified with the Quantikine M murine IL-4 enzyme-linked immunosorbent assay (ELISA) kit, and serum IFN-γ and IL-10 concentrations were quantified with Duo-Set ELISA kits (all from R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Statistical analysis.Unless otherwise stated, results shown are from one experiment representative of three independent experiments. The statistical significance of results was determined by the unpaired Student t test, and survival data were analyzed with the log-rank test by using GraphPad Prism 4 software (GraphPad Software, Inc., La Jolla, CA). Results were considered statistically significant with P values of less than 0.05.

Virulence of C. albicans in a murine model of hematogenously disseminated infection.Prior to the virulence studies, we determined the generation time for each strain in synthetic defined and complex media at 30° and 37°C and the ability to form hyphae in serum- or N-acetylglucosamine-containing media. No yeast growth defects were observed under the conditions tested (similar generation times), and filamentous growth was similar to that of wild-type (WT) strain SC5314 under the hypha-inducing conditions analyzed (data not shown).

To explore the role of the aspartyl proteinases Sap1 to Sap6 as virulence factors in the course of hematogenously disseminated candidiasis, BALB/c mice were initially infected i.v. with 5 × 10⁵ and 1 × 10⁵ cells of C. albicans WT strain SC5314 and two independent series of homozygous-deletion triple-mutant strains lacking the SAP1 to SAP3 genes (SAP123MS4C and SAP123MS4D) or lacking the SAP4 to SAP6 genes (SAP456MS4A and SAP456MS4B) (29). The independent mutant strains behaved indistinctively (data not shown), and therefore, the results presented below correspond to those obtained with strains SAP123MS4C and SAP456MS4A.

Mice infected with the highest inoculum showed 100% mortality for every group by day 6 after infection, and no differences could be observed (data not shown).

No differences in survival time were observed after i.v. infection of BALB/c mice with 1 × 10⁵ CFU of the WT or Δsap123 mutant strain SAP123MS4C (P = 0.5698 by log-rank test) (Fig. 1A). Mice infected with Δsap456 mutant strain SAP456MS4A had an extended overall survival time compared to that of their WT-infected counterparts (Fig. 1). The median survival times of SAP456MS4A-infected mice were 13 and 16 versus 10 and 12 days for WT-infected mice, as determined for the independent experiments shown in Fig. 1A and B, respectively. All mice injected with either the WT or the triple-mutant strain SAP123MS4C succumbed to candidal infection within 17 days. In contrast, at the end of the experimental period (30 days), 15 to 20% of the mice infected with the Δsap456 mutant survived infection. Nevertheless, the kidneys of the surviving mice had Candida microabscesses and granulomas revealed by histopathological analysis (data not shown). Despite these differences, statistical comparisons of the survival curves revealed that the survival of mice infected with the WT strain did not differ significantly from that of mice infected with the Δsap456 mutant (P = 0.1405 and P = 0.1331) (Fig. 1A and B, respectively).

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FIG. 1.

Influence of SAP1 to SAP6 on C. albicans virulence in a murine model of hematogenously disseminated candidiasis. Male BALB/c mice were i.v. injected with 1 × 10⁵ cells of C. albicans WT strain SC5314 and mutant strains SAP123MS4C and SAP456MS4A (A); C. albicans WT strain SC5314 and mutant strains SAP4MS4A, SAP5MS4A, SAP6MS4A, and SAP456MS4A (B); and C. albicans WT strains SC5314 and CAF2-1 and mutant strains SAP123MS4C, M119, SAP456MS4A, and DSY459 (C). Each strain was injected into seven mice per group, per experiment, and survival was monitored twice daily for 30 days (A and B) or for 60 days (C). Results are representative of two to three independent experiments.

To ascertain the contribution of each of the deleted genes of the triple Δsap456 mutant to the slightly extended survival time observed, Δsap4, Δsap5, and Δsap6 single-mutant strains were used. As shown in Fig. 1B, the survival time of mice infected with strains SAP4MS4A, SAP5MS4A, and SAP6MS4A was statistically equivalent to that of their WT-infected counterparts (P = 0.6265, P = 0.2121, and P = 0.9465, respectively). The observed median survival time was, however, consistently longer for mice infected with mutants lacking SAP5, such as with strains SAP5MS4A and SAP456MS4A. The survival curves of mice injected with these two strains were similar (P = 0.8621).

These results are in apparent contrast with previous studies, with a similar model of murine hematogenously disseminated candidiasis, using single-mutant strains deficient in SAP1, SAP2, or SAP3 (20) and a triple mutant lacking the SAP4 to SAP6 genes (43), constructed with the Ura-blaster technique (17). In those studies, mutant strains deficient in each of the SAP1 to SAP3 genes and Δsap456 triple-mutant strain DSY459 were reported to survive longer than WT-infected mice, and the latter mutant was reported to have significantly decreased mean CFU in the kidneys (43). Therefore, parallel experiments with the SAT1-flipping mutants and Ura-blaster triple-mutant Δsap123 strain M119 (23) and Δsap456 strain DSY459 (43) were performed by using the same batch of BALB/c mice and the same experimental conditions. Strain CAF2-1 (17) was also included. As shown in Fig. 1C, significant differences in survival times were observed between mice infected with Δsap123 strain M119 and Δsap456 strain DSY459 and control strain CAF2-1 (P = 0.0089 and P = 0.0065, respectively). Mice infected with the Ura-blaster mutants survived significantly longer than mice infected with the equivalent SAT1-flipping mutants. The median survival times of mice infected with mutants lacking SAP1 to SAP3 were 21 days for the Ura-blaster-constructed Δsap123 mutant strain M119 and 10 days for Δsap123 mutant strain SAP123MS4C (P = 0.0127). Similar results were found when comparing the triple mutants lacking the SAP4 to SAP6 genes. The median survival times were 51 days for mice infected with the Ura-blaster mutant and 13 days for mice infected with the SAT1-flipping mutant (P = 0.0096). The survivals of mice infected with WT strain SC5314 and with URA3 heterozygous strain CAF2-1 were similar (P = 0.7005).

The evaluation of the fungal ability to invade the kidneys has been frequently used to measure the virulence of C. albicans strains (30, 38). The numbers of C. albicans CFU in the kidneys of mice infected with either the SAT1-flipping or the Ura-blaster mutants as well as with the respective control strains were similar, except for mice infected with Δsap456 strain DSY459 (Fig. 2). Seven days after infection, CFU numbers were significantly reduced in mice infected with the latter mutant (P = 0.0149 for CAF2-1 versus Δsap456 strain DSY459).

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FIG. 2.

Kidney fungal burden of BALB/c mice 3 and 7 days after i.v. infection with 5 × 10⁴ CAF2-1 (⧫), M119 (▴), DSY459 (○), SC5314 (□), SAP123MS4C (▵), and SAP456MS4A (•) C. albicans cells. Data are representative of two independent experiments. Each symbol represents an individual mouse, and horizontal bars are means of CFU numbers for each group.

The two sets of mutants tested behaved distinctly in the same experimental model, suggesting that the observed differences could be due to the effect of the ectopic URA3 insertion and not caused by the disruption of SAP genes. However, the limited impact of the SAP gene deletion in C. albicans virulence could be due to a compensatory expression of the nondeleted SAP genes, as reported previously for Ura-blaster mutants (34, 45). The expression of SAP1 to SAP10 in SC5314 and in SAT1-flipping triple mutants was evaluated by qRT-PCR in kidney samples 3 and 7 days after infection. Only the results for the latter time point analyzed are presented, since after 3 days, the fungal burden was often insufficient to obtain reproducible results. The expression levels were always inferior or similar to the level of ACT1 expression in the WT strain, except for SAP7.

No significant differences in SAP1 to SAP10 expression were observed for either mutant compared with SC5314. However, mRNA levels of SAP4 were higher in the Δsap123 mutant, and the levels of expression of SAP1, SAP2, and SAP3 trended higher in the Δsap456 mutant (Fig. 3). Thus, the virulence phenotypes observed do not seem to be due to significant compensatory upregulation.

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FIG. 3.

Compensatory upregulation of SAP1 to SAP10 in sap triple-mutant strains SAP123MS4C and SAP456MS4A from kidney homogenates 7 days after i.v. infection with 1 × 10⁵ cells. Results are presented as fold differences in expression relative to that of WT SC5314 from infected mice.

Although not significant, a reduction in virulence was consistently seen for the Δsap456 mutant. Therefore, the impact of a deficiency of SAP4 to SAP6 on C. albicans virulence was further evaluated.

The abilities of WT and Δsap456 triple-mutant strains to infect and injure several organs were assessed by histopathological analysis of the kidneys, liver, lungs, and brain 3 and 7 days after infection. As shown in Fig. 4, similar C. albicans cell morphologies and invasive abilities of the WT and mutant strains were observed. The kidneys of both mouse groups showed moderate multifocal renal medullary interstitial neutrophilic infiltration, with small areas of ductular necrosis. Intralesional PAS-positive organisms both in yeast and with septated, branched hyphal morphology were detected 3 days after C. albicans i.v. infection with strains SC5314 (Fig. 4A) and SAP456MS4A (Fig. 4B). At the later time point tested, 7 days after infection, analysis of the kidneys of WT-infected (Fig. 4C) and mutant-infected (Fig. 4D) mice showed moderate to severe, focally extensive to coalescing, renal medullary interstitial neutrophilic infiltration surrounding numerous PAS-positive organisms. These organisms were present mainly as septated, branched hyphal structures, which largely effaced the medulla and invaded the urothelium. Invasion of liver, lungs, and brain was not consistently seen (data not shown). Altogether, these results suggest that the SAP4 to SAP6 genes are not essential for the invasion of the kidneys during hematogenously disseminated candidiasis.

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FIG. 4.

Representative photomicrographs of histological sections of kidneys from BALB/c mice infected with 5 × 10⁴ SC5314 (A and C) and SAP456MS4A (B and D) C. albicans cells. At 3 days post-i.v. infection, PAS-positive hyphae and yeast-like organisms were present in the renal medulla, partially effacing the renal tubuli and eliciting moderate neutrophilic infiltration in both WT-infected (A) and mutant-infected (B) mice. At 7 days post-i.v. infection, numerous PAS-positive hyphae and yeast-like organisms were present in the renal medulla, extensively effacing the renal tubuli, eliciting intense neutrophilic infiltration, and invading the urothelium (arrows), with no clear differences between the WT (C) and mutant (D) strains. P, renal pelvis. Bar, 100 μm.

Host immune response to hematogenously disseminated candidiasis.To determine the effect of the disruption of SAP4 to SAP6 on the immune response elicited by C. albicans systemic infection, BALB/c mice were infected i.v. with 5 × 10⁴C. albicans yeast cells of the WT and Δsap456 mutant strains. At days 3 and 7 upon infection, absolute numbers and phenotypes of different splenic leukocyte populations were determined by flow cytometric analysis. Macrophages and neutrophils represent the first line of host immune defense when C. albicans cells infect the bloodstream or the endothelia (47, 58). Macrophages typically express the F4/80 cell surface marker, whereas neutrophils have a Gr-1^high surface phenotype. Murine splenic cells expressing both antigens with either inflammatory or immunosuppressive function have also been described in the context of C. albicans infections (31, 56). According to the expression of these two surface markers, three cell populations were analyzed in this study: F4/80^high Gr-1^neg, F4/80^high Gr-1^high, and F4/80^neg/low Gr-1^high. These cell populations were designated macrophages, inflammatory monocytes, and neutrophils, respectively (55). An extensive recruitment of neutrophils and inflammatory monocytes into the spleen could be observed for infected mice compared to noninfected controls at 3 and, more markedly, 7 days after infection. Higher numbers of spleen macrophages were also detected in the infected mice at 7 days after infection. The total numbers of these myeloid cell populations in WT-infected mice were equivalent to the ones in mice infected with the Δsap456 triple mutant (Fig. 5A).

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FIG. 5.

(A) Scatter plots of the total numbers of neutrophils (F4/80^neg/low Gr-1^high), inflammatory monocytes (F4/80^high Gr-1^high), macrophages (F4/80^high Gr-1^neg), cDC (CD11c^high), B cells (B220⁺), Treg cells (CD4⁺ CD25⁺ Foxp3⁺), and Teffector cells (CD4⁺ CD25⁺ Foxp3⁻), as indicated, observed 3 and 7 days after infection in the spleens of noninfected control mice (open triangles) and mice challenged i.v. with 5 × 10⁴ WT (open squares) and SAP456MS4A (filled circles) C. albicans cells. Data are representative of three independent experiments. Each symbol represents an individual mouse, and horizontal bars are means of cell numbers for each group (n = 3 for control and n = 4 to 5 for infected mouse groups). Statistically significant differences between controls and C. albicans-infected mice are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Relative proportions of splenic Teffector (black bars) and Treg (white bars) cells in the spleens of noninfected controls (PBS) and SC5314 or SAP456MS4A i.v. infected mice 3 and 7 days after challenge.

Dendritic cells were previously shown to play a major role in the induction of the cell-mediated immune response to C. albicans infection (9, 14) and to directly influence the infection outcome (6). Therefore, the numbers and surface maturation markers of splenic conventional dendritic cells (cDC), defined as CD11c^high cells, were assessed upon C. albicans infection. High numbers of splenic cDC, compared to noninfected controls, were observed at the earlier time point analyzed. The cDC surface expression of the costimulatory molecules CD40 and CD80 remained practically unchanged after C. albicans i.v. infection, as evaluated by flow cytometry and recorded as the mean fluorescence intensities (MFIs). In contrast, the costimulatory molecule CD86 was upregulated on the surface of splenic cDC from C. albicans-infected mice 3 and 7 days after infection. The cDC surface expression of MHC class II molecules was slightly downregulated in infected mice 3 days after infection and upregulated at day 7 postinfection compared to that of noninfected controls. No significant differences of cDC numbers (Fig. 5A) and surface expression of any of the assessed costimulatory and antigen-presenting molecules were detected between WT- and SAP456MS4A-infected mice at the two tested time points (Fig. 6).

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FIG. 6.

Expression of CD40, CD80, CD86, and MHC class II molecules on the surface of spleen conventional dendritic cells (cDC) and B cells of BALB/c mice 3 and 7 days after i.v. injection with PBS (controls, gray histograms) or i.v. infection with 5 × 10⁴ SC5314 (solid lines) and SAP456MS4A (dashed lines) C. albicans yeast cells. Staining with the respective isotypic controls was omitted for simplicity. Numbers below histograms represent means ± 1 standard deviation of the mean fluorescence intensities of antibody stainings for noninfected controls (top) and SC5314-infected (middle) or SAP456MS4A-infected (bottom) mice (n = 3 for control and n = 5 for infected mouse groups). Statistically significant differences between controls and C. albicans-infected mice are indicated (*, P < 0.05; **, P < 0.01). Results shown are from one experiment representative of three independent experiments.

B cells have been shown to mediate host resistance to i.v. established C. albicans systemic infection (61). B-cell numbers were significantly increased 7 days after infection in either WT- or SAP456MS4A-challenged mice (Fig. 5A). As observed for splenic cDC, an upregulation of the costimulatory molecule CD86 was observed 3 and 7 days postinfection on the surface of B cells of the infected mice compared to noninfected controls. The expression of CD80 and MHC class II molecules on the surface of B cells was observed to be upregulated only at day 7 after challenge. Challenge with the mutant deficient in SAP4 to SAP6 did not result in any differences compared to the WT strain, regarding either B-cell numbers or costimulatory molecule expression levels (Fig. 5A and 6).

Although CD4⁺ T cells have been reported to have little influence on survival and on fungal burden during acute systemic candidiasis (3, 24), the CD4⁺ T-cell subset of naturally occurring regulatory T (Treg) cells was shown to promote host susceptibility to C. albicans (36, 53) and to limit tissue damage and/or enhance healing but not to directly augment the clearance of the organism from infected tissues (32). To ascertain the impact of a deficiency of SAP4 to SAP6 on the immune response mediated by CD4⁺ T-lymphocyte cell populations, the numbers of CD4⁺, CD4⁺ CD25⁺, and CD4⁺ CD25⁺ Foxp3⁺ (Treg) cells and also of CD4⁺ CD25⁺ Foxp3⁻ (Teffector) cells in the spleens of the infected BALB/c mice were assessed. The numbers of splenic CD4⁺ and CD4⁺ CD25⁺ T cells of infected mice were not significantly different from those of noninfected controls (data not shown). However, as assessed by Foxp3 expression within the CD4⁺ CD25⁺ T-cell population, reduced percentages and numbers of Treg cells were observed 7 days after C. albicans infection. This correlated with higher splenic percentages and numbers of Teffector cells in these mice (Fig. 5A), resulting in higher Teffector/Treg ratios than those for noninfected controls (Fig. 5B). The percentages and numbers of both Teffector and Treg cells were not significantly different between the two C. albicans-infected groups.

To better elucidate the effector function of the CD4⁺ T cells from WT- and SAP456MS4A-infected mice, the proportion of splenic CD4⁺ T cells producing IFN-γ, IL-4, and IL-10 was determined by intracytoplasmic cytokine staining analysis. An increased frequency of CD4⁺ T cells expressing IFN-γ or IL-4 was observed for C. albicans-infected mice compared to the noninfected controls. Although the frequency of cells producing either cytokine increased upon infection in the spleens of C. albicans-challenged mice, a bias toward a Th1-type response was observed (high IFN-γ/IL-4 ratio). The frequency of CD4⁺ T cells expressing IL-10 was also increased in the spleens of infected mice (Fig. 7). The intracellular expression of IFN-γ and IL-4 was also evaluated in kidney CD4⁺ T cells, as it is associated with the outcome of infection (48). As observed for the spleen, the frequency of CD4⁺ T cells expressing the cytokines IFN-γ and IL-4 increased in infected mice. Although a trend toward extended survival was observed for mice infected with mutant strain SAP456MS4A, the percentages of CD4⁺ T cells producing the cytokine IFN-γ or IL-4 in the kidneys of either group of infected mice were similar, resulting in equivalent Th1/Th2 cell ratios (Fig. 7). No serum IFN-γ, IL-4, and IL-10 was detected by ELISA 3 and 7 days after infection of either infected or uninfected mice (data not shown).

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FIG. 7.

Cytokine production in the spleen and kidneys of BALB/c mice infected i.v. with 5 × 10⁴ SC5314 and SAP456MS4A C. albicans cells. (A) Representative examples of flow cytometric analysis of intracellular IFN-γ and IL-4 expression on gated splenic and renal CD4⁺ T cells, as indicated. Numbers inside dot plot regions represent means ± 1 standard deviation of the frequency of IFN-γ⁺ or IL-4⁺ CD4⁺ T cells. (B) IFN-γ⁺/IL-4⁺ CD4⁺ T-cell ratio, normalized considering the ratio of noninfected controls as the basal value (zero value). (C) Dot plots showing the percentages of splenic CD4⁺ T cells producing IL-10. Numbers inside dot plots represent means ± 1 standard deviation of the frequency of IL-10⁺ CD4⁺ T cells. Data are representative of two independent experiments (n = 4 for noninfected control and n = 7 for infected mouse groups). Statistically significant differences between controls and C. albicans-infected mice are indicated (*, P < 0.05; **, P < 0.01).

Overall, these results indicate that a deficiency of SAP4 to SAP6 does not have a significant impact on the immune response elicited in the spleen and kidneys of BALB/c mice hematogenously challenged with C. albicans.