candida glabrata persistence in mice does not depend on ... · candida glabrata has emerged as an...

INFECTION AND IMMUNITY, Mar. 2010, p. 1066–1077 Vol. 78, No. 30019-9567/10/$12.00 doi:10.1128/IAI.01244-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Candida glabrata Persistence in Mice Does Not Depend on HostImmunosuppression and Is Unaffected by Fungal

Amino Acid Auxotrophy!

I. D. Jacobsen,1 S. Brunke,1 K. Seider,1 T. Schwarzmuller,2 A. Firon,3,4†C. d’Enfert,3,4 K. Kuchler,2 and B. Hube1,5*

Department for Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, 07745 Jena,1and Friedrich Schiller University Jena, Jena,5 Germany; Medical University of Vienna, Christian Doppler Laboratory for

Infection Biology, Max F. Perutz Laboratories, Campus Vienna Biocenter, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria2;and Institut Pasteur, Unite Biologie et Pathogenicite Fongiques, Departement Genomes et Genetique,3

and INRA USC2019,4 F-75015 Paris, France

Received 5 November 2009/Returned for modification 30 November 2009/Accepted 4 December 2009

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infec-tions in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, evenwith high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leadsto fungal burdens that are transiently increased over those in immunocompetent mice. However, even immu-nocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immuneresponses 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 acidauxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, ortryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in richmedium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parentalwild-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 thegeneration of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfullydeveloped immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Candida glabrata is a commensal yeast that can be isolatedfrom the mucosal layers of healthy individuals (10, 32). How-ever, as an opportunistic pathogen, it can also cause mucosaland severe, life-threatening invasive infections (10). In theUnited States, C. glabrata is the second most common cause ofcandidemia, representing about 20% of all Candida blood-stream isolates (reviewed in reference 34). C. glabrata is lesscommonly isolated in Europe but still accounts for !10% ofcandidemia cases (reviewed in reference 34). As with C. albi-cans, risk factors for the development of invasive C. glabratainfections in human patients include mucosal colonization byCandida spp., indwelling vascular catheters, antibiotic therapy,gastrointestinal surgery, cancer chemotherapy, and neutrope-nia (12, 26, 35). Despite antimycotic treatment and partiallydue to the naturally high resistance of C. glabrata to severalantifungal drugs (33), systemic C. glabrata infections often re-sult 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,p47phox"/" knockout mice and mice treated with a combina-tion of cyclophosphamide and 5-fluorouracil are susceptible tolethal C. glabrata infections (15, 28). p47phox"/" knockout miceare deficient in the phagocyte oxidative burst and serve as amodel for human chronic granulomatous disease (14). Cyclo-phosphamide and 5-fluorouracil are cytotoxic agents used forcancer treatment and affect replicating cells, such as myeloidimmune cell progenitors. Furthermore, complement (C3)-de-ficient mice were recently shown to be highly susceptible to C.glabrata infections (46). These findings suggest that immuno-suppression might be a key factor affecting the outcome ofsystemic C. glabrata infections in mice, consistent with therelevance of immunosuppression as a risk factor for humanpatients. However, animals treated with cyclophosphamidealone do not succumb to systemic C. glabrata infection, eventhough cyclophosphamide induces leukopenia. In the absenceof mortality, the fungal burden has been used as a parameterfor virulence and the efficacy of antifungal treatment for cy-clophosphamide-treated mice (4, 41). Similarly, fungal burdenshave been determined for immunocompetent mice systemi-cally infected with C. glabrata (6, 16, 43); however, differencesin mouse strains, animal age and gender, the C. glabrata strainused, and infectious doses hamper direct comparison of pub-lished data from immunocompetent and immunosuppressedmice. Thus, the influence of immunosuppression on systemic

* Corresponding author. Mailing address: Department of MicrobialPathogenicity Mechanisms, Leibniz Institute for Natural Product Re-search and Infection Biology, Hans Knoell Institute Jena (HKI), Beu-tenbergstraße 11a, D-07745 Jena, Germany. Phone: 49 (0) 3641 5321401. Fax: 49 (0) 3641 532 0810. E-mail: [email protected].

† Present address: Institut Pasteur, Unite de Recherche Biologie desBacteries pathogenes a Gram-positif, Departement Microbiologie,F-75015 Paris, France.

! Published ahead of print on 14 December 2009.

1066

C. glabrata infections in mice is still unclear. In this study, weaimed to clarify the importance of immunosuppression for thepathogenesis of systemic C. glabrata infections. Therefore, wecompared the fungal burden, the clinical course of infection,and histopathological alterations in immunocompetent, cyclo-phosphamide-treated, and dexamethasone-treated mice sys-temically infected with C. glabrata.

Both natural and genetically engineered auxotrophies in mi-croorganisms have been successfully developed as genetic toolsfor the investigation of virulence. However, some auxotrophiescan have detrimental effects on the virulence or survival ofpathogens within the host. For example, uracil auxotrophyleads to attenuation of Aspergillus fumigatus (9) and reducedsurvival of Saccharomyces cerevisiae in mice (11), and uridineauxotrophy affects the adhesion and virulence of Candida al-bicans (3, 17, 44). Amino acid auxotrophies seem to have lessimpact on virulence, since methionine, arginine, histidine, andleucine auxotrophic mutants of C. albicans are fully virulent(17, 27, 31). The finding of Goldstein and McCusker thatleucine auxotrophy in S. cerevisiae strongly affects survival inmice (11), however, demonstrates that similar auxotrophiesmight have different effects on virulence, survival, and persis-tence depending on the fungal species. Here, we investigatedthe question of whether histidine, leucine, and tryptophan aux-otrophies, alone or in combination, affect in vitro fitness and invivo survival and competitive fitness of C. glabrata in immuno-competent mice. Our results suggest only a minor influence ofhost immunosuppression and fungal auxotrophy on the sur-vival, persistence, and virulence of C. glabrata within mouseorgans. Based on these findings, we established a mouse modelsuitable for identifying the C. glabrata genes required for fit-ness in vivo.

MATERIALS AND METHODS

Strains and culture conditions. The strains used in this study are listed inTable 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 forselective reisolation of auxotrophic strains from tissue homogenates, minimalmedium (MM; 1# yeast nitrogen base with ammonium sulfate [BD Bioscience,Heidelberg, Germany] and 2% glucose), supplemented with the appropriateamino acids (0.3 mM histidine [Sigma-Aldrich, Taufkirchen, Germany], 0.8 mMleucine [Roth, Karlsruhe, Germany], 0.5 mM tryptophan [Roth]) if necessary,

was used. Inocula for infection experiments were harvested from 50-ml culturesgrown overnight at 200 rpm and 37°C. Cells were washed three times withphosphate-buffered saline (PBS), enumerated with a hemocytometer, and di-luted to the desired cell concentration with PBS. The viable counts in the inoculawere verified by plating serial dilutions on YPD agar.

Construction of auxotrophic strains. The dominant recyclable nourseothricinresistance marker SAT1 (37) was used to generate a set of auxotrophic C.glabrata strains in the ATCC 2001 genetic background. Homologous flankingregions of 500 bp were amplified from genomic DNA and ligated into plasmidpSFS2a by using the ApaI/XhoI and SacII/SacI restriction sites, thereby creatingrecyclable gene deletion cassettes for the C. glabrata HIS3 (CgHIS3), CgLEU2,and CgTRP1 genes. The deletion cassettes were obtained by ApaI and SacIrestriction and were used for transformation of the ATCC 2001 recipient strainby an electroporation protocol as described previously (37). Correct transfor-mants 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 theSAT1 cassette, leaving only one FRT site behind. Dilutions were then plated onYPD. The resulting colonies were tested for nourseothricin sensitivity andscreened by PCR. Repeated use of the appropriate deletion constructs yielded allcombinations of double-auxotrophic strains and the triple-auxotrophic strain. Allstrains were verified by Southern blot analysis for correct genomic integration.

Analysis of in vitro fitness and competition indices (CIs) of auxotrophicstrains. Growth curves were performed in 96-well plates in a Tecan InfiniteM200 microplate reader. Cells were grown in 100 $l YPD at 30°C (6 replicates)or 37°C (8 replicates). Doubling times (D) were calculated as the differencebetween the time at a certain optical density at 600 nm (OD600), occurring duringexponential growth, and the time 2 generations before, divided by 2 (45).

In vitro competition indices were determined in YPD and MM supplementedwith histidine, leucine, and tryptophan. Overnight cultures of ATCC 2001, thetriple-auxotrophic strain, and the single-auxotrophic strains were washed twicewith sterile water and enumerated with a CASY cell counter, and cultures wereadjusted to the same cell number. Cell pools were inoculated at a total cellnumber of 1 # 106/ml in 15 ml of medium and were grown with shaking (220rpm) at 30°C. After 5 h, serial dilutions were prepared, and cell suspensions ofthe input and output pools were plated on YPD agar and incubated at 30°C for24 h. Colonies were then replica plated on YPD (growth of all strains), MMplates without supplementation (growth of the wild type only), and MM platessupplemented with either histidine, leucine, or tryptophan (growth of the wildtype and the respective auxotrophic strain) in order to quantify each strain.Replica-plated colonies were counted after 48 h of incubation at 30°C. Thecompetition indices were calculated as the output ratio of the CFU of the mutantstrain to that of the wild type divided by the input ratio of the CFU of the mutantstrain 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 animalswere housed in groups of five in individually ventilated cages and were cared forin accordance with the principles outlined in the European Convention for theProtection of Vertebrate Animals Used for Experimental and Other Scientific Pur-poses (http://conventions.coe.int/Treaty/en/Treaties/Html/123.htm). All animalexperiments were in compliance with the German animal protection law andwere approved by the responsible Federal State authority and ethics committee

TABLE 1. Fungal strains and plasmids used in this study

Strain or plasmid Genotype or descriptiona Source or reference

C. glabrata strainsATCC 2001 C. glabrata wild-type strain American Type Culture Collection;

received from Ken Hayneshis3% mutant Histidine-auxotrophic derivative of ATCC 2001; his3%::FRT This study; H. Jungwirth and S. Lechnerleu2% mutant Leucine-auxotrophic derivative of ATCC 2001; leu2%::FRT This study; H. Jungwirth and S. Lechnertrp1% mutant Tryptophan-auxotrophic derivative of ATCC 2001; trp1%::FRT This study; H. Jungwirth and S. Lechnerhis% leu% trp% mutant Triple mutant auxotrophic for histidine, leucine, and tryptophan;

his3%::FRT leu2%::FRT trp1%::FRTThis study; H. Jungwirth and S. Lechner

PlasmidspSFS2a SAT1 marker, recombinase; AmpR; SAT1-caFLP 37pSFS2a-HIS3 For deletion of CgHIS3; AmpR; 5&-CgHIS3-SAT1-FLP-3&-CgHIS3 This study; H. Jungwirth and S. LechnerpSFS2a-LEU2 For deletion of CgLEU2; AmpR; 5&-CgLEU2-SAT1-FLP-3&-CgLEU2 This study; H. Jungwirth and S. LechnerpSFS2a-TRP1 For deletion of CgTRP1; AmpR; 5&-CgTRP1-SAT1-FLP-3&-CgTRP1 This study; H. Jungwirth and S. Lechnera For plasmids, the description includes the marker and insert.

VOL. 78, 2010 C. GLABRATA SYSTEMIC MURINE INFECTION MODEL 1067

(permit no. 03-008/07). For immunosuppression, either cyclophosphamide (150mg/kg; Sigma) or dexamethasone (100 mg/kg; Rotexmedica, Germany) was ap-plied intraperitoneally on days "3, 0, 7, 14, and 21. The effect of immunosup-pression was determined by comparing differential blood cell counts beforeimmunosuppression to those on the day of postmortem analysis. Mice werechallenged on day 0 with 5 # 107 CFU in 200 $l PBS via the lateral tail vein. Fivemice per group were sacrificed on days 2, 7, 14, and 28 postinfection (p.i.) foranalysis of macroscopic and histological changes, fungal burden, and changes inthe levels of blood marker enzymes.

Clinical parameters, blood enzymes, and pathology. A veterinarian performedphysical examinations twice daily. Body weight and body surface temperature(used as a noninvasive surrogate for internal body temperature [47]) were mea-sured once a day. To assess tissue damage to parenchymal organs, liver markerenzyme activities, such as alanine aminotransferase (ALT) and aspartate ami-notransferase (AST), as well as levels of urea in the blood (blood urea nitrogen),were determined from serum samples by using the EuroLyser CCA 180 Vetsystem (QinLAB Diagnostik, Martinsried, Germany) according to standardmethods recommended by the International Federation of Clinical Chemistry.Pooled samples (five animals per pool) collected on day "6 were analyzed ascontrols; for samples from infected animals, mice were anesthetized with anoverdose 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 sec-tions 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 sterilePBS, weighed, and placed in 1 ml (heart, lungs, kidneys, spleen) or 3 ml (liver andbrain) sterile PBS on ice. The organs were aseptically homogenized using a IkaT10 basic Ultra-Turrax homogenizer (Ika, Staufen, Germany). Serial dilutions ofhomogenates were plated on YPD plates. Colonies were counted after 48 h ofincubation at 30°C. The fungal burden was calculated as CFU per gram of tissue.To depict negative culture results on logarithmic scales, negative cultures werecounted as 1 CFU/g, thus appearing as a zero on log10 scales.

Pool experiments and competition indices. For the inoculum of the poolexperiment, independent cultures of the different strains were grown and treatedas 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 wasverified by plating serial dilutions on YPD agar. In addition, dilutions wereplated on MM without supplementation (growth of the wild type only) and onMM supplemented with either histidine, leucine, or tryptophan (growth of thewild type and the respective auxotrophic strain) in order to quantify each strain.Fifteen immunocompetent mice were infected as described above; five mice eachwere sacrificed on days 2, 7, and 28 postinfection. Fungal burdens were quanti-fied as described above with the following modification: Homogenates wereplated on YPD, MM lacking any supplementation, and MM supplemented witheither histidine, leucine, or tryptophan in order to quantify each strain. If fewerthan 100 colonies were expected per organ, homogenates were directly platedonly on YPD, and single colonies were replica plated on MM plates and on MMsupplemented with either histidine, leucine, or tryptophan. In vivo competitionindices (CIs) were calculated from the CFU of every individual animal as theoutput ratio of the strain-specific CFU to total CFU divided by the input ratio ofthe strain-specific CFU to total CFU. The CIs of five mice per time point wereused to generate the mean CI for each strain, organ, and time point.

Quantification of MPO and cytokines from tissue homogenates. Tissue ho-mogenates of immunocompetent mice infected with the wild-type strain ATCC2001 and the auxotrophic his3% and his% leu% trp% mutants as described abovewere diluted 1:1 to 1:7 in tissue lysis buffer (200 mM NaCl, 5 mM EDTA, 10 mMTris, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 $g/ml leu-peptin, and 28 $g/ml aprotinin [pH 7.4]) and centrifuged twice (1,500 # g, 15min, 4°C), and the supernatants were stored at "80°C until measurement. My-eloperoxidase (MPO) and cytokine levels were determined by commerciallyavailable murine enzyme-linked immunosorbent assay (ELISA) kits (for MPO,the Mouse MPO ELISA kit [Hycult Biotechnology, Uden, the Netherlands]; formultiple cytokines, the Mouse Inflammatory Cytokines Multi-Analyte ELISAr-ray kit [SABiosciences, Frederick, MD]; for interleukin 1' (IL-1'), the MouseILA SingleAnalyte ELISArray [SABiosciences]; and for IL-1(, IL-2, IL-6, tumornecrosis factor alpha [TNF-'], and granulocyte-macrophage colony-stimulatingfactor [GM-CSF], ELISA Ready SET Go! [eBioscience, Hatfield, United King-dom]) according to the manufacturer’s recommendations.

Statistical analysis. Data were plotted and statistically analyzed without logtransformation using GraphPad Prism, version 5.00 for Windows (GraphPadSoftware, 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 ofvariance (ANOVA) followed by Dunnett’s multiple-comparison test. Fungalburdens are shown as box plots (box, inner quartiles; whiskers, minimum andmaximum value in data set; line, median). One-way ANOVA was performed ondata sets from the same day after infection, followed by a Tukey-Kramer test toidentify which groups were significantly different. Myeloperoxidase and cytokinedata were analyzed per time point by 1-way ANOVA followed by the Tukey-Kramer test.

RESULTS

Influence of immunosuppression on fungal burden andpathological alterations during the course of infection. In or-der to determine the effect of immunosuppression on the out-come of C. glabrata infection in mice, we infected mice treatedwith either cyclophosphamide or dexamethasone, or left un-treated (immunocompetent), with 5 # 107 CFU C. glabrataATCC 2001 by lateral tail vein injection. Cyclophosphamidewas chosen because it is the drug most commonly used toinduce leukopenia in mouse models. Immunosuppression withdexamethasone was chosen because (i) dexamethasone is usedas an anti-inflammatory compound in combination therapy ofcancer and transplant patients, and (ii) immunosuppressionwith corticosteroids has been shown to render mice susceptibleto invasive aspergillosis by affecting macrophage and neutro-phil function (13). Five mice per treatment group were ana-lyzed on days 2, 7, 14, and 28 postinfection (p.i.). As expected,treatment with cyclophosphamide led to profound panleuko-penia ()5 # 103 leukocytes/ml) and absence of polymorpho-nuclear granulocytes (PMNs) on day 2 p.i. White blood cellcounts recovered during the first 2 weeks after infection (datanot shown). Animals treated with dexamethasone showedgranulocytosis from day 2 onward. In contrast, immunocom-petent mice showed normal differential white blood cell countson day 2 p.i. All groups presented monocytosis on days 14 and28 p.i. (data not shown). Mice treated with cyclophosphamideshowed a moderate weight loss (4.00 g * 1.00 g) from day "4(beginning of immunosuppressive treatment) to day 2. Nota-bly, all animals remained clinically healthy and even gainedweight over the full course of the experiment. Fever was notdetected 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 thistime after infection, the only detectable pathology was signif-icant spleen enlargement in immunocompetent animals (Table2). Histological analysis revealed the presence of fungal cells inthe red pulp, occasionally associated with mononuclear phago-cytes (data not shown). The splenic parenchyma was hyper-emic and showed minor follicular hyperplasia. On day 7 p.i.,significant splenomegaly and more-pronounced follicular hy-perplasia with germination centers were observed in all groups(Table 2 and Fig. 2A to C), indicating an active immune re-sponse toward the infection. The fungal burden in the spleendecreased over time in all groups, and fungal cells could beonly sporadically detected by histology on day 7 p.i. However,CFU counts in the spleens of cyclophosphamide-treated micewere significantly higher on days 7 and 14 than those for im-munocompetent animals (Fig. 1A).

The livers of infected mice appeared macroscopically nor-mal at all time points. However, mild liver damage can occurwithout macroscopic changes. Therefore, we measured the lev-

1068 JACOBSEN ET AL. INFECT. IMMUN.

els of two liver marker enzymes, alanine aminotransferase(ALT) and aspartate aminotransferase (AST), in serum beforeand after infection. These enzymes have been successfully usedto assess liver damage in mice infected intraperitoneally withC. albicans (20). ALT levels were significantly elevated in allgroups on day 7 p.i. Similarly, AST levels were elevated in allgroups on day 7 p.i.; however, the increase was statisticallysignificant 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, toa lesser degree, in dexamethasone-treated mice, small areaswith mononuclear infiltrates surrounded fungal cells. The sizesof the infiltrates increased to day 7 p.i., and infiltrates were alsopresent in cyclophosphamide-treated mice on day 7 p.i. (Fig.2G and H). During the same time, the fungal burden declinedabout 100-fold in immunocompetent and dexamethasone-treated mice but only 10-fold in cyclophosphamide-treated an-imals (Fig. 1B). Thus, the occurrence of immune cell infiltrates

FIG. 1. Fungal burdens in different organs of immunocompetent and immunosuppressed mice infected with C. glabrata ATCC 2001. Mice wereintravenously infected with 5 # 107 CFU on day zero. Fungal burdens were determined by culture from tissue homogenates of five animals pertreatment 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. Symbolsindicate significant (P ) 0.05) increases in fungal burdens over those for immunocompetent mice (#) or over those for immunocompetent anddexamethasone-treated mice (*).


coincided both with detectable liver damage and with signifi-cant reductions in fungal burdens. The CFU count in the liverdeclined further over time in all groups but was significantlyhigher in cyclophosphamide-treated mice on days 7 and 14 p.i.(Fig. 1B).

In contrast to those in the liver and spleen, the fungal bur-den 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 bymononuclear cells in immunocompetent animals and to alesser degree in dexamethasone-treated but not in cyclophos-phamide-treated mice. A significant reduction in CFU countsin the brain was observed from day 7 to day 14. The fungalburden in the brain was significantly higher in cyclophospha-mide-treated mice, suggesting that the reduction might be dueto the host response (Fig. 1C). However, we did not observeincreased inflammation in immunocompetent mice (data notshown). The absence of gross inflammation in the brain wasconsistent with the absence of neurological symptoms.

The fungal burdens in the lung and heart decreased steadilyover time (Fig. 1D and E). Fungal cells could be only sporad-ically detected in these organs on days 2 and 7 p.i. From day7 p.i. onward, mononuclear cell infiltrates were found in fewindividual animals of all groups.

The kidney is known to be one of the main target organs forsystemic C. glabrata infection in mice. Consistent with previousstudies demonstrating that C. glabrata can persist for at least 3weeks in the kidney (6), fungal burdens remained compara-tively stable throughout the experiment (Fig. 1F). Fungal cellswere present in histological sections of all treatment groups atall time points (Fig. 2N to T). While C. glabrata was associatedwith glomeruli on day 2 p.i. (Fig. 2R), fungal cells were found

predominantly within or surrounding tubuli at later timepoints. As in the other organs, mononuclear infiltrates werefound from day 7 p.i. onward. However, in contrast to otherorgans, 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. Thesealterations were histologically confirmed to be extensivemononuclear infiltrates containing C. glabrata cells (Fig. 2T).Despite the presence of macroscopic kidney alterations, therenal function of these animals was not significantly altered.However, in these mice, less than 50% of the kidney tissue wasaffected. Since only gross kidney lesions affecting 70% or moreof functional parenchyma cause increased urea levels in theblood, it was not surprising that no animal showed increasedblood urea levels (Table 2).

Construction of auxotrophic strains and in vitro analysis. Inorder to analyze whether a lack of auxotrophic marker genescan influence growth and fitness, we used SAT1 flipper tech-nology to generate single C. glabrata mutants auxotrophic forhistidine (his3%), leucine (leu2%), or tryptophan (trp1%), aswell as the triple-auxotrophic strain (his% leu% trp%). Genomicintegration was confirmed by colony PCR and growth on syn-thetic complete (SC) medium lacking the respective aminoacids. The mutants showed normal susceptibility to nourseo-thricin, indicating that the SAT1 construct was successfullyexcised. Southern blot analysis of all strains verified correctintegration of the gene deletion cassette, as well as loss of theSAT1 flipper cassette after recombinase induction in maltose-supplemented medium (data not shown).

In order to analyze whether the deletion of auxotrophicmarker genes had an influence on growth and fitness in vitro,the doubling times of the auxotrophic mutants were compared

TABLE 2. Liver marker enzymes, blood urea content, and spleen weights in immunosuppressed and immunocompetent CD-1 micesystemically infected with C. glabrata ATCC 2001

Time p.i. and groupaLiver marker enzyme level (U/liter)b

Blood ureacontent (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 2Immunocompetent 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 * 14.4Cyclophosphamide 75.6 * 19.8 126.0 * 101.2 19.2 * 3.6 60.5 * 11.5

Day 7Immunocompetent 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 14Immunocompetent 154.2 * 88.1 184.8 * 77.9 21.0 * 2.9 137.1 * 21.6Dexamethasone 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 28Immunocompetent 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*a Five 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).c Serum sampled on day "6 from animals within the experiment; 6 pools of 5 samples each were measured.d Immunocompetent, 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. Micewere 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 fungalcells. (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 onday 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.


upon growth in rich medium at 30°C and 37°C. The in vitrodoubling times of the wild-type and auxotrophic strains werenot significantly different at 30°C (67.7 * 2.6 min for the wildtype, 66.1 * 2.2 min for the his3% mutant, 65.7 * 1.8 min forthe leu2% mutant, 65.8 * 1.8 min for the trp1% mutant, and65.1 * 2.8 min for the his% leu% trp% mutant) and 37°C (53.2 *3.3 min for the wild type, 51.7 * 2.2 min for the his3% mutant,49.6 * 2.1 min for the leu2% mutant, 51.8 * 2.2 min for thetrp1% mutant, and 52.8 * 2.3 min for the his% leu% trp1%mutant). Likewise, in vitro competition in YPD or in supple-mented minimal medium revealed no disadvantage of auxo-trophic mutants relative to the wild type (CIs, 0.92 * 0.31 and0.82 * 0.17 for the his3% mutant; 1.08 * 0.35 and 1.45 * 0.91for the leu2% mutant; 0.96 * 0.35 and 1.19 * 0.59 for the trp1%mutant; and 0.96 * 0.27 and 1.65 * 0.41 for the his% leu% trp%mutant).

In vivo competition of wild-type and auxotrophic C. glabratastrains. In order to determine whether infection of mice couldserve as a negative selection system to identify mutants whosesurvival within the host is impaired, we performed an infectionexperiment with a pool consisting of the wild type (ATCC2001) and four auxotrophic strains (the his3%, leu2%, trp1%,and his% leu% trp% strains). The auxotrophic strains were cho-sen because they provide a versatile background for futuregenetic manipulations. However, auxotrophies have beenshown 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 parentalbackground for virulence studies. Our initial experimentshowed that immunosuppression led to increased fungal bur-dens on days 7 and 14 p.i.; however, negative selection withina host is stronger in the presence of an intact immune system.Furthermore, deficiencies of a mutant strain in withstandingthe host’s immune response might not be detectable in animmunocompromised host. Therefore, we decided to use im-munocompetent mice as hosts in an in vivo competition exper-iment.

Mice were infected with pools of 5 # 107 CFU consisting ofATCC 2001 and the his3%, leu2%, trp1%, and his% leu% trp%mutants. The inoculum was prepared to contain equal amountsof each strain; the strain proportions within the inoculum werecontrolled by plating serial dilutions on selective media andwere found to be 15.4% ATCC 2001, 13.5% his3% mutant,24.4% leu2% mutant, 13.3% trp1% mutant, and 33.3% his% leu%trp% mutant. Five mice each were sacrificed on days 2, 7, and28 p.i. to determine the in vivo competition indices (CIs). Therelative pool composition of auxotrophic fungal cells in theliver and spleen remained stable over time, with less than2-fold changes in the CIs (Table 3). In contrast, a 10-folddecrease in the CI of the his3% mutant was observed on day28 p.i. in the brain. The his% leu% trp% triple mutant wasvirtually absent from the brain on day 28 p.i. (Table 3). In thelung, the leu% mutant CI was 10-fold decreased on day 28 p.i.,as was the his3% mutant CI in the heart (Table 3). The CIs for

TABLE 3. In vivo competition indices of ATCC 2001 and auxotrophic mutant strains

Organand day

Competition indexa for the following strain:

ATCC 2001 his3% mutant leu2% mutant trp1% mutant his% leu%trp% mutant

SpleenDay 2 1.04 * 0.19 1.32 * 0.45 0.85 * 0.25 1.49 * 0.19 0.83 * 0.28Day 7 1.25 * 0.38 0.83 * 0.68 0.76 * 0.48 1.97 * 1.28 0.87 * 0.78Day 28 1.59 * 0.47 0.81 * 0.73 0.80 * 0.30 1.66 * 1.09 0.75 * 0.54

LiverDay 2 1.29 * 0.34 1.03 * 0.50 0.81 * 0.20 1.10 * 0.47 1.29 * 0.34Day 7 1.19 * 0.30 1.48 * 0.28 0.96 * 0.19 1.85 * 0.57 0.48 * 0.30Day 28 1.75 * 0.24 1.03 * 0.34 0.68 * 0.20 1.57 * 0.38 0.71 * 0.16

BrainDay 2 2.85 * 0.50 1.79 * 1.07 0.72 * 0.30 1.73 * 1.16 0.34 * 0.36Day 7 2.18 * 0.83 0.79 * 0.75 0.60 * 0.50 2.03 * 0.39 0.50 * 0.59Day 28 4.88 * 1.83 0.10 * 0.19 0.53 * 1.03 0.98 * 1.83 0.00 * 0.00

LungDay 2 1.16 * 0.12 1.23 * 0.44 0.94 * 0.42 1.79 * 0.61 0.63 * 0.38Day 7 1.17 * 0.45 0.67 * 0.78 0.66 * 0.56 1.01 * 0.6 1.36 * 0.76Day 28 4.00 * 2.79 1.54 * 1.58 0.08 * 1.26 0.92 * 1.38 0.18 * 0.35

HeartDay 2 1.14 * 0.26 0.63 * 0.48 0.58 * 0.25 1.10 * 0.43 1.4 * 0.30Day 7 1.70 * 0.65 1.08 * 0.93 0.92 * 0.33 1.99 * 0.47 0.41 * 0.39Day 28 1.74 * 1.37 0.00 * 0.00 1.09 * 0.88 2.52 * 2.27 0.45 * 0.53

KidneyDay 2 1.95 * 0.87 1.75 * 2.06 1.47 * 1.30 2.58 * 1.72 0.47 * 0.50Day 7 1.38 * 0.64 1.03 * 0.92 0.86 * 0.52 3.04 * 3.08 0.31 * 0.46Day 28 1.82 * 1.37 0.04 * 0.09 0.06 * 0.07 1.78 * 1.72 1.44 * 1.07a Data 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.


the his% leu% trp% triple mutant were mildly reduced in theseorgans at the late time point (5-fold and 2-fold, respectively).However, the total numbers of fungal cells in the heart andlung on day 28 p.i. were low (101 to 102), thus limiting theaccuracy of CIs in these organs at late time points. In thekidney, both the his3% and the leu2% mutant were stronglyunderrepresented in the output pools on day 28 p.i., whereasthe his% leu% trp% mutant was readily reisolated (Table 3).Analysis of output pools in the kidneys of individual mice onday 28 p.i. revealed that in 4 out of 5 mice, two strains domi-nated the output pool (Fig. 3).

Single-strain infections. To confirm the results of the in vivocompetition experiment, the survival of the wild-type strainATCC 2001 and the his3% and his% leu% trp% auxotrophicmutants in immunocompetent mice was tested in a single-strain infection experiment. In agreement with the competitionexperiment, no strain-specific differences in fungal burdenwere observed in any organs on days 2 and 7 p.i. (Fig. 4).

Likewise, no differences between the strains were observed onday 28 p.i. in the liver and spleen. In the brain, heart, and lung,we observed slightly higher fungal burdens in mice infectedwith the wild-type strain; however, these differences were notstatistically significant. Similarly, mice infected with the wild-type strain had higher fungal burdens in the kidney than miceinfected with either the his3% or the his% leu% trp% mutant(Fig. 4). This result was not anticipated, since the in vivocompetition experiment did not show any impairment of his%leu% trp% mutant survival in the kidneys (Table 3 and Fig. 3).

Myeloperoxidase content and cytokine response in infectedorgans. PMNs play a crucial role as immune effector cellsduring candidemia. They have been shown to be involved incontrolling C. albicans but also contribute to tissue damageduring infection. However, immune cell infiltrates in organs ofC. glabrata-infected mice consisted mainly of mononuclearcells. Since quantification of immune cells in histological sec-tions is difficult, we used the PMN marker enzyme myeloper-oxidase (MPO) (19, 23) to quantify PMN infiltration in tissuehomogenates of immunocompetent mice infected with ATCC2001 or the his3% or his% leu% trp% mutant. Upon infection, theMPO content significantly increased in all organs to day 7 p.i.and then, with the exception of the brain, decreased to day28 p.i., without significant differences between the strains (Ta-ble 4). In order to evaluate the cytokine responses of miceinfected with the different C. glabrata strains, we analyzed thelevels of the proinflammatory cytokines IL-1', IL-1(, IL-2,IL-6, GM-CSF, and TNF-' in the liver, kidney, and brain byELISA. No C. glabrata strain-specific differences were ob-served for any cytokine. The IL-1' and IL-6 contents of allorgans were unaltered at all time points. Levels of IL-1( were2-fold increased in the brain on day 7 p.i. but were unaltered atother time points. No differences from levels in control animalswere observed for IL-1( in the liver and kidney. IL-2 showeda 1.5-fold increase in the kidney on day 28 p.i. GM-CSF levelswere 2-fold increased in the kidney and liver on day 2, but were4-fold decreased in the liver on days 7 and 28 p.i., relative tothe levels in noninfected controls. TNF-' was barely detectablein the kidneys of both control and infected animals (detectionlimit, 75 pg/g). It was not detectable in the brain (detectionlimit, 75 pg/g). The TNF-' contents in the liver were the samefor infected and control mice.

DISCUSSION

Due to the medical significance of systemic C. glabrata in-fections in humans, both the virulence traits of C. glabrata andthe efficacy of antifungal treatments are increasingly studied inmurine models. Although clinical data clearly show that im-munosuppression is a risk factor for C. glabrata infections inhumans, it is not a prerequisite for C. glabrata candidiasis (38).In mice, certain aspects of the host defense are clearly involvedin 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 require-ment of immunosuppression for the establishment of persis-tent colonization of internal organs in mice. While Atkinson etal. (2) reported that fungal burdens were very low or absent inimmunocompetent mice, Kaur et al. (16) demonstrated that C.glabrata could be recovered after 7 days; Srikantha et al. (43)

FIG. 3. Strain ratios in the kidneys of individual mice 28 days afterinfection with a pool of C. glabrata ATCC 2001 (wild type [wt])andauxotrophic mutants. Mice were intravenously infected with a total of5 # 107 CFU on day zero. The input pool consisted of ATCC 2001(15.4%), the his3% mutant (13.5%), the leu2% mutant (24.4%), thetrp1% mutant (13.5%), and the his% leu% trp% mutant (33.3%).


successfully reisolated C. glabrata 14 days p.i.; and Brielandet al. (6) showed persistent recovery of C. glabrata for 21 daysp.i. Thus, it has been clearly shown that C. glabrata survives inimmunocompetent mice for at least 21 days. However, to ourknowledge, no study directly comparing the course of diseaseand C. glabrata fungal burdens in immunocompetent and im-munosuppressed mice has been published to date. Therefore,we infected both untreated and immunosuppressed mice in-travenously and compared the tissue distribution, histopatho-logical changes, and clinical course of infection over 28 days.Our data clearly show that C. glabrata can survive for unex-pectedly prolonged periods in all organs tested, despite thepresence of a functional host immune response, supporting

previous observations by others (6, 16, 43). In agreement withhistological observations by others (6, 15), immunocompetentanimals showed a rapid but mild immunological response to C.glabrata infection, characterized by splenomegaly and mono-nuclear immune cell infiltrations in affected organs. Likewise,monocytosis was observed in the blood of infected animals.However, the amounts of the proinflammatory cytokines IL-1', IL-1(, IL-2, IL-6, GM-CSF, and TNF-' in the kidneys,livers, and brains of infected immunocompetent mice wereeither unaltered or only marginally increased over those inuninfected controls. The only cytokine upregulated on day2 p.i. was GM-CSF. One of the functions of GM-CSF is therecruitment of macrophages to sites of infection (30). These

FIG. 4. Fungal burden in different organs of immunocompetent mice infected with C. glabrata ATCC 2001 (wild type) or the his3% or his% leu%trp% strain. Mice were intravenously infected with 5 # 107 CFU on day zero. Fungal burdens were determined by culture from tissue homogenatesof five animals per treatment group and time point and are shown as box plots (box: inner quartiles; whiskers: minimum and maximum value indata set; line: median). One-way ANOVA was performed on data sets from the same day after infection, followed by Tukey-Kramer test. Nosignificant (P ) 0.05) differences between strains were observed.


observations are consistent with the findings of Li et al. (21, 22)and Schaller et al. (39) for C. glabrata-infected epithelial mod-els, where GM-CSF is likewise upregulated, while the overallcytokine response is low. Thus, the observed mononuclear cellinfiltrates are likely the result of increased local GM-CSF con-centrations. Interestingly, GM-CSF has been shown to be pro-tective in murine models of cryptococcosis (8) and aspergillosis(7, 36) and has been implied to have a protective effect insystemic murine C. albicans infections (25). Therefore, thedownregulation of GM-CSF at later time points in the liverand kidney, which we observed in C. glabrata infections, mightbe beneficial for the persistence of the fungus. Of the othercytokines investigated in this study, only IL-1( and IL-2showed any alterations due to infection. However, the levels ofboth cytokines were only marginally and independently in-creased in single organs at single time points. Cytokine induc-tion within the first 48 h of infection was not determined in thisstudy, but Brieland et al. (6) showed that TNF-', gammainterferon (IFN-+), and IL-12 levels are increased only withinthe first 24 h after infection. Thus, our results, in combinationwith published data, show that C. glabrata infection in micedoes not lead to a significant proinflammatory cytokine re-sponse.

Although pronounced neutrophil infiltration could not beobserved by histology, increases in MPO contents were ob-served in infected organs. However, MPO is not exclusivelyproduced by neutrophils but is also, although to a lesser extent,produced by monocytes (18). Whether the observed MPO in-crease reflects neutrophils or monocyte infiltration remainsunclear. However, it is noteworthy that the highest MPO levelsmeasured in C. glabrata-infected kidneys were 10-fold lowerthan those in the kidneys of mice 10 days after infection with asublethal dose of C. albicans, which contained comparablenumbers of CFU (CFU count, 4.7 # 103 * 2.1 # 103; MPOcontent, 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 responseinvolving neutrophils, could not be detected in mice infectedwith C. glabrata. This suggests that neutrophil recruitment doesnot play a major role in the immune response to C. glabrata inmice. Consistent with this hypothesis, impairment of neutro-phil function by dexamethasone treatment did not significantlyinfluence fungal burdens. In this respect, C. glabrata clearlydiffers from C. albicans, where massive neutrophil infiltration iscommonly observed and is believed to contribute significantlyto host tissue destruction (25, 42). Thus, the absence of neu-trophil recruitment might be one crucial factor contributing tothe lower virulence of C. glabrata than of C. albicans in murinemodels.

Mononuclear immune cell infiltrates were the only alterationsobserved in the liver. Since the appearance of infiltrates coincideswith subclinical liver damage, determined by the levels of themarker enzymes ALT and AST in serum, the immune responsemight have caused subclinical organ damage.

Macrophage recruitment was delayed in cyclophosphamide-treated mice and coincided with the recovery of white bloodcell counts. The delayed immune response could explain theobserved 1- to 2-log increase in fungal burdens on day 7 and/orday 14 postinfection (p.i.) in cyclophosphamide-treated mice.Consistently, upon full recovery of white blood cell counts in thechronic phase of infection (day 28), no differences were observed.The rapid recovery of white blood cells counts despite the ob-served efficient depletion after two cyclophosphamide doses (day2) and weekly applications of cyclophosphamide was surprisingand unexpected. A higher cyclophosphamide dose and more-frequent dosing might render mice leukopenic for a more pro-longed period. However, because frequent, high-dose treatmentwith cyclophosphamide also affects other organs, leading to se-vere side effects, it was not compatible with our study outline of 4weeks of observation after infection.

C3-deficient mice are rapidly killed by C. glabrata within afew days (46). However, the complement system is unaffectedby the immunosuppressive drugs used in our study. Togetherwith our findings that fungal burdens in the peracute phase(day 2) are not influenced by leukopenia, this suggests thatother, noncellular mechanisms possibly mediate protectionagainst lethal C. glabrata fungemia. On the other hand, whilemice in our study did not develop clinical disease, C. glabratacould still be reisolated from all organs, even from those ofimmunocompetent mice, as late as 28 days p.i. Thus, even afully functional immune system is not capable of fully clearingthe infection over several weeks. In addition, no strong induc-tion of a proinflammatory cytokine response could be ob-served. These observations strongly suggest that immune eva-sion is a strategy used by C. glabrata to survive in the host. Incontrast, the virulent C. albicans strain CAI4-CIp10 induces afulminant, rapid cytokine response and extensive neutrophilinfiltration (24). However, the attenuated C. albicans pmr1%strain induces lower cytokine levels and no mortality, and fun-gal burdens do not increase over time (5, 24). Therefore, it hasbeen suggested that the immune response contributes to sepsisand host death in systemic murine C. albicans infection (24,42). Given that systemic murine C. glabrata infections appearto resemble infection with the C. albicans pmr1% strain, weconclude that the absence of a strong cytokine and neutrophil

TABLE 4. Myeloperoxidase content in livers, kidneys, and brains ofmice infected with ATCC 2001 and auxotrophic mutant strains

Organand day

Myeloperoxidase content (ng/g)a in organs of mice:

ControlbInfected with the following strain:

ATCC 2001 his3% mutant his% leu% trp%mutant

LiverDay 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

KidneyDay 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*

BrainDay 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

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

b Five uninfected, age-matched control mice.c Below the detection limit of 12 pg/g.d One mouse with a value below the detection limit.e Two mice with values below the detection limit.


response is one factor that determines the benign outcome ofC. 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 conse-quences of fungal amino acid auxotrophy for the persistence ofC. glabrata. Fungal burdens in immunocompetent mice aresufficiently high during the first week of infection to allow invivo fitness comparison of different C. glabrata strains. Addi-tionally, we assumed that immunocompetent mice would pro-vide the most stringent negative selection to assess C. glabratasurvival inside the host. Thus, we analyzed the fitness of theauxotrophic mutants in this model in a competition experi-ment. Kaur et al. (16) and Srikantha et al. (43) demonstratedthat this approach can be successfully used for comparing awild type with one mutant strain. We extended the pool size tofour mutants plus the wild type, combining the wild type, thehis3%, leu2%, and trp1% single mutants, and the his% leu% trp%triple mutant in the infection inoculum. All auxotrophic mu-tants showed normal or only marginally impaired fitness in thedifferent organs during this observation period, implying thatthese auxotrophies do not affect survival within the host. Be-cause we were also interested in the question of whether pro-longed survival was affected by auxotrophy, analysis was addi-tionally performed 28 days p.i. At this late time point, reducedsurvival of some of the mutants was observed in the brain,heart, and lung. Surprisingly, leucine auxotrophy had no effecton survival in the brain, in contrast to the situation in S.cerevisiae (11). However, due to the low fungal burden in theseorgans at the late time point, we considered these results un-reliable. Furthermore, the fact that CFU counts of his3% andleu2% mutants were strongly reduced in the kidneys 28 daysp.i., while the kidneys of one animal were colonized solely withthe his% leu% trp% triple mutant, was difficult to explain byauxotrophy alone. Therefore, we decided to verify these ob-servations by determining the survival of the his3% mutant andthe his% leu% trp% mutant in comparison to that of the wildtype in a single-strain infection experiment. Single-strain infec-tions confirmed the observations of the competition experi-ment: Fungal burdens on days 2 and 7 were undistinguishablebetween strains. A tendency toward lower fungal burdens ofmutant strains was observed in the brain, lung, and heart onday 28. However, survival reduction was less pronounced thanin the competition experiment. Whether this is an indicationfor competition between strains in the host or a random ob-servation due to low fungal numbers at the late time pointremains unclear. While the in vitro growth kinetics of the mu-tant strains did not differ from those of the wild-type strain, itis possible that the in vivo replication of the auxotrophic mu-tants is impaired in certain organs in the chronic phase ofinfection. Thus, altered in vivo growth might be responsible forthe slightly reduced fungal burdens in the brain, lung, andheart on day 28. In conclusion, histidine, leucine, and trypto-phan auxotrophies, even in combination, did not have a signif-icant effect on C. glabrata fitness in vitro and survival in mice.Thus, his3%, leu2%, and trp1% mutations provide suitable ge-netic backgrounds for constructing deletion strains for viru-lence studies.

In summary, our data indicate that depletion of the cellularimmune system is not a prerequisite for the establishment ofsystemic infection and for prolonged survival of C. glabrata in

mice. Mononuclear immune cells are recruited to the site ofinfection and appear to control the fungus. The absence of astrong proinflammatory cytokine response, which could lead tolethal septic shock, could be one mechanism contributing tothe benign outcome of systemic C. glabrata infection in mice.In addition, we demonstrated that systemic infection of immu-nocompetent mice is a suitable model for testing the compet-itive in vivo fitness of five C. glabrata strains in one pool, evenin the absence of clinical disease and mortality. Finally, fungalhistidine, leucine, and tryptophan auxotrophy does not have astrong effect on the ability of C. glabrata to colonize and persistin different organs.

ACKNOWLEDGMENTS

This work was supported by the German Federal Ministry of Edu-cation and Health (BMBF, project 0313931B), the French AgenceNationale de la Recherche (ANR-06-PATHO-005-01), and the Aus-trian Science Foundation (FWF-API-0125-B09) as part of ERA-NETPathoGenoMics project 0313931B FunPath, “Genomic approaches tounravel the molecular mechanisms of pathogenicity in the humanfungal pathogen Candida glabrata.” K.K. was additionally supported bya grant from the Christian Doppler Society.

We thank Birgit Weber and Ursula Stockel for excellent technicalhelp and Duncan Wilson for critical reading of the manuscript. We areindebted to Helmut Jungwirth and Stefan Lechner for help with C.glabrata mutant strain construction.

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