Candida glabrata Persistence in Mice Does Not Depend on Host Immunosuppression and Is Unaffected by Fungal Amino Acid Auxotrophy

Abstract

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Candida glabrata is a commensal yeast that can be isolated from the mucosal layers of healthy individuals (10, 32). However, as an opportunistic pathogen, it can also cause mucosal and severe, life-threatening invasive infections (10). In the United States, C. glabrata is the second most common cause of candidemia, representing about 20% of all Candida bloodstream isolates (reviewed in reference 34). C. glabrata is less commonly isolated in Europe but still accounts for ∼10% of candidemia cases (reviewed in reference 34). As with C. albicans, risk factors for the development of invasive C. glabrata infections in human patients include mucosal colonization by Candida spp., indwelling vascular catheters, antibiotic therapy, gastrointestinal surgery, cancer chemotherapy, and neutropenia (12, 26, 35). Despite antimycotic treatment and partially due to the naturally high resistance of C. glabrata to several antifungal drugs (33), systemic C. glabrata infections often result in high mortality (29, 40).

In contrast, experimental intravenous infection of laboratory animals with C. glabrata generally does not cause mortality. Among mice, the most commonly used model species, p47^phox−/− knockout mice and mice treated with a combination of cyclophosphamide and 5-fluorouracil are susceptible to lethal C. glabrata infections (15, 28). p47^phox−/− knockout mice are deficient in the phagocyte oxidative burst and serve as a model for human chronic granulomatous disease (14). Cyclophosphamide and 5-fluorouracil are cytotoxic agents used for cancer treatment and affect replicating cells, such as myeloid immune cell progenitors. Furthermore, complement (C3)-deficient mice were recently shown to be highly susceptible to C. glabrata infections (46). These findings suggest that immunosuppression might be a key factor affecting the outcome of systemic C. glabrata infections in mice, consistent with the relevance of immunosuppression as a risk factor for human patients. However, animals treated with cyclophosphamide alone do not succumb to systemic C. glabrata infection, even though cyclophosphamide induces leukopenia. In the absence of mortality, the fungal burden has been used as a parameter for virulence and the efficacy of antifungal treatment for cyclophosphamide-treated mice (4, 41). Similarly, fungal burdens have been determined for immunocompetent mice systemically infected with C. glabrata (6, 16, 43); however, differences in mouse strains, animal age and gender, the C. glabrata strain used, and infectious doses hamper direct comparison of published data from immunocompetent and immunosuppressed mice. Thus, the influence of immunosuppression on systemic C. glabrata infections in mice is still unclear. In this study, we aimed to clarify the importance of immunosuppression for the pathogenesis of systemic C. glabrata infections. Therefore, we compared the fungal burden, the clinical course of infection, and histopathological alterations in immunocompetent, cyclophosphamide-treated, and dexamethasone-treated mice systemically infected with C. glabrata.

Both natural and genetically engineered auxotrophies in microorganisms have been successfully developed as genetic tools for the investigation of virulence. However, some auxotrophies can have detrimental effects on the virulence or survival of pathogens within the host. For example, uracil auxotrophy leads to attenuation of Aspergillus fumigatus (9) and reduced survival of Saccharomyces cerevisiae in mice (11), and uridine auxotrophy affects the adhesion and virulence of Candida albicans (3, 17, 44). Amino acid auxotrophies seem to have less impact on virulence, since methionine, arginine, histidine, and leucine auxotrophic mutants of C. albicans are fully virulent (17, 27, 31). The finding of Goldstein and McCusker that leucine auxotrophy in S. cerevisiae strongly affects survival in mice (11), however, demonstrates that similar auxotrophies might have different effects on virulence, survival, and persistence depending on the fungal species. Here, we investigated the question of whether histidine, leucine, and tryptophan auxotrophies, alone or in combination, affect in vitro fitness and in vivo survival and competitive fitness of C. glabrata in immunocompetent mice. Our results suggest only a minor influence of host immunosuppression and fungal auxotrophy on the survival, persistence, and virulence of C. glabrata within mouse organs. Based on these findings, we established a mouse model suitable for identifying the C. glabrata genes required for fitness in vivo.

MATERIALS AND METHODS

Strains and culture conditions.

The strains used in this study are listed in Table 1 and were routinely cultured in YPD (1% yeast extract, 1% peptone, 2% dextrose) at 37°C. For solid media, 2% agar was added. To confirm auxotrophy, for determination of individual auxotrophic strains in pooled inocula, and for selective reisolation of auxotrophic strains from tissue homogenates, minimal medium (MM; 1× yeast nitrogen base with ammonium sulfate [BD Bioscience, Heidelberg, Germany] and 2% glucose), supplemented with the appropriate amino acids (0.3 mM histidine [Sigma-Aldrich, Taufkirchen, Germany], 0.8 mM leucine [Roth, Karlsruhe, Germany], 0.5 mM tryptophan [Roth]) if necessary, was used. Inocula for infection experiments were harvested from 50-ml cultures grown overnight at 200 rpm and 37°C. Cells were washed three times with phosphate-buffered saline (PBS), enumerated with a hemocytometer, and diluted to the desired cell concentration with PBS. The viable counts in the inocula were verified by plating serial dilutions on YPD agar.

TABLE 1.

Fungal strains and plasmids used in this study

Strain or plasmid	Genotype or descriptiona	Source or reference
C. glabrata strains
ATCC 2001	C. glabrata wild-type strain	American Type Culture Collection; received from Ken Haynes
his3Δ mutant	Histidine-auxotrophic derivative of ATCC 2001; his3Δ::FRT	This study; H. Jungwirth and S. Lechner
leu2Δ mutant	Leucine-auxotrophic derivative of ATCC 2001; leu2Δ::FRT	This study; H. Jungwirth and S. Lechner
trp1Δ mutant	Tryptophan-auxotrophic derivative of ATCC 2001; trp1Δ::FRT	This study; H. Jungwirth and S. Lechner
hisΔ leuΔ trpΔ mutant	Triple mutant auxotrophic for histidine, leucine, and tryptophan; his3Δ::FRT leu2Δ::FRT trp1Δ::FRT	This study; H. Jungwirth and S. Lechner
Plasmids
pSFS2a	SAT1 marker, recombinase; AmpR; SAT1-caFLP	37
pSFS2a-HIS3	For deletion of CgHIS3; AmpR; 5′-CgHIS3-SAT1-FLP-3′-CgHIS3	This study; H. Jungwirth and S. Lechner
pSFS2a-LEU2	For deletion of CgLEU2; AmpR; 5′-CgLEU2-SAT1-FLP-3′-CgLEU2	This study; H. Jungwirth and S. Lechner
pSFS2a-TRP1	For deletion of CgTRP1; AmpR; 5′-CgTRP1-SAT1-FLP-3′-CgTRP1	This study; H. Jungwirth and S. Lechner

^aFor plasmids, the description includes the marker and insert.

Construction of auxotrophic strains.

The dominant recyclable nourseothricin resistance marker SAT1 (37) was used to generate a set of auxotrophic C. glabrata strains in the ATCC 2001 genetic background. Homologous flanking regions of 500 bp were amplified from genomic DNA and ligated into plasmid pSFS2a by using the ApaI/XhoI and SacII/SacI restriction sites, thereby creating recyclable gene deletion cassettes for the C. glabrata HIS3 (CgHIS3), CgLEU2, and CgTRP1 genes. The deletion cassettes were obtained by ApaI and SacI restriction and were used for transformation of the ATCC 2001 recipient strain by an electroporation protocol as described previously (37). Correct transformants were grown in YP (1% yeast extract, 1% peptone) supplemented with 2% maltose for 2 h to induce expression of the recombinase, driving excision of the SAT1 cassette, leaving only one FRT site behind. Dilutions were then plated on YPD. The resulting colonies were tested for nourseothricin sensitivity and screened by PCR. Repeated use of the appropriate deletion constructs yielded all combinations of double-auxotrophic strains and the triple-auxotrophic strain. All strains were verified by Southern blot analysis for correct genomic integration.

Analysis of in vitro fitness and competition indices (CIs) of auxotrophic strains.

Growth curves were performed in 96-well plates in a Tecan Infinite M200 microplate reader. Cells were grown in 100 μl YPD at 30°C (6 replicates) or 37°C (8 replicates). Doubling times (D) were calculated as the difference between the time at a certain optical density at 600 nm (OD₆₀₀), occurring during exponential growth, and the time 2 generations before, divided by 2 (45).

In vitro competition indices were determined in YPD and MM supplemented with histidine, leucine, and tryptophan. Overnight cultures of ATCC 2001, the triple-auxotrophic strain, and the single-auxotrophic strains were washed twice with sterile water and enumerated with a CASY cell counter, and cultures were adjusted to the same cell number. Cell pools were inoculated at a total cell number of 1 × 10⁶/ml in 15 ml of medium and were grown with shaking (220 rpm) at 30°C. After 5 h, serial dilutions were prepared, and cell suspensions of the input and output pools were plated on YPD agar and incubated at 30°C for 24 h. Colonies were then replica plated on YPD (growth of all strains), MM plates without supplementation (growth of the wild type only), and MM plates supplemented with either histidine, leucine, or tryptophan (growth of the wild type and the respective auxotrophic strain) in order to quantify each strain. Replica-plated colonies were counted after 48 h of incubation at 30°C. The competition indices were calculated as the output ratio of the CFU of the mutant strain to that of the wild type divided by the input ratio of the CFU of the mutant strain to that of wild type. Competition experiments were performed in triplicate.

Mouse model.

Female specific-pathogen-free outbred CD-1 mice 6 weeks old (18 to 22 g; Charles River, Germany) were used for all experiments. The animals were housed in groups of five in individually ventilated cages and were cared for in accordance with the principles outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (http://conventions.coe.int/Treaty/en/Treaties/Html/123.htm). All animal experiments were in compliance with the German animal protection law and were approved by the responsible Federal State authority and ethics committee (permit no. 03-008/07). For immunosuppression, either cyclophosphamide (150 mg/kg; Sigma) or dexamethasone (100 mg/kg; Rotexmedica, Germany) was applied intraperitoneally on days −3, 0, 7, 14, and 21. The effect of immunosuppression was determined by comparing differential blood cell counts before immunosuppression to those on the day of postmortem analysis. Mice were challenged on day 0 with 5 × 10⁷ CFU in 200 μl PBS via the lateral tail vein. Five mice per group were sacrificed on days 2, 7, 14, and 28 postinfection (p.i.) for analysis of macroscopic and histological changes, fungal burden, and changes in the levels of blood marker enzymes.

Clinical parameters, blood enzymes, and pathology.

A veterinarian performed physical examinations twice daily. Body weight and body surface temperature (used as a noninvasive surrogate for internal body temperature [47]) were measured once a day. To assess tissue damage to parenchymal organs, liver marker enzyme activities, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as levels of urea in the blood (blood urea nitrogen), were determined from serum samples by using the EuroLyser CCA 180 Vet system (QinLAB Diagnostik, Martinsried, Germany) according to standard methods recommended by the International Federation of Clinical Chemistry. Pooled samples (five animals per pool) collected on day −6 were analyzed as controls; for samples from infected animals, mice were anesthetized with an overdose of ketamine and xylazine prior to blood collection by heart puncture. Gross pathological alterations were recorded during necropsy. For histology, parts of organs were fixed with buffered formalin, and paraffin-embedded sections were stained with hematoxylin-eosin (HE) or periodic acid-Schiff (PAS) stain according to standard protocols.

Quantification of C. glabrata in infected tissue.

The spleen, liver, kidneys, heart, lung, and brain were removed aseptically at necropsy, rinsed with sterile PBS, weighed, and placed in 1 ml (heart, lungs, kidneys, spleen) or 3 ml (liver and brain) sterile PBS on ice. The organs were aseptically homogenized using a Ika T10 basic Ultra-Turrax homogenizer (Ika, Staufen, Germany). Serial dilutions of homogenates were plated on YPD plates. Colonies were counted after 48 h of incubation at 30°C. The fungal burden was calculated as CFU per gram of tissue. To depict negative culture results on logarithmic scales, negative cultures were counted as 1 CFU/g, thus appearing as a zero on log₁₀ scales.

Pool experiments and competition indices.

For the inoculum of the pool experiment, independent cultures of the different strains were grown and treated as described above. Cell numbers were adjusted to 2.5 × 10⁸/ml for each strain; equal volumes were combined for the inoculum. The actual infectious dose was verified by plating serial dilutions on YPD agar. In addition, dilutions were plated on MM without supplementation (growth of the wild type only) and on MM supplemented with either histidine, leucine, or tryptophan (growth of the wild type and the respective auxotrophic strain) in order to quantify each strain. Fifteen immunocompetent mice were infected as described above; five mice each were sacrificed on days 2, 7, and 28 postinfection. Fungal burdens were quantified as described above with the following modification: Homogenates were plated on YPD, MM lacking any supplementation, and MM supplemented with either histidine, leucine, or tryptophan in order to quantify each strain. If fewer than 100 colonies were expected per organ, homogenates were directly plated only on YPD, and single colonies were replica plated on MM plates and on MM supplemented with either histidine, leucine, or tryptophan. In vivo competition indices (CIs) were calculated from the CFU of every individual animal as the output ratio of the strain-specific CFU to total CFU divided by the input ratio of the strain-specific CFU to total CFU. The CIs of five mice per time point were used to generate the mean CI for each strain, organ, and time point.

Quantification of MPO and cytokines from tissue homogenates.

Tissue homogenates of immunocompetent mice infected with the wild-type strain ATCC 2001 and the auxotrophic his3Δ and hisΔ leuΔ trpΔ mutants as described above were diluted 1:1 to 1:7 in tissue lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml leupeptin, and 28 μg/ml aprotinin [pH 7.4]) and centrifuged twice (1,500 × g, 15 min, 4°C), and the supernatants were stored at −80°C until measurement. Myeloperoxidase (MPO) and cytokine levels were determined by commercially available murine enzyme-linked immunosorbent assay (ELISA) kits (for MPO, the Mouse MPO ELISA kit [Hycult Biotechnology, Uden, the Netherlands]; for multiple cytokines, the Mouse Inflammatory Cytokines Multi-Analyte ELISArray kit [SABiosciences, Frederick, MD]; for interleukin 1α (IL-1α), the Mouse ILA SingleAnalyte ELISArray [SABiosciences]; and for IL-1β, IL-2, IL-6, tumor necrosis factor alpha [TNF-α], and granulocyte-macrophage colony-stimulating factor [GM-CSF], ELISA Ready SET Go! [eBioscience, Hatfield, United Kingdom]) according to the manufacturer's recommendations.

Statistical analysis.

Data were plotted and statistically analyzed without log transformation using GraphPad Prism, version 5.00 for Windows (GraphPad Software, San Diego, CA). Spleen weights and blood enzyme levels for each day of analysis were compared to those of noninfected controls by 1-way analysis of variance (ANOVA) followed by Dunnett's multiple-comparison test. Fungal burdens are shown as box plots (box, inner quartiles; whiskers, minimum and maximum value in data set; line, median). One-way ANOVA was performed on data sets from the same day after infection, followed by a Tukey-Kramer test to identify which groups were significantly different. Myeloperoxidase and cytokine data were analyzed per time point by 1-way ANOVA followed by the Tukey-Kramer test.

RESULTS

Influence of immunosuppression on fungal burden and pathological alterations during the course of infection.

In order to determine the effect of immunosuppression on the outcome of C. glabrata infection in mice, we infected mice treated with either cyclophosphamide or dexamethasone, or left untreated (immunocompetent), with 5 × 10⁷ CFU C. glabrata ATCC 2001 by lateral tail vein injection. Cyclophosphamide was chosen because it is the drug most commonly used to induce leukopenia in mouse models. Immunosuppression with dexamethasone was chosen because (i) dexamethasone is used as an anti-inflammatory compound in combination therapy of cancer and transplant patients, and (ii) immunosuppression with corticosteroids has been shown to render mice susceptible to invasive aspergillosis by affecting macrophage and neutrophil function (13). Five mice per treatment group were analyzed on days 2, 7, 14, and 28 postinfection (p.i.). As expected, treatment with cyclophosphamide led to profound panleukopenia (<5 × 10³ leukocytes/ml) and absence of polymorphonuclear granulocytes (PMNs) on day 2 p.i. White blood cell counts recovered during the first 2 weeks after infection (data not shown). Animals treated with dexamethasone showed granulocytosis from day 2 onward. In contrast, immunocompetent mice showed normal differential white blood cell counts on day 2 p.i. All groups presented monocytosis on days 14 and 28 p.i. (data not shown). Mice treated with cyclophosphamide showed a moderate weight loss (4.00 g ± 1.00 g) from day −4 (beginning of immunosuppressive treatment) to day 2. Notably, all animals remained clinically healthy and even gained weight over the full course of the experiment. Fever was not detected in any animal.

Two days after infection, high fungal burdens of 10⁵ to 10⁷ CFU/g were detected in all organs analyzed (Fig. 1). At this time after infection, the only detectable pathology was significant spleen enlargement in immunocompetent animals (Table 2). Histological analysis revealed the presence of fungal cells in the red pulp, occasionally associated with mononuclear phagocytes (data not shown). The splenic parenchyma was hyperemic and showed minor follicular hyperplasia. On day 7 p.i., significant splenomegaly and more-pronounced follicular hyperplasia with germination centers were observed in all groups (Table 2 and Fig. 2A to C), indicating an active immune response toward the infection. The fungal burden in the spleen decreased over time in all groups, and fungal cells could be only sporadically detected by histology on day 7 p.i. However, CFU counts in the spleens of cyclophosphamide-treated mice were significantly higher on days 7 and 14 than those for immunocompetent animals (Fig. 1A).

FIG. 1.

Fungal burdens in different organs of immunocompetent and immunosuppressed mice infected with C. glabrata ATCC 2001. Mice were intravenously infected with 5 × 10⁷ CFU on day zero. Fungal burdens were determined by culture from tissue homogenates of five animals per treatment group and time point. Fungal burdens are shown as box plots (box, inner quartiles; whiskers, minimum and maximum value in data set; line, median). One-way ANOVA was performed on data sets from the same day after infection, followed by the Tukey-Kramer test. Symbols indicate significant (P < 0.05) increases in fungal burdens over those for immunocompetent mice (#) or over those for immunocompetent and dexamethasone-treated mice (*).

TABLE 2.

Liver marker enzymes, blood urea content, and spleen weights in immunosuppressed and immunocompetent CD-1 mice systemically infected with C. glabrata ATCC 2001

Time p.i. and groupa	Liver marker enzyme level (U/liter)b		Blood urea content (mg/dl)	Spleen wt (mg)b
	ALT	AST
Uninfected control	80.2 ± 6.9c	140.2 ± 30.7c	18.7 ± 2.9c	77.0 ± 37.0d
Day 2
Immunocompetent	118.6 ± 39.0	189.8 ± 56.1	19.2 ± 0.8	129.7 ± 26.0*
Dexamethasone	97.8 ± 19.9	118.3 ± 35.2	19.0 ± 2.6	83.8 ± 14.4
Cyclophosphamide	75.6 ± 19.8	126.0 ± 101.2	19.2 ± 3.6	60.5 ± 11.5
Day 7
Immunocompetent	145.0 ± 41.8*	170.2 ± 55.1	16.8 ± 0.8	265.5 ± 36.2*
Dexamethasone	286.0 ± 109.2*	313.4 ± 109.1*	23.5 ± 4.4	254.4 ± 12.1*
Cyclophosphamide	249.0 ± 47.2*	256.8 ± 100.1	20.3 ± 1.3	262.1 ± 58.1*
Day 14
Immunocompetent	154.2 ± 88.1	184.8 ± 77.9	21.0 ± 2.9	137.1 ± 21.6
Dexamethasone	69.2 ± 20.1	134.2 ± 44.4	16.8 ± 5.4	188.5 ± 54.9*
Cyclophosphamide	123.6 ± 46.0	192.0 ± 48.9	23.3 ± 5.6	267.1 ± 49.2*
Day 28
Immunocompetent	57.4 ± 9.6	99.2 ± 38.2	17.6 ± 2.9	150.3 ± 26.1*
Dexamethasone	79.2 ± 43.8	238.0 ± 150.6	19.0 ± 0.7	113.1 ± 22.1*
Cyclophosphamide	79.5 ± 46.8	149.5 ± 38.2	23.0 ± 1.2	161.4 ± 24.7*

^aFive mice were sampled for each group at each time point.

^b*, significant increase over the value for the control (P < 0.05 by 1-way ANOVA and Dunnett's multiple-comparison test).

^cSerum sampled on day −6 from animals within the experiment; 6 pools of 5 samples each were measured.

^dImmunocompetent, noninfected, age-matched (day zero) control animals.

FIG. 2.

Histological findings for different organs of immunocompetent and immunosuppressed mice infected with C. glabrata ATCC 2001. Mice were intravenously infected with 5 × 10⁷ CFU on day zero. (A to P) The treatment group is given at the top. (A to C) Spleen tissues on day 7 p.i., stained with HE; magnification, ×100. (D to F) Liver tissues on day 2 p.i., stained with PAS stain; magnification, ×630. Arrows indicate fungal cells. (G to I) Liver tissues on day 7 p.i., stained with HE; magnification, ×200. Arrows indicate immune cell infiltrates. (K to M) Brain tissues on day 7 p.i., stained with PAS stain; magnification, ×630. Arrows indicate fungal cells. (N to P) Kidney tissues on day 14 p.i., stained with PAS stain; magnification, ×630. Arrows indicate fungal cells. (R to S) Kidney tissues from cyclophosphamide-treated mice on days 2 (R), 7 (S), and 28 (T) p.i. Tissues were stained with PAS stain; magnification, ×630. Arrows indicate fungal cells.

The livers of infected mice appeared macroscopically normal at all time points. However, mild liver damage can occur without macroscopic changes. Therefore, we measured the levels of two liver marker enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum before and after infection. These enzymes have been successfully used to assess liver damage in mice infected intraperitoneally with C. albicans (20). ALT levels were significantly elevated in all groups on day 7 p.i. Similarly, AST levels were elevated in all groups on day 7 p.i.; however, the increase was statistically significant only for mice treated with dexamethasone (Table 2). In histological sections, fungal cells were readily found in liver sinusoids (Fig. 2D to F). In immunocompetent animals and, to a lesser degree, in dexamethasone-treated mice, small areas with mononuclear infiltrates surrounded fungal cells. The sizes of the infiltrates increased to day 7 p.i., and infiltrates were also present in cyclophosphamide-treated mice on day 7 p.i. (Fig. 2G and H). During the same time, the fungal burden declined about 100-fold in immunocompetent and dexamethasone-treated mice but only 10-fold in cyclophosphamide-treated animals (Fig. 1B). Thus, the occurrence of immune cell infiltrates coincided both with detectable liver damage and with significant reductions in fungal burdens. The CFU count in the liver declined further over time in all groups but was significantly higher in cyclophosphamide-treated mice on days 7 and 14 p.i. (Fig. 1B).

In contrast to those in the liver and spleen, the fungal burden in the brain did not decrease from day 2 to day 7 p.i. (Fig. 1C). Fungal cells were readily detected in histological sections (Fig. 2K to M). As in the liver, C. glabrata was surrounded by mononuclear cells in immunocompetent animals and to a lesser degree in dexamethasone-treated but not in cyclophosphamide-treated mice. A significant reduction in CFU counts in the brain was observed from day 7 to day 14. The fungal burden in the brain was significantly higher in cyclophosphamide-treated mice, suggesting that the reduction might be due to the host response (Fig. 1C). However, we did not observe increased inflammation in immunocompetent mice (data not shown). The absence of gross inflammation in the brain was consistent with the absence of neurological symptoms.

The fungal burdens in the lung and heart decreased steadily over time (Fig. 1D and E). Fungal cells could be only sporadically detected in these organs on days 2 and 7 p.i. From day 7 p.i. onward, mononuclear cell infiltrates were found in few individual animals of all groups.

The kidney is known to be one of the main target organs for systemic C. glabrata infection in mice. Consistent with previous studies demonstrating that C. glabrata can persist for at least 3 weeks in the kidney (6), fungal burdens remained comparatively stable throughout the experiment (Fig. 1F). Fungal cells were present in histological sections of all treatment groups at all time points (Fig. 2N to T). While C. glabrata was associated with glomeruli on day 2 p.i. (Fig. 2R), fungal cells were found predominantly within or surrounding tubuli at later time points. As in the other organs, mononuclear infiltrates were found from day 7 p.i. onward. However, in contrast to other organs, macroscopic alterations were evident on day 28 p.i. (two out of five animals in each treatment group) as white, nodular areas ranging from 1 to 3 mm in diameter. These alterations were histologically confirmed to be extensive mononuclear infiltrates containing C. glabrata cells (Fig. 2T). Despite the presence of macroscopic kidney alterations, the renal function of these animals was not significantly altered. However, in these mice, less than 50% of the kidney tissue was affected. Since only gross kidney lesions affecting 70% or more of functional parenchyma cause increased urea levels in the blood, it was not surprising that no animal showed increased blood urea levels (Table 2).

Construction of auxotrophic strains and in vitro analysis.

In order to analyze whether a lack of auxotrophic marker genes can influence growth and fitness, we used SAT1 flipper technology to generate single C. glabrata mutants auxotrophic for histidine (his3Δ), leucine (leu2Δ), or tryptophan (trp1Δ), as well as the triple-auxotrophic strain (hisΔ leuΔ trpΔ). Genomic integration was confirmed by colony PCR and growth on synthetic complete (SC) medium lacking the respective amino acids. The mutants showed normal susceptibility to nourseothricin, indicating that the SAT1 construct was successfully excised. Southern blot analysis of all strains verified correct integration of the gene deletion cassette, as well as loss of the SAT1 flipper cassette after recombinase induction in maltose-supplemented medium (data not shown).

In order to analyze whether the deletion of auxotrophic marker genes had an influence on growth and fitness in vitro, the doubling times of the auxotrophic mutants were compared upon growth in rich medium at 30°C and 37°C. The in vitro doubling times of the wild-type and auxotrophic strains were not significantly different at 30°C (67.7 ± 2.6 min for the wild type, 66.1 ± 2.2 min for the his3Δ mutant, 65.7 ± 1.8 min for the leu2Δ mutant, 65.8 ± 1.8 min for the trp1Δ mutant, and 65.1 ± 2.8 min for the hisΔ leuΔ trpΔ mutant) and 37°C (53.2 ± 3.3 min for the wild type, 51.7 ± 2.2 min for the his3Δ mutant, 49.6 ± 2.1 min for the leu2Δ mutant, 51.8 ± 2.2 min for the trp1Δ mutant, and 52.8 ± 2.3 min for the hisΔ leuΔ trp1Δ mutant). Likewise, in vitro competition in YPD or in supplemented minimal medium revealed no disadvantage of auxotrophic mutants relative to the wild type (CIs, 0.92 ± 0.31 and 0.82 ± 0.17 for the his3Δ mutant; 1.08 ± 0.35 and 1.45 ± 0.91 for the leu2Δ mutant; 0.96 ± 0.35 and 1.19 ± 0.59 for the trp1Δ mutant; and 0.96 ± 0.27 and 1.65 ± 0.41 for the hisΔ leuΔ trpΔ mutant).

In vivo competition of wild-type and auxotrophic C. glabrata strains.

In order to determine whether infection of mice could serve as a negative selection system to identify mutants whose survival within the host is impaired, we performed an infection experiment with a pool consisting of the wild type (ATCC 2001) and four auxotrophic strains (the his3Δ, leu2Δ, trp1Δ, and hisΔ leuΔ trpΔ strains). The auxotrophic strains were chosen because they provide a versatile background for future genetic manipulations. However, auxotrophies have been shown to affect virulence or survival within the host. Thus, testing of survival fitness within the host is an essential step in assessing the suitability of auxotrophic strains as the parental background for virulence studies. Our initial experiment showed that immunosuppression led to increased fungal burdens on days 7 and 14 p.i.; however, negative selection within a host is stronger in the presence of an intact immune system. Furthermore, deficiencies of a mutant strain in withstanding the host's immune response might not be detectable in an immunocompromised host. Therefore, we decided to use immunocompetent mice as hosts in an in vivo competition experiment.

Mice were infected with pools of 5 × 10⁷ CFU consisting of ATCC 2001 and the his3Δ, leu2Δ, trp1Δ, and hisΔ leuΔ trpΔ mutants. The inoculum was prepared to contain equal amounts of each strain; the strain proportions within the inoculum were controlled by plating serial dilutions on selective media and were found to be 15.4% ATCC 2001, 13.5% his3Δ mutant, 24.4% leu2Δ mutant, 13.3% trp1Δ mutant, and 33.3% hisΔ leuΔ trpΔ mutant. Five mice each were sacrificed on days 2, 7, and 28 p.i. to determine the in vivo competition indices (CIs). The relative pool composition of auxotrophic fungal cells in the liver and spleen remained stable over time, with less than 2-fold changes in the CIs (Table 3). In contrast, a 10-fold decrease in the CI of the his3Δ mutant was observed on day 28 p.i. in the brain. The hisΔ leuΔ trpΔ triple mutant was virtually absent from the brain on day 28 p.i. (Table 3). In the lung, the leuΔ mutant CI was 10-fold decreased on day 28 p.i., as was the his3Δ mutant CI in the heart (Table 3). The CIs for the hisΔ leuΔ trpΔ triple mutant were mildly reduced in these organs at the late time point (5-fold and 2-fold, respectively). However, the total numbers of fungal cells in the heart and lung on day 28 p.i. were low (10¹ to 10²), thus limiting the accuracy of CIs in these organs at late time points. In the kidney, both the his3Δ and the leu2Δ mutant were strongly underrepresented in the output pools on day 28 p.i., whereas the hisΔ leuΔ trpΔ mutant was readily reisolated (Table 3). Analysis of output pools in the kidneys of individual mice on day 28 p.i. revealed that in 4 out of 5 mice, two strains dominated the output pool (Fig. 3).

TABLE 3.

In vivo competition indices of ATCC 2001 and auxotrophic mutant strains

Organ and day	Competition indexa for the following strain:
	ATCC 2001	his3Δ mutant	leu2Δ mutant	trp1Δ mutant	hisΔ leuΔ trpΔ mutant
Spleen
Day 2	1.04 ± 0.19	1.32 ± 0.45	0.85 ± 0.25	1.49 ± 0.19	0.83 ± 0.28
Day 7	1.25 ± 0.38	0.83 ± 0.68	0.76 ± 0.48	1.97 ± 1.28	0.87 ± 0.78
Day 28	1.59 ± 0.47	0.81 ± 0.73	0.80 ± 0.30	1.66 ± 1.09	0.75 ± 0.54
Liver
Day 2	1.29 ± 0.34	1.03 ± 0.50	0.81 ± 0.20	1.10 ± 0.47	1.29 ± 0.34
Day 7	1.19 ± 0.30	1.48 ± 0.28	0.96 ± 0.19	1.85 ± 0.57	0.48 ± 0.30
Day 28	1.75 ± 0.24	1.03 ± 0.34	0.68 ± 0.20	1.57 ± 0.38	0.71 ± 0.16
Brain
Day 2	2.85 ± 0.50	1.79 ± 1.07	0.72 ± 0.30	1.73 ± 1.16	0.34 ± 0.36
Day 7	2.18 ± 0.83	0.79 ± 0.75	0.60 ± 0.50	2.03 ± 0.39	0.50 ± 0.59
Day 28	4.88 ± 1.83	0.10 ± 0.19	0.53 ± 1.03	0.98 ± 1.83	0.00 ± 0.00
Lung
Day 2	1.16 ± 0.12	1.23 ± 0.44	0.94 ± 0.42	1.79 ± 0.61	0.63 ± 0.38
Day 7	1.17 ± 0.45	0.67 ± 0.78	0.66 ± 0.56	1.01 ± 0.6	1.36 ± 0.76
Day 28	4.00 ± 2.79	1.54 ± 1.58	0.08 ± 1.26	0.92 ± 1.38	0.18 ± 0.35
Heart
Day 2	1.14 ± 0.26	0.63 ± 0.48	0.58 ± 0.25	1.10 ± 0.43	1.4 ± 0.30
Day 7	1.70 ± 0.65	1.08 ± 0.93	0.92 ± 0.33	1.99 ± 0.47	0.41 ± 0.39
Day 28	1.74 ± 1.37	0.00 ± 0.00	1.09 ± 0.88	2.52 ± 2.27	0.45 ± 0.53
Kidney
Day 2	1.95 ± 0.87	1.75 ± 2.06	1.47 ± 1.30	2.58 ± 1.72	0.47 ± 0.50
Day 7	1.38 ± 0.64	1.03 ± 0.92	0.86 ± 0.52	3.04 ± 3.08	0.31 ± 0.46
Day 28	1.82 ± 1.37	0.04 ± 0.09	0.06 ± 0.07	1.78 ± 1.72	1.44 ± 1.07

^aData are means and standard deviations for five mice per time point. Competition indices were calculated as the output ratio of the strain-specific CFU to total CFU divided by the input ratio of the strain-specific CFU to total CFU.

FIG. 3.

Strain ratios in the kidneys of individual mice 28 days after infection with a pool of C. glabrata ATCC 2001 (wild type [wt])and auxotrophic mutants. Mice were intravenously infected with a total of 5 × 10⁷ CFU on day zero. The input pool consisted of ATCC 2001 (15.4%), the his3Δ mutant (13.5%), the leu2Δ mutant (24.4%), the trp1Δ mutant (13.5%), and the hisΔ leuΔ trpΔ mutant (33.3%).

Single-strain infections.

To confirm the results of the in vivo competition experiment, the survival of the wild-type strain ATCC 2001 and the his3Δ and hisΔ leuΔ trpΔ auxotrophic mutants in immunocompetent mice was tested in a single-strain infection experiment. In agreement with the competition experiment, no strain-specific differences in fungal burden were observed in any organs on days 2 and 7 p.i. (Fig. 4). Likewise, no differences between the strains were observed on day 28 p.i. in the liver and spleen. In the brain, heart, and lung, we observed slightly higher fungal burdens in mice infected with the wild-type strain; however, these differences were not statistically significant. Similarly, mice infected with the wild-type strain had higher fungal burdens in the kidney than mice infected with either the his3Δ or the hisΔ leuΔ trpΔ mutant (Fig. 4). This result was not anticipated, since the in vivo competition experiment did not show any impairment of hisΔ leuΔ trpΔ mutant survival in the kidneys (Table 3 and Fig. 3).

FIG. 4.

Fungal burden in different organs of immunocompetent mice infected with C. glabrata ATCC 2001 (wild type) or the his3Δ or hisΔ leuΔ trpΔ strain. Mice were intravenously infected with 5 × 10⁷ CFU on day zero. Fungal burdens were determined by culture from tissue homogenates of five animals per treatment group and time point and are shown as box plots (box: inner quartiles; whiskers: minimum and maximum value in data set; line: median). One-way ANOVA was performed on data sets from the same day after infection, followed by Tukey-Kramer test. No significant (P < 0.05) differences between strains were observed.

Myeloperoxidase content and cytokine response in infected organs.

PMNs play a crucial role as immune effector cells during candidemia. They have been shown to be involved in controlling C. albicans but also contribute to tissue damage during infection. However, immune cell infiltrates in organs of C. glabrata-infected mice consisted mainly of mononuclear cells. Since quantification of immune cells in histological sections is difficult, we used the PMN marker enzyme myeloperoxidase (MPO) (19, 23) to quantify PMN infiltration in tissue homogenates of immunocompetent mice infected with ATCC 2001 or the his3Δ or hisΔ leuΔ trpΔ mutant. Upon infection, the MPO content significantly increased in all organs to day 7 p.i. and then, with the exception of the brain, decreased to day 28 p.i., without significant differences between the strains (Table 4). In order to evaluate the cytokine responses of mice infected with the different C. glabrata strains, we analyzed the levels of the proinflammatory cytokines IL-1α, IL-1β, IL-2, IL-6, GM-CSF, and TNF-α in the liver, kidney, and brain by ELISA. No C. glabrata strain-specific differences were observed for any cytokine. The IL-1α and IL-6 contents of all organs were unaltered at all time points. Levels of IL-1β were 2-fold increased in the brain on day 7 p.i. but were unaltered at other time points. No differences from levels in control animals were observed for IL-1β in the liver and kidney. IL-2 showed a 1.5-fold increase in the kidney on day 28 p.i. GM-CSF levels were 2-fold increased in the kidney and liver on day 2, but were 4-fold decreased in the liver on days 7 and 28 p.i., relative to the levels in noninfected controls. TNF-α was barely detectable in the kidneys of both control and infected animals (detection limit, 75 pg/g). It was not detectable in the brain (detection limit, 75 pg/g). The TNF-α contents in the liver were the same for infected and control mice.

TABLE 4.

Myeloperoxidase content in livers, kidneys, and brains of mice infected with ATCC 2001 and auxotrophic mutant strains

Organ and day	Myeloperoxidase content (ng/g)a in organs of mice:
	Controlb	Infected with the following strain:
		ATCC 2001	his3Δ mutant	hisΔ leuΔ trpΔ mutant
Liver
Day 2	93.62 ± 29.2	277.2 ± 71.1*	424.2 ± 192.6*	267.3 ± 80.2*
Day 7		605.8 ± 230.6*	614.8 ± 250.8*	950.1 ± 393.8*
Day 28		287.0 ± 122.1*	242.6 ± 97.3*	189.2 ± 89.9
Kidney
Day 2	114.6 ± 51.4	270.1 ± 104.9	355.1 ± 176.6*	304.2 ± 135.1*
Day 7		656.9 ± 110.9*	541.4 ± 119.7*	884.4 ± 324.8*
Day 28		349.3 ± 105.0*	134.7 ± 52.78	320.8 ± 158.1*
Brain
Day 2	<12 pg/gc	<12 pg/gc	<12 pg/gc	<12 pg/gc
Day 7		63.4 ± 53.9d	26.1 ± 22.1e	33.8 ± 18.9e
Day 28		63.2 ± 12.3d	41.4 ± 8.2	55.7 ± 19.8

^aData are means and standard deviations for five mice per time point. *, significantly (P < 0.05) greater than the control value by 1-way ANOVA followed by a Tukey-Kramer test.

^bFive uninfected, age-matched control mice.

^cBelow the detection limit of 12 pg/g.

^dOne mouse with a value below the detection limit.

^eTwo mice with values below the detection limit.

DISCUSSION

Due to the medical significance of systemic C. glabrata infections in humans, both the virulence traits of C. glabrata and the efficacy of antifungal treatments are increasingly studied in murine models. Although clinical data clearly show that immunosuppression is a risk factor for C. glabrata infections in humans, it is not a prerequisite for C. glabrata candidiasis (38). In mice, certain aspects of the host defense are clearly involved in C. glabrata survival in systemic infections (15, 46). However, mortality is rarely observed, even in leukopenic models (1, 2). Conflicting evidence has been published regarding the requirement of immunosuppression for the establishment of persistent colonization of internal organs in mice. While Atkinson et al. (2) reported that fungal burdens were very low or absent in immunocompetent mice, Kaur et al. (16) demonstrated that C. glabrata could be recovered after 7 days; Srikantha et al. (43) successfully reisolated C. glabrata 14 days p.i.; and Brieland et al. (6) showed persistent recovery of C. glabrata for 21 days p.i. Thus, it has been clearly shown that C. glabrata survives in immunocompetent mice for at least 21 days. However, to our knowledge, no study directly comparing the course of disease and C. glabrata fungal burdens in immunocompetent and immunosuppressed mice has been published to date. Therefore, we infected both untreated and immunosuppressed mice intravenously and compared the tissue distribution, histopathological changes, and clinical course of infection over 28 days. Our data clearly show that C. glabrata can survive for unexpectedly prolonged periods in all organs tested, despite the presence of a functional host immune response, supporting previous observations by others (6, 16, 43). In agreement with histological observations by others (6, 15), immunocompetent animals showed a rapid but mild immunological response to C. glabrata infection, characterized by splenomegaly and mononuclear immune cell infiltrations in affected organs. Likewise, monocytosis was observed in the blood of infected animals. However, the amounts of the proinflammatory cytokines IL-1α, IL-1β, IL-2, IL-6, GM-CSF, and TNF-α in the kidneys, livers, and brains of infected immunocompetent mice were either unaltered or only marginally increased over those in uninfected controls. The only cytokine upregulated on day 2 p.i. was GM-CSF. One of the functions of GM-CSF is the recruitment of macrophages to sites of infection (30). These observations are consistent with the findings of Li et al. (21, 22) and Schaller et al. (39) for C. glabrata-infected epithelial models, where GM-CSF is likewise upregulated, while the overall cytokine response is low. Thus, the observed mononuclear cell infiltrates are likely the result of increased local GM-CSF concentrations. Interestingly, GM-CSF has been shown to be protective in murine models of cryptococcosis (8) and aspergillosis (7, 36) and has been implied to have a protective effect in systemic murine C. albicans infections (25). Therefore, the downregulation of GM-CSF at later time points in the liver and kidney, which we observed in C. glabrata infections, might be beneficial for the persistence of the fungus. Of the other cytokines investigated in this study, only IL-1β and IL-2 showed any alterations due to infection. However, the levels of both cytokines were only marginally and independently increased in single organs at single time points. Cytokine induction within the first 48 h of infection was not determined in this study, but Brieland et al. (6) showed that TNF-α, gamma interferon (IFN-γ), and IL-12 levels are increased only within the first 24 h after infection. Thus, our results, in combination with published data, show that C. glabrata infection in mice does not lead to a significant proinflammatory cytokine response.

Although pronounced neutrophil infiltration could not be observed by histology, increases in MPO contents were observed in infected organs. However, MPO is not exclusively produced by neutrophils but is also, although to a lesser extent, produced by monocytes (18). Whether the observed MPO increase reflects neutrophils or monocyte infiltration remains unclear. However, it is noteworthy that the highest MPO levels measured in C. glabrata-infected kidneys were 10-fold lower than those in the kidneys of mice 10 days after infection with a sublethal dose of C. albicans, which contained comparable numbers of CFU (CFU count, 4.7 × 10³ ± 2.1 × 10³; MPO content, 6,402 ± 3,247 ng/g; n = 20) (I. D. Jacobsen et al., unpublished data). Furthermore, neutrophilia and left shift, which are clear indications of an active inflammatory response involving neutrophils, could not be detected in mice infected with C. glabrata. This suggests that neutrophil recruitment does not play a major role in the immune response to C. glabrata in mice. Consistent with this hypothesis, impairment of neutrophil function by dexamethasone treatment did not significantly influence fungal burdens. In this respect, C. glabrata clearly differs from C. albicans, where massive neutrophil infiltration is commonly observed and is believed to contribute significantly to host tissue destruction (25, 42). Thus, the absence of neutrophil recruitment might be one crucial factor contributing to the lower virulence of C. glabrata than of C. albicans in murine models.

Mononuclear immune cell infiltrates were the only alterations observed in the liver. Since the appearance of infiltrates coincides with subclinical liver damage, determined by the levels of the marker enzymes ALT and AST in serum, the immune response might have caused subclinical organ damage.

Macrophage recruitment was delayed in cyclophosphamide-treated mice and coincided with the recovery of white blood cell counts. The delayed immune response could explain the observed 1- to 2-log increase in fungal burdens on day 7 and/or day 14 postinfection (p.i.) in cyclophosphamide-treated mice. Consistently, upon full recovery of white blood cell counts in the chronic phase of infection (day 28), no differences were observed. The rapid recovery of white blood cells counts despite the observed efficient depletion after two cyclophosphamide doses (day 2) and weekly applications of cyclophosphamide was surprising and unexpected. A higher cyclophosphamide dose and more-frequent dosing might render mice leukopenic for a more prolonged period. However, because frequent, high-dose treatment with cyclophosphamide also affects other organs, leading to severe side effects, it was not compatible with our study outline of 4 weeks of observation after infection.

C3-deficient mice are rapidly killed by C. glabrata within a few days (46). However, the complement system is unaffected by the immunosuppressive drugs used in our study. Together with our findings that fungal burdens in the peracute phase (day 2) are not influenced by leukopenia, this suggests that other, noncellular mechanisms possibly mediate protection against lethal C. glabrata fungemia. On the other hand, while mice in our study did not develop clinical disease, C. glabrata could still be reisolated from all organs, even from those of immunocompetent mice, as late as 28 days p.i. Thus, even a fully functional immune system is not capable of fully clearing the infection over several weeks. In addition, no strong induction of a proinflammatory cytokine response could be observed. These observations strongly suggest that immune evasion is a strategy used by C. glabrata to survive in the host. In contrast, the virulent C. albicans strain CAI4+CIp10 induces a fulminant, rapid cytokine response and extensive neutrophil infiltration (24). However, the attenuated C. albicans pmr1Δ strain induces lower cytokine levels and no mortality, and fungal burdens do not increase over time (5, 24). Therefore, it has been suggested that the immune response contributes to sepsis and host death in systemic murine C. albicans infection (24, 42). Given that systemic murine C. glabrata infections appear to resemble infection with the C. albicans pmr1Δ strain, we conclude that the absence of a strong cytokine and neutrophil response is one factor that determines the benign outcome of C. glabrata infection in mice.

In addition to the influence of the host immune status on C. glabrata infection in mice, we were interested in the consequences of fungal amino acid auxotrophy for the persistence of C. glabrata. Fungal burdens in immunocompetent mice are sufficiently high during the first week of infection to allow in vivo fitness comparison of different C. glabrata strains. Additionally, we assumed that immunocompetent mice would provide the most stringent negative selection to assess C. glabrata survival inside the host. Thus, we analyzed the fitness of the auxotrophic mutants in this model in a competition experiment. Kaur et al. (16) and Srikantha et al. (43) demonstrated that this approach can be successfully used for comparing a wild type with one mutant strain. We extended the pool size to four mutants plus the wild type, combining the wild type, the his3Δ, leu2Δ, and trp1Δ single mutants, and the hisΔ leuΔ trpΔ triple mutant in the infection inoculum. All auxotrophic mutants showed normal or only marginally impaired fitness in the different organs during this observation period, implying that these auxotrophies do not affect survival within the host. Because we were also interested in the question of whether prolonged survival was affected by auxotrophy, analysis was additionally performed 28 days p.i. At this late time point, reduced survival of some of the mutants was observed in the brain, heart, and lung. Surprisingly, leucine auxotrophy had no effect on survival in the brain, in contrast to the situation in S. cerevisiae (11). However, due to the low fungal burden in these organs at the late time point, we considered these results unreliable. Furthermore, the fact that CFU counts of his3Δ and leu2Δ mutants were strongly reduced in the kidneys 28 days p.i., while the kidneys of one animal were colonized solely with the hisΔ leuΔ trpΔ triple mutant, was difficult to explain by auxotrophy alone. Therefore, we decided to verify these observations by determining the survival of the his3Δ mutant and the hisΔ leuΔ trpΔ mutant in comparison to that of the wild type in a single-strain infection experiment. Single-strain infections confirmed the observations of the competition experiment: Fungal burdens on days 2 and 7 were undistinguishable between strains. A tendency toward lower fungal burdens of mutant strains was observed in the brain, lung, and heart on day 28. However, survival reduction was less pronounced than in the competition experiment. Whether this is an indication for competition between strains in the host or a random observation due to low fungal numbers at the late time point remains unclear. While the in vitro growth kinetics of the mutant strains did not differ from those of the wild-type strain, it is possible that the in vivo replication of the auxotrophic mutants is impaired in certain organs in the chronic phase of infection. Thus, altered in vivo growth might be responsible for the slightly reduced fungal burdens in the brain, lung, and heart on day 28. In conclusion, histidine, leucine, and tryptophan auxotrophies, even in combination, did not have a significant effect on C. glabrata fitness in vitro and survival in mice. Thus, his3Δ, leu2Δ, and trp1Δ mutations provide suitable genetic backgrounds for constructing deletion strains for virulence studies.

In summary, our data indicate that depletion of the cellular immune system is not a prerequisite for the establishment of systemic infection and for prolonged survival of C. glabrata in mice. Mononuclear immune cells are recruited to the site of infection and appear to control the fungus. The absence of a strong proinflammatory cytokine response, which could lead to lethal septic shock, could be one mechanism contributing to the benign outcome of systemic C. glabrata infection in mice. In addition, we demonstrated that systemic infection of immunocompetent mice is a suitable model for testing the competitive in vivo fitness of five C. glabrata strains in one pool, even in the absence of clinical disease and mortality. Finally, fungal histidine, leucine, and tryptophan auxotrophy does not have a strong effect on the ability of C. glabrata to colonize and persist in different organs.