Elevated Cell Wall Chitin in Candida albicans Confers EchinocandinResistance In Vivo

Keunsook K. Lee, Donna M. MacCallum, Mette D. Jacobsen, Louise A. Walker, Frank C. Odds, Neil A. R. Gow, and Carol A. Munro

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom

Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this studywas to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/cmice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspo-fungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspo-fungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions.In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fun-gal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantlyreduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further in-crease in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered cloneshad acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known toconfer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest thatthe efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acqui-sition of FKS1 point mutations.

Echinocandins have made a major contribution to antifungaltherapy and represent the first class of clinically useful antifun-

gal drugs to inhibit synthesis of the fungal cell wall. The echino-candin target is the catalytic subunit of �(1,3)-glucan synthase,Fks1, which produces �(1,3)-glucan. Echinocandins have fungi-cidal activity against the majority of Candida spp. and are effectiveagainst Candida isolates which are resistant to other antifungaldrugs, such as azoles (16, 51, 54). In Aspergillus fumigatus, echino-candins can be cidal or static agents. Although the current inci-dence of echinocandin resistance is low (53, 55, 56), significantnumbers of clinical cases of echinocandin therapy failure havebeen reported (2, 28, 29, 31, 44, 57, 69). To date, all isolates whichacquire resistance to the echinocandins have been shown to havepoint mutations in the FKS1 gene (51), which generally occurwithin two hot-spot (HS) regions, HS1 and HS2. The HS1 regionof Candida albicans Fks1 (CaFks1) lies between amino acid resi-dues 641 and 649 and HS2 lies between residues 1345 and 1365 (3,50). Point mutations in the FKS1 gene of Candida spp. have beenreported in isolates from patients with breakthrough infectionsduring echinocandin therapy (23, 69). The most frequent pointmutation resulting in resistance to caspofungin occurs at Ser645 inthe HS1 region (36, 50). FKS2 encodes an alternative Fks subunitin Saccharomyces cerevisiae and other fungi (19, 50). Point muta-tions within FKS2 of C. glabrata can also lead to reducedsusceptibility to the echinocandins (23). Some intrinsicallyechinocandin-resistant fungal species, such as Neurospora crassa,Fusarium solani, Fusarium graminearum, Fusarium verticillioides,and Magnaporthe grisea, have the same echinocandin-resistantFks1 alleles (32). The prevalence of FKS1 HS mutations was as-sessed in a bank of 133 Candida spp. with variable echinocandinMIC values collected during 2006 and 2007 from worldwide loca-tions (7). In this study, only 2.9% of isolates contained point mu-tations in FKS1 HS regions, suggesting that during this period theoccurrence of these mutations was low. In vitro, an effect termed

“paradoxical growth” or the “Eagle effect” has been observedwhen some fungal isolates respond less well to high echinocandinconcentrations than to lower concentrations of drug (11, 66, 73).One C. albicans clinical isolate which demonstrated the paradox-ical growth phenomenon had a 9-fold increase in chitin content atechinocandin concentrations where paradoxical growth occurred(67). This indicates that alterations in cell wall compositionand architecture may also lead to reduced susceptibility to caspo-fungin.

The cell wall is a dynamic structure, and inhibition of one cellwall component can lead to a compensatory increase in another(59). In C. albicans, inhibition of �(1,3)-glucan by caspofunginresults in a compensatory increase in chitin synthesis (70). In C.albicans, this increase in chitin synthesis is mediated via multiplecell wall integrity pathways, such as the protein kinase C (PKC),Ca2�/calcineurin, and high-osmolarity glycerol (HOG) responsepathways (47, 61, 70). Treating wild-type C. albicans cells withCa2� and calcofluor white (CFW), which activates the Ca2�/cal-cineurin and PKC pathways, respectively, leads to a 3-fold increasein chitin content and reduced susceptibility to caspofungin (47,70). Cell wall mutants with higher basal chitin contents are alsoless susceptible to caspofungin (58). Therefore, production of ex-cess chitin has been highlighted to be a possible mechanism of

Received 17 May 2011 Returned for modification 13 July 2011Accepted 23 September 2011

Published ahead of print 10 October 2011

Address correspondence to Carol A. Munro, c.a.munro@abdn.ac.uk, or Neil A. R.Gow, n.gow@abdn.ac.uk.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.00683-11

The authors have paid a fee to allow immediate free access to this article.

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reduced susceptibility to echinocandins in vitro. However, the po-tential of increased chitin synthesis as an echinocandin resistancemechanism has not been determined in vivo. Here, we investigatethe effect of elevated chitin synthesis on in vivo susceptibility tocaspofungin in a murine model of systemic candidiasis. We dem-onstrate that high-chitin C. albicans cells are less susceptible tocaspofungin in vivo and that treatment of mice with caspofunginelevates the chitin content of C. albicans cells in infected kidneys.We also present evidence for the in vivo accumulation of fks1 pointmutations that are not dependent on prior exposure to echino-candins.

MATERIALS AND METHODSC. albicans strain and growth conditions. C. albicans clinical isolateSC5314, which is the most common parent for null mutant construction,was used in this study (21). C. albicans was maintained on Sabouraud agar(1% [wt/vol] mycological peptone, 4% [wt/vol] glucose, and 2% [wt/vol]agar) and incubated at 30°C in a static incubator. Experimental inoculawere prepared from YPD (1% [wt/vol] yeast extract, 2% [wt/vol] myco-logical peptone, 2% [wt/vol] glucose) broth cultures incubated at 30°Covernight with shaking at 200 rpm. For cells with increased chitin content,cells were grown in YPD containing 0.2 M calcium chloride (CaCl2) and100 �g of CFW ml�1.

Caspofungin MIC testing: CLSI M27-A2 method. MICs of caspofun-gin against C. albicans cells were determined according to the guidelines inCLSI (formerly NCCLS) document M27-A2 using modified (or supple-mented) RPMI 1640 broth (48) as described previously (70). Caspofunginconcentrations ranged from 0.016 �g ml�1 to 16 �g ml�1.

Cell wall composition analysis. Cell walls were extracted as describedpreviously (46). Briefly, C. albicans cells were grown in YPD broth withand without Ca2� and CFW for 16 h at 30°C with shaking at 200 rpm.Cells were collected by centrifugation at 3,000 � g for 5 min, washed oncewith chilled deionized water, resuspended in deionized water, and physi-cally fractured with glass beads in a FastPrep machine (Qbiogene). Thedisrupted cells were collected and centrifuged at 5,000 � g for 5 min. Thepellet, containing the cell debris and walls, was washed five times with 1 MNaCl, resuspended in buffer (500 mM Tris-HCl buffer, pH 7.5, 2% [wt/vol] SDS, 0.3 M �-mercaptoethanol, and 1 mM EDTA), boiled at 100°Cfor 10 min, and freeze-dried. For quantification of glucan, mannan, andchitin, cell walls were acid hydrolyzed with 2 M trifluoroacetic acid at100°C for 3 h. The acid was evaporated at 60 to 65°C, and the samples werewashed with deionized water and resuspended again in deionized water.The hydrolyzed samples were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD) in a carbohydrate analyzer system from Dionex (Surrey,United Kingdom) as described previously (58). The total concentration ofeach cell wall component was expressed as �g per mg of dried cell wall,determined by calibration from the standard curves of glucosamine, glu-cose, and mannose monomers, and converted to a percentage of the totalcell wall.

Animal models. Female BALB/c mice (weight range, 18 to 23 g; Har-lan Laboratories, Bicester, United Kingdom) were maintained in groupsof up to 9 animals per cage and supplied with food and water ad libitumunder conditions approved by the United Kingdom Home Office labora-tory animals inspectorate. Cells were prepared for injection by growth inYPD or YPD containing Ca2� and CFW under the conditions describedabove, collected by centrifugation, washed twice, and resuspended in ster-ile saline (Baxter Healthcare Ltd., Norfolk, United Kingdom).

For the 16-day virulence model, C. albicans (3 � 104 CFU per g bodyweight) was administrated intravenously (i.v.) via the lateral tail vein ofgroups of 6 mice. Animals were treated i.v. with daily doses of sterile saline(placebo) or caspofungin solution (1 mg kg�1; Merck Research Labora-tories, NJ) for up to 7 days beginning at 24 h postinfection. Caspofunginwas diluted in saline and administered as a 200-�g ml�1 solution. Animals

were monitored daily for up to 16 days postchallenge. Animals that be-came immobile or otherwise showed signs of severe illness were humanelyterminated by cervical dislocation and recorded as dying on the followingday. The kidney burdens and percent change of body weight for eachanimal were measured as described previously (40–42).

For investigation of early infection stages, a 4-day virulence model wasadopted (40, 42); 2 � 104 CFU per g body weight was injected i.v. into thetail veins of groups of 12 mice. The animals were treated i.v. with sterilesaline placebo or caspofungin (Merck Research Laboratories) at a dailydose of 1 mg kg�1 for up to 4 days starting 24 h after the day of challenge.Mice from each group (n � 3) were sacrificed each day for 4 days postin-fection to investigate disease development and cell wall chitin levels dur-ing initial stages of infection. Disease development in each animal wasmonitored by kidney burdens and body weight change.

To investigate the frequency of acquisition of the FKS1 hot-spot mu-tation, 10 different high-chitin inocula were prepared as described earlier,with each inoculum used to intravenously infect a female BALB/c mouse(1.3 � 104 to 2.0 � 104 CFU per g body weight). Mice were sacrificed at 24h postinfection, and kidney organ burdens were determined, with cellsfrom colonies saved for sequence analysis of the FKS1 gene.

Ethics statement. All animal experimentation in this study conformsto the University of Aberdeen Ethical Review Committee and currentUnited Kingdom Home Office legislation requirements.

Measurement of chitin content by fluorescence microscopy. Chitincontents were measured as described previously (70). Briefly, to visualizechitin in the C. albicans cell wall, samples of homogenized infected kid-neys were fixed with 10% (vol/vol) neutral buffered formalin (Sigma-Aldrich). Fixed cells were stained with CFW (25 �g ml�1), and fluores-cence was preserved with Vectashield mounting medium (VectorLaboratories, Peterborough, United Kingdom). All samples were exam-ined by differential interference contrast (DIC) and fluorescence micros-copy (456 nm) with a Zeiss Axioplan 2 microscope. Images were capturedby a C4742-95 digital camera (Hamamatsu Photonics, Hamamatsu, Ja-pan) and analyzed with the Openlab (version 4.04) software (Improvi-sion, Coventry, United Kingdom). CFW fluorescence was quantified forindividual yeast cells using region-of-interest measurements. Mean fluo-rescence intensities were then calculated, with the number of individualcells analyzed ranging from 96 to 254, and expressed as arbitrary units.

Semiquantitative histopathological analysis. For animals harvestedat 24 h and 72 h postinfection, kidneys were fixed with 10% (vol/vol)neutral buffered formalin and embedded in paraffin, and 5-�m longitu-dinal sections were cut. Deparaffinized sections were stained with periodicacid-Schiff (PAS) reagent and counterstained with hematoxylin. C. albi-cans density (number of PAS-positive pixels mm�2) and infiltrate density(number of host infiltrate pixels mm�2) were measured as described pre-viously (40) as a semiquantitative indicator of fungal burden.

Caspofungin sensitivity testing on agar plates. Kidneys were har-vested from euthanized mice at 24 h and 48 h postchallenge and homog-enized with sterile saline, and serial 10-fold dilutions of homogenate weremade to determine viable counts. Diluted homogenate was spotted ontoYPD plates containing caspofungin at concentrations ranging from 0.031�g ml�1 to 0.5 �g ml�1 and incubated at 30°C for 24 h. Colonies werecounted for each caspofungin concentration, and results were expressedas a percentage of the number of colonies on control YPD plates. The samekidney homogenates were also regrown in YPD medium and used asinocula for broth microdilution susceptibility testing by CLSI methodM27-A2.

DNA sequence analysis of FKS1 hot-spot regions. FKS1 HS regionswere analyzed as described previously (50). Briefly, genomic DNA wasextracted according to the protocol of Hoffman and Winston (26) fromindividual colonies from normal- and high-chitin cells in vitro (n � 22),from whole in vitro-grown cultures (n � 3), and from colonies (n � 1 to120) recovered from homogenized mouse kidneys. HS regions were am-plified with the forward (F) and reverse (R) primers (1 �M) 5=-TTT ATTCAA ATT CTT GCC-3= (HS1-F), 5=-AAT GCC ATG ATG AGA GGT

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GG-3= (HS2-F), 5=-GGA ATG CCA TTG TTA TTT CC-3= (HS1-R), and5=-GGT ACA GTT TCT CAT TGG CA-3= (HS2-R) (50). PCRs were per-formed using Extensor master mix (Abgene) and involved an initial 5-mindenaturation step at 94°C, followed by 30 cycles of 94°C for 30 s, 53°C for1 min, and 68°C for 1 min, with a 5-min final extension step at 68°C. PCRproducts were purified according to the protocol of Rosenthal et al. (62).

Methods for DNA sequence determinations have been described pre-viously (5). Briefly, purified PCR fragments were sequenced on bothstrands using 0.7 �M HS-F and HS-R primers. Sequencing reactions wereperformed with an ABI Prism BigDye Terminator cycle sequencing readyreaction kit (Applied Biosystems) according to the manufacturer’s recom-mendations. Sequences were analyzed with an ABI Prism DNA 3730xlanalyzer (Applied Biosystems) by Gene Service, Oxford, United King-dom. The sequence of FKS1/GSC1 (orf19.2929) from the Candida Ge-nome Database (CGD; http://www.candidagenome.org) was used as areference (1).

Statistical analysis. All data sets were analyzed with PASW (version18.0) statistical software. Organ burdens and body weight changes werecompared statistically by the Mann-Whitney U test. Survival data wereanalyzed by the Kaplan-Meier log rank test. In addition, the chitin mea-surement by fluorescence intensity was analyzed by the Mann-Whitney Utest, with a level of significance between groups set as a P value of �0.05.The data are presented as mean � standard error of the mean (SEM).

RESULTSTreatment of C. albicans with Ca2� and CFW increases chitincontent but does not affect glucan or mannan levels. Growth ofC. albicans yeast cells in the presence of Ca2� and CFW results inelevated chitin content (47, 70). However, as this treatment couldpotentially induce other changes in cell wall composition, we in-vestigated its effect on chitin levels of the other cell wall polysac-charides, glucan and mannan. The cell walls of untreated (normal-chitin) and Ca2�/CFW-treated (high-chitin) C. albicans cells wereextracted, acid hydrolyzed, and analyzed by high-performanceliquid chromatography (HPLC; HPAEC-PAD). No significantchanges in relative glucan and mannan levels were found upontreatment (Fig. 1B). HPLC data also indicated that growth of cellswith Ca2� and CFW resulted in a nearly 3-fold increase in cell wallchitin content (Fig. 1B). Supporting this, fluorescence images ofCFW-stained high-chitin cells showed considerably higher inten-sity than normal-chitin cells (Fig. 1A), indicating elevated chitincontent. Analysis of chitin content by fluorescence intensity con-firmed an approximately 4-fold increase in high-chitin cells com-pared to chitin-normal cells (data not shown), consistent withprevious work (47, 70).

C. albicans cells with elevated chitin content are less suscep-tible to caspofungin in a murine model of systemic candidiasis.Prior to the virulence assays, the growth rate and hyphal elonga-tion rates of C. albicans SC5314, which had been pregrown withCa2� and CFW, were determined. These were comparable tothose of untreated cells (data not shown); thus, treatment of cellswith Ca2� and CFW had no marked effect on the growth of yeastcells and hyphae.

Mice were infected with chitin-normal and high-chitin C. al-bicans cells. Caspofungin treatment was initiated at day 1 postin-fection and repeated daily for 7 days. All animals of the chitin-normal-infected group treated with caspofungin survived untilthe end of the experiment (day 16), indicating that caspofungintreatment was successful (Fig. 2A). However, all saline-treatedmice infected with chitin-normal cells died within 13 days (Fig.2A). Kaplan-Meier log rank tests showed these survival differencesto be significant (P � 0.05). In contrast, there was no significant

difference in the survival of mice inoculated with high-chitin cellswhether treated with caspofungin or saline. In addition, the high-chitin-infected mice treated with saline had significantly longersurvival than the saline-treated, normal-chitin-infected group(P � 0.05). Kidney fungal burdens in the normal-chitin-infectedgroup treated with caspofungin were reduced by 3 orders of mag-nitude compared to the saline-treated, normal-chitin-infectedgroup (P � 0.05) (Fig. 2B). In contrast, caspofungin treatment didnot reduce kidney fungal burdens of mice challenged with high-chitin cells, and these mice had longer mean survival times (Fig.2B). Kidney fungal burdens in the high-chitin-infected groupstreated with caspofungin were comparable to those of theplacebo-treated, normal-chitin-infected group. This suggestedthat the high-chitin cells were tolerated better than the chitin-normal cells or were less capable of causing disease. There was oneexample where no fungal cells were detected at the time of death inthe kidney of a caspofungin-treated mouse infected with chitin-normal cells. Considered together, the results showed that miceinfected with high-chitin cells were able to survive longer thanmice inoculated with chitin-normal cells regardless of caspofun-gin therapy.

According to the survival curves shown in Fig. 2, 50% of miceinoculated with high-chitin cells and treated with caspofungindied by day 3 and the remaining 50% survived until the end of theexperiment. To further investigate the caspofungin response ofhigh-chitin cells at the earlier stages of infection, a 4-day systemicinfection model was used. Mice were challenged i.v. with normal-or high-chitin cells at 2 � 104 CFU per g body weight, with eithercaspofungin or saline administered daily from 24 h postinfection.Each day, 3 mice were sacrificed and their kidney fungal burdensand weight change were determined. The kidney fungal burden

FIG 1 Changes in cell wall components when cells were grown in the presenceof Ca2�/CFW in vitro. (A) Untreated (i) and Ca2�/CFW-pretreated (ii) cellsshowing intensity of CFW staining of nascent chitin in the cell wall. Bars � 10�m. (B) Cell walls of untreated (normal-chitin) and pretreated (high-chitin)cells were acid hydrolyzed, and released monosaccharide was detected byHPAEC-PAD using a CarboPac PA10 analytical column. Results are expressedas a percentage of dried cell wall (�g mg�1). �, P � 0.05, mean � SEM (n � 5).

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from mice infected with chitin-normal cells was significantly re-duced after the initial dose of caspofungin compared to the saline-treated mice. The kidney fungal burden of chitin-normal-infectedmice treated with caspofungin remained significantly lower thanthat of the other groups for the remainder of the experiment,although no further reduction in burden was measured (P � 0.05)(Fig. 3A). In the other groups of mice, kidney fungal burdensremained essentially unchanged (Fig. 3A). Interestingly, at day 4postinfection (the black arrow in Fig. 3A), the kidney fungal bur-den from mice infected with high-chitin cells and treated withcaspofungin appeared to increase compared to the other threegroups, but this was not statistically significant. The fungal burdenresults were reflected in altered body weight, where mice infectedwith normal-chitin cells and treated with caspofungin were theonly group not to lose weight over the 4-day virulence model (Fig.3B). The results suggested that high-chitin cells have a comparableability to cause disease during early infection, even though in theprevious 16-day virulence model the mean survival time of miceinfected with high-chitin cells was longer than that for mice in-fected with normal-chitin cells. In addition, the efficacy of caspo-fungin against high-chitin cells was compromised, suggesting thatthe compensatory elevation in chitin was associated with reducedechinocandin susceptibility in vivo.

In vivo caspofungin treatment stimulates chitin biosynthesisin high-chitin cells. We investigated, by measuring the CFW flu-orescence of individual cells, whether the high chitin levels in-duced during inoculum preparation were maintained during in-

fection and whether caspofungin therapy caused furtheralterations in chitin levels in vivo (Fig. 4). At day 1 postchallenge,the CFW fluorescence (802 � 86) of high-chitin cells recoveredfrom infected kidneys was approximately 1.6-fold higher than thatof chitin-normal cells (496 � 31) (P � 0.05) (Fig. 4). Therefore,after 24 h of growth in vivo, high-chitin cells retained an elevatedchitin content. Likewise, at 2 days postchallenge, fungal chitinlevels were increased in cells recovered from saline-treated, high-chitin-infected animals (963 � 45) compared to cells from saline-treated, chitin-normal-infected animals (755 � 215) (Fig. 4). Forcaspofungin-treated animals, quantitative comparison of chitinlevels of cells from infected kidneys could not be performed be-cause too few fungal cells were recovered from kidneys of animalsinfected with normal-chitin cells. However, the mean fluores-cence intensity of fungal cells from high-chitin-infected,caspofungin-treated mice (1425 � 144) was significantly highereven than that of cells from the high-chitin-infected, saline-treated animals, suggesting that caspofungin treatment stimulatedfurther chitin synthesis in the high-chitin cells in vivo (Fig. 4).

Increased colonization of kidney tissue by high-chitin C. al-bicans cells. It has been shown that the kidney is the major targetorgan for C. albicans infection in the mouse i.v. challenge model(43). In addition, the severe kidney damage in mice generally con-tributes to the overall pathology of disease. Semiquantification oflesions in the kidney has been used to assess differences in thevirulence of C. albicans strains (8, 40). We investigated threevirulence-associated parameters (the number of lesions, Candida

FIG 2 Comparison of the caspofungin response of C. albicans normal- and high-chitin cells in a mouse systemic infection model. (A) Kaplan-Meier survivalcurves of mice infected with 3 � 104 CFU per g body weight of normal-chitin (NC) or high-chitin (HC) cells. Mice were treated i.v. either with saline or 1 mg perkg of caspofungin (CFN) daily for 7 days (n � 6). (B) Mean fungal burdens of kidneys harvested from each group of mice at the time of death. �, P � 0.05compared to the normal-chitin plus saline group. Results represent mean � SEM (n � 6).

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cell density, and immune infiltrate density mm�2), assessed bypixel counting, using Photoshop, of color images of stained kid-ney lesions, to gain a more in-depth understanding of the responseof high-chitin cells to caspofungin compared to untreated chitin-normal cells. We used samples collected on day 1 (prior to initia-tion of therapy) and on day 3 postchallenge (when the greatestdifference in fungal burden was observed).

Kidney sections were stained with PAS and hematoxylin todifferentiate C. albicans cells and immune infiltrates (Fig. 5). Atday 1 postchallenge, both hyphae and yeasts were observed inkidney sections from mice infected with chitin-normal or high-chitin cells (Fig. 5A). In the semiquantitative data, at day 1 post-challenge, there were no statistically significant differences in thenumber of kidney lesions mm�2, Candida density, or infiltratedensity between mice infected with chitin-normal and mice in-fected with high-chitin cells (Table 1). The results suggested thatkidney infections by chitin-normal and high-chitin cells progressin a similar manner up to 24 h after challenge. At day 3 postinfec-tion, examination of kidney sections from mice treated withcaspofungin revealed very few PAS-positive C. albicans elementsfrom chitin-normal-infected kidney lesions compared to thosefrom mice infected with high-chitin cells (Fig. 5B). The mean C.albicans-associated pixel density of high-chitin cells in kidneys ofplacebo-treated mice increased nearly 5-fold on day 3 comparedto day 1 (Table 1). In addition, after 2 days of caspofungin therapythe mean high-chitin cell density was 1.5-fold increased in day 3kidneys compared to day 1 kidneys (Table 1) and provides furtherevidence that high-chitin cells were resistant to caspofungin invivo. This corresponds well with data evaluating kidney fungalburdens determined by the numbers of CFU per g of kidney (Fig.3A). The mean infiltrate density of kidneys infected with high-chitin cells with and without caspofungin treatment increased3.1- and 3.5-fold, respectively, from day 1 to day 3 (Table 1). Incontrast, at day 3 the mean C. albicans pixel density of the chitin-

FIG 3 Caspofungin responses in vivo. Mice were infected with 2 � 104 CFUper g body weight of normal-chitin (NC) or high-chitin (HC) cells and treatedi.v. daily with either placebo or 1 mg per kg caspofungin (CFN), started at day1 postchallenge. (A) Mice (n � 3) were sacrificed daily, and the fungal load ofthe kidney burdens was determined. �, P � 0.05 compared to the normal-chitin plus saline group. Results represent mean � SEM. A black arrow indi-cates increase of the fungal burden in the high-chitin plus caspofungin group.(B) Weight change of each mouse group. �, P � 0.05, comparing the normal-chitin plus saline group to the others. Results represent mean � SEM (n � 3).

FIG 4 Chitin contents of cells from infected kidneys. Cells from homogenizedkidneys were fixed with 10% formalin and stained with 25 �g ml�1 of CFW.Chitin contents were determined by analyzing CFW fluorescence intensity ofindividual cells (n � 96 to 254). �, P � 0.05, mean � SEM (kidneys collectedfrom 3 mice at day 1 and 2 mice at day 2); ‡, no or few cells detected.

FIG 5 Histological stained kidney sections of mice infected with normal- andhigh-chitin cells with or without caspofungin therapy. (A) PAS-hematoxylin-stained kidney sections from day 1 postchallenge with C. albicans and infiltratedensities of each group of mice infected with normal- or high-chitin cells priorto the caspofungin therapy. (B) At day 3 postinfection, stained kidney sectionsof each group treated with placebo or caspofungin. Bars � 50 �m. Blackarrows, PAS-positive fungal cells.

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normal group with caspofungin treatment had decreased 0.6-foldand the mean infiltrate density had increased only 1.4-fold com-pared to the values at day 1 (Table 1). Without caspofungin treat-ment, C. albicans and infiltrate densities of the placebo-treatedchitin-normal group were increased 28.1- and 8.6-fold, respec-tively, over the 2-day period (Table 1). Therefore, the efficacy ofcaspofungin against high-chitin cells was reduced in vivo.

High-chitin cells recovered from infected kidneys retainedreduced susceptibility to caspofungin. High-chitin cells main-tained elevated chitin levels up to 48 h postinfection compared tochitin-normal cells, and chitin content was significantly increasedby treatment with caspofungin in vivo (Fig. 4). We next deter-mined the caspofungin susceptibility of C. albicans cells culturedfrom kidney homogenates by agar plate spot assays. The numberof colonies at each caspofungin concentration was counted andused to generate caspofungin dose-response susceptibility curves(Fig. 6). On day 1, prior to echinocandin treatment, fungi fromhigh-chitin-infected mice were less susceptible, with growth un-affected at all caspofungin concentrations tested. In contrast, fun-gal cells from chitin-normal-infected animals were progressivelyinhibited by increasing caspofungin concentrations. Fungal cellsrecovered on day 2 from mice infected with high-chitin cells andtreated with caspofungin were not inhibited by any of the caspo-fungin concentrations tested. Fungi recovered on day 2 fromhigh-chitin-infected, saline-treated mice showed intermediatecaspofungin susceptibility (Fig. 6).

Homogenates of kidneys were diluted, regrown in YPD me-

dium, and used as inocula for broth microdilution MIC tests. Theoverall 50% inhibitory concentrations (IC50s; concentrations re-sulting in 50% of cell growth compared to the control) of caspo-fungin for chitin-normal cells recovered from day 1 and 2 were0.13 to 0.25 �g ml�1 (Table 2). The IC50 for high-chitin cellsrecovered from saline-treated mice at day 2 was 0.13 �g ml�1. Incomparison, the IC50s of caspofungin for high-chitin cells recov-ered from day 1 and 2 caspofungin-treated animals had increasedup to 32-fold compared to the chitin-normal cells (Table 2).

Caspofungin-resistant cells with elevated chitin acquire mu-tations in Fks1 hot spot 1. The FKS1 sequence of cells that hadbeen passaged in vivo was compared with that of cells grown ex-clusively in the laboratory under chitin-inducing conditions(growth in the presence of Ca2� and CFW). Analysis of theSC5314 inoculum used to infect the mice indicated the presence ofheterozygosities in HS2 of FKS1 relative to the reference sequenceof FKS1 (CGD). Heterozygous single nucleotide polymorphismswere detected at positions 1410 (Y � C or T) and 1465 (R � A orG) in both the normal- and high-chitin inocula. These were silentmutations. However, at 24 h postinfection high-chitin cells (86%,12 out of 14) and chitin-normal cells (6%, 1 out of 16) had lost theheterozygosity at these positions.

Treatment of SC5314 with Ca2� and CFW in vitro did notinduce alterations in the Fks1 amino acid sequence (Table 3). Inaddition to sequencing the FKS1 HS regions of individual coloniesderived from the in vitro-grown cultures, genomic DNA was iso-lated from the entire culture; however, no FKS1 HS1 mutationswere detected within the entire in vitro population by sequenceanalysis. This suggests that high-chitin cells in vitro retained re-duced susceptibility to caspofungin due to elevated chitin levels inthe cell wall, as shown in Fig. 1, rather than alterations in the HSregions of Fks1. However, some FKS1 mutations were found inthe in vivo-passaged cultures. Even before initiation of caspofun-

TABLE 1 Semiquantitative histological analyses of kidney lesionsa

Day postinfection Inoculum TreatmentNo. of kidneylesions mm�2

Density (no. of pixels mm�2 [10�4])

C. albicans Infiltrate

1 Normal chitin No 1.89 � 0.26 0.60 � 0.09 3.3 � 0.38High chitin 1.71 � 0.11 0.86 � 0.13 2.6 � 0.22

3 Normal chitin Saline 3.87 � 0.99 16.78 � 14.31 28.86 � 13.38High chitin 2.92 � 0.58 4.09 � 2.56 9.23 � 2.64Normal chitin Caspofungin 2.16 � 0.47 0.34 � 0.05 4.72 � 0.99High chitin 2.60 � 0.10 1.30 � 0.24 8.23 � 1.39

a Formalin-fixed and paraffin-embedded kidneys were sectioned and stained with PAS and hematoxylin. Photoshop software was used to analyze PAS-positive fungal and infiltratematerials. Results presented are mean � SEM, and n is an average of 30 � 4 regions per kidney.

FIG 6 Caspofungin MICs of C. albicans recovered from infected kidneys.Homogenized infected kidney samples were serially diluted and plated on YPDcontaining a range of concentrations from 0.031 to 0.5 �g ml�1 caspofungin.The number of colonies was counted at each caspofungin concentration, andresults are expressed as a percentage of the colonies grown on control YPDplates lacking drug. The results for the normal-chitin cells treated with caspo-fungin (NC � CFN) group are not shown as insufficient cells were recoveredfor this test.

TABLE 2 Caspofungin sensitivities of C. albicans cells recovered frominfected kidneysa

Inoculum Source kidney CFN IC50 (�g ml�1)

Normal chitin Day 1 0.13High chitin 4Normal chitin Day 2, saline treated 0.25High chitin 0.13Normal chitin Day 2, CFN treated —b

High chitin 4a Cells recovered from kidney burdens were regrown in YPD, washed, and inoculated inRPMI 1640 for caspofungin (CFN) sensitivity testing using the CLSI method.b —, insufficient cells were recovered for this test

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gin therapy, 85% of high-chitin cells (17 out of 20 colonies) recov-ered at day 1 from infected kidneys were resistant to caspofunginand had an S645Y substitution in Fks1 HS1. Interestingly somemice were infected with a mixed population of fungal cells, withsome with an Fks1 S645Y substitution and some without (Table3). This substitution arose from homozygous FKS1 point muta-tions, suggesting the possibility of homozygosis of the chromo-some. S645Y is a common mutation in Fks1 HS1 associated withdecreased sensitivity to echinocandins (50, 51). At day 1, isolatesthat had acquired the FKS1 point mutation were harvested from 3different mice infected with high-chitin cells. Isolates with FKS1point mutations were recovered from 2 mice treated with caspo-fungin and 2 saline-treated mice at day 2. In contrast, no Fks1substitutions were found in chitin-normal cells (n � 20 colonies)recovered from infected kidneys. Interestingly, the remaining col-onies recovered from infected kidneys that had not acquired theHS1 Fks1 substitution at position Ser645 remained sensitive tocaspofungin (data not shown). Therefore, there may be variabilityin the mechanism of echinocandin resistance that evolves whenchitin-normal and high-chitin samples are exposed to echinocan-dins in vivo and in vitro.

At day 2 postinfection, all five colonies recovered from thefungal burden plates of mice infected with high-chitin cells andtreated with caspofungin had homozygous S645Y HS1 Fks1 sub-stitutions (Table 3). In comparison, 67% (four out of six colonies)of high-chitin cells recovered from the drug-free mice had thismutation. One colony from high-chitin cells recovered from theplacebo-treated mouse group with no substitution in HS1 Fks1had reduced susceptibility and was able to grow in 0.5 �g ml�1

caspofungin (data not shown). Therefore, this HS mutation seemsto be selected only in high-chitin cells in vivo in this study.

Further experiments were performed to investigate the fre-quency of acquisition of FKS1 HS point mutations in high-chitincells in vivo in 10 parallel cultures that were used to inoculate 10mice. Conditions of the 4-day infection model were used, exceptthat all mice were sacrificed at 24 h postinfection. Independentlygrown inocula of high-chitin cells (2 � 104 cells per g body weight)were each injected i.v. into individual mice. At 24 h postinfection,mice were sacrificed and kidney homogenates were cultured invitro. Genomic DNA was extracted from 12 isolates from the fun-gal burden plates of each mouse. Genomic DNA was also ex-tracted from the 10 independently grown inocula (�5 � 109

cells). No point mutations in FKS1 HS1 were found in the in vitroand in vivo samples (Table 3). Therefore, the acquisition of theFKS1 HS1 point mutation was only rarely observed in high-chitincells in vivo (once in 11 parallel experiments).

DISCUSSION

Echinocandins are the newest class of antifungal drugs introducedinto clinical use. They show less toxicity than other systemic anti-fungal agents and have fungicidal activity against C. albicans (16).In the majority of patients, echinocandin treatment is highly ef-fective; however, in recent years clinical failures have also beenreported (44, 57, 69). The majority of these cases involved pro-longed exposure to echinocandins and the acquisition of pointmutations in the FKS1 gene resulting in echinocandin insensi-tivity.

Fungi can remodel damaged cell walls by increasing chitin con-tent to maintain cell wall integrity (47, 59, 69). We observed pre-viously that treatment of C. albicans in vitro with sub-MICs ofechinocandins could result in a compensatory increase in chitincontent (70). This response to echinocandin exposure has alsobeen seen for A. fumigatus (22; L. A. Walker, J. Anjos, K. K. Lee,C. A. Munro, and N. A. Gow, unpublished data). Therefore, ele-vated chitin biosynthesis is a potential mechanism of resistance ortolerance to echinocandins. As yet there is no direct evidence thatthis phenomenon leads to clinical failures when treating Candidainfections with echinocandins. This study demonstrates that C.albicans cells with increased chitin levels in their cell walls hadreduced susceptibility to caspofungin in a mouse model of sys-temic candidiasis. Therefore, elevated cell wall chitin confers areduced susceptibility of C. albicans to echinocandins in vivo aswell as in vitro.

The cell wall signaling network, including the PKC, HOG, andCa2�/calcineurin pathways, activates CHS expression in responseto echinocandins, cations, or CFW (11, 22, 33, 35, 47, 70). In C.albicans, activation of the Ca2�/calcineurin and PKC pathwayswith a combination of Ca2� and CFW leads to a 3-fold increase inchitin content and reduced susceptibility to caspofungin (70). Inour experiments, high-chitin cells are primed to activate the cellwall regulatory networks prior to inoculation into mice. There-fore, the basal activity of these pathways may differ between cellswith high or normal chitin levels. Because the chitin content of theinoculum was further increased when exposed to caspofungin in

TABLE 3 Sequence analysis of C. albicans Fks1 hot spots

Daypostinfection Group Treatment

Mutation

Hot spot 1(residues 641-649)

Hot spot 2(residues 1345-1365)

0 SC5314 (normal chitin) No None (0/25)a None (0/24)SC5314 (high chitin) CaCl2/CFW None (0/25), (0/10)b None (0/25)

1c Normal chitin No None (0/20) None (0/20)High chitin No S645Y (17/20), (0/120)b None (0/20)

2d Normal chitin Saline None (0/11) None (0/11)Caspofungin None (0/1) None (0/1)

High chitin Saline S645Y (4/6) None (0/5)Caspofungin S645Y (5/5) None (0/5)

a Data in parentheses represent the number of colonies that have a mutation in Fks1/total number of colonies tested.b Data are for samples from 10 inocula (day 0) and 12 individual colonies from 10 separate in vivo experiments (day 1).c Samples were collected from three individual mice at 24 h postinfection.d Samples were collected from two individual mice at 48 h postinfection.

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vivo, it is clear that these signaling pathways were not fully acti-vated in the cells of the inoculum. A variety of stress pathways havebeen shown to be activated by echinocandins, including the oxi-dative stress tolerance pathway, which is important for C. albicansto survive killing by host macrophages (68). It has been demon-strated that Cap1, a key component of the C. albicans oxidativestress adaptation pathway, and Hog1 were activated by 0.19 �gml�1 of caspofungin (33). In this study, pretreatment of cells with0.5 mM hydrogen peroxide resulted in decreased sensitivity tocaspofungin (33). The heat shock protein 90 (Hsp90) is a keyelement of the heat stress and other stress responses and plays acritical role in azole resistance by regulating the stability of itsclient protein, calcineurin (12–14, 64). A synergistic effect of mi-cafungin with an Hsp90 and with a calcineurin inhibitor was ob-served in isolates harboring the F641S substitution in Fks1 (64).In addition, Hsp90 regulates C. albicans morphology in a tem-perature-dependent way via the Ras1-PKA (cyclic AMP-proteinkinase A) pathway (63). Therefore, echinocandins influence anti-fungal drug sensitivity and virulence via multiple direct and indi-rect effects that can involve modulation of a range of stress re-sponses.

“Paradoxical growth,” or the “Eagle effect,” is a phenomenonby which clinical fungal isolates respond less well in vitro to highdrug concentrations than to lower drug concentrations (10, 20,60, 73). The clinical relevance of the paradoxical growth responseof C. albicans to echinocandins has not been demonstrated in vivo.A survey of bloodstream Candida isolates, which included C. al-bicans, C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata,showed that the occurrence of the paradoxical effect is echinocan-din specific and species related (9, 27). Furthermore, it has beenreported that A. fumigatus infections in vivo can exhibit paradox-ical growth in response to echinocandin treatment, with the fun-gal burdens of caspofungin-treated animals being comparable tothose of animals receiving no antifungal treatment. However, thisdid not translate into a difference in mortality (52). Stevens et al.showed that the in vitro paradoxical effect of a C. albicans clinicalisolate was associated with an increase in cell wall chitin content(67). These data support the hypothesis that the echinocandinparadoxical effect may be determined by cell wall chitin content,although the clinical significance of this effect remains to be de-termined.

The possible role of C. albicans chitin in virulence and hostinteractions has been addressed previously in a number of studies(reviewed in reference 39). For example, the virulence of theCachs3� chitin synthase mutant with a 70% reduction in chitincontent was highly attenuated in a mouse systemic infectionmodel, but the mutant was still able to colonize kidneys (6). Fur-thermore, isolates with fks1 point mutations have thicker walls,elevated chitin levels, and reduced virulence in a murine candidi-asis model and in Toll-deficient Drosophila melanogaster (4). Chi-tin and �(1,3)-glucan are located in the inner layer of the C. albi-cans cell wall buried beneath the outer mannan-rich layer (30, 34).However, during in vivo infection and in response to caspofungintreatment, �(1,3)-glucan becomes unmasked in both hyphae andyeast cells, making it more accessible to the �-glucan host receptordectin-1 (71, 72). Unmasking of �(1,3)-glucan in C. albicans stim-ulates a strong immune response and increases levels of cytokinessuch as tumor necrosis factor alpha, interleukin-6 (IL-6), IL-10,and gamma interferon (25, 71). Recent studies have drawn atten-tion to chitin in fungal cell recognition and immune responses

(15, 37–39). Chitin and chitin derivatives can act as pathogen-associated molecular patterns and stimulate innate immune cells(37, 38). However, chitin has also recently been shown to blockimmune recognition of C. albicans by mononuclear cells (45). Theimmunological effects of chitin seem to also depend critically onthe size of the chitin-containing particle (37). The unmasking of�(1,3)-glucan may also lead to increased exposure of chitin on thecell surface and thus contribute to the observed elevation in CFWfluorescence intensity of caspofungin-treated high-chitin cells re-covered from infected kidneys (45) (Fig. 4). It is not knownwhether the high-chitin cells used in this study affect �(1,3)-glucan or chitin exposure or alter immune cell recognition in away which may result in their attenuated virulence and persistencein the kidneys. However, the finding that chitin may attenuateimmune recognition (45) is compatible with the observations re-ported here. In addition, fks1 mutants with increased chitin con-tents had a reduced inflammatory response mediated via dectin-1(4), strengthening the idea that chitin may dampen the innateimmune response. This merits further investigation.

Unexpectedly, high-chitin cells but not cells with normal chitinlevels acquired FKS1 mutations with a low frequency in vivo inseveral of the mice tested. Our results are consistent with clinicaldata that indicate that the acquisition of FKS1 mutations in clini-cal isolates of Candida species is a rare and stochastic event (7). At24 h postinfection, there was evidence for selection of homozy-gous point mutations in FKS1 in high-chitin cells even withoutexposure to caspofungin (Table 3). Fungal cells recovered at 24 hpostinfection from mice infected with high-chitin inocula werealso resistant to caspofungin (Table 2). Therefore, in vivo, specificpoint mutations in FKS1, when they occurred, were rapidly se-lected and were associated with subsequent reduced echinocandinsusceptibility.

A small number of C. albicans clinical isolates with FKS1 pointmutations have been tested in the murine models of systemic can-didiasis and showed differences in virulence (4, 74). However,clinical isolates are known to vary widely in their ability to causedisease in the systemic candidiasis model (41). Strains selected forin vitro echinocandin resistance that contain an Fks1 Ser645 muta-tion had very few virulence differences, as measured by fungalburdens (65). Therefore, more in-depth studies with higher num-bers of isolates and with strains engineered to contain only FKS1mutations, to rule out other possible mechanisms, are required todetermine whether these mutations convey altered fitness in vivo.

Previous studies showed that the Km of Fks1 from echino-candin-resistant isolates was not altered but the maximum rate ofreaction (Vmax) was reduced (49). The lower Vmax value suggestslower production of �(1,3)-glucan, which may result in activationof the cell wall integrity pathways that induce the compensatorychitin biosynthesis response. Defects in cell wall �(1,3)-glucandue to mutations, such as gas1, are known to activate chitin bio-synthesis, and S. cerevisiae fks1 null mutants had increased chitinsynthase activity and chitin content (17, 24). Furthermore, C. al-bicans strains with FKS1 hot-spot mutations have increased chitincontent compared to wild-type strains (4, 70). In this study, at day2 postinfection, high-chitin cells recovered from infected kidneyshad increased chitin content in the presence of caspofungin treat-ment compared to the cells recovered from drug-free mice (Fig.4). Also, the cell wall composition of high-chitin cells recoveredfrom two individual mice at day 1 postinfection was analyzed byHPLC. These clones harbored an S645Y Fks1 mutation and had

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higher chitin levels, as measured by HPLC, than normal-chitincells (unpublished data). An in vitro study showed that anechinocandin-resistant homozygous fks1 point mutant (18) hadno further increase in chitin in the presence of caspofungin at asub-MIC (70). These studies show that there are two nonexclusivemechanisms that can affect echinocandin sensitivity: (i) low-frequency acquisition of FKS1 mutations and (ii) elevation of chi-tin content via the stimulation of cell wall integrity pathways.

We observed that high-chitin, echinocandin-naive cells thatacquired an Fks1 substitution were selected during the first day ofinfection, sustained a higher chitin content, and were resistant tocaspofungin in a systemic candidiasis mouse model. The meansurvival time of mice infected with high-chitin cells was consider-ably longer than that of mice infected with normal-chitin cells.Our experiments also highlight the general conclusion that a de-tailed understanding of cell wall remodeling processes during in-fection is critical to decipher host-pathogen interactions at themolecular and cellular level. This in turn may provide valuableinsight into improving drug efficacy and identifying drug targetsfor novel or combination therapies.

ACKNOWLEDGMENTS

We thank Gillian Milne from the University of Aberdeen EM and histol-ogy facility and Luis Castillo for assistance with the histological analysis.We thank Cameron Douglas for helpful comments on the manuscript.

We also thank Gilead Sciences Ltd. (Cambridge, United Kingdom),the Wellcome Trust (080088 and 086827), and the EC (ALLFUN andAriadne) consortia for financial support and Merck Research Laborato-ries (NJ) for provision of caspofungin.

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