Characterization of Virulence-Related Phenotypes in Candida Speciesof the CUG Clade

Shelby J. Priest,a,b Michael C. Lorenza

Department of Microbiology and Molecular Genetics and the Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston,Texas, USAa; Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USAb

Candida species cause a variety of mucosal and invasive infections and are, collectively, the most important human fungalpathogens in the developed world. The majority of these infections result from a few related species within the “CUG clade,” sonamed because they use a nonstandard translation for that codon. Some members of the CUG clade, such as Candida albicans,present significant clinical problems, whereas others, such as Candida (Meyerozyma) guilliermondii, are uncommon in patients.The differences in incidence rates are imperfectly correlated with virulence in animal models of infection, but comparative anal-yses that might provide an explanation for why some species are effective pathogens and others are not have been rare or incom-plete. To better understand the phenotypic basis for these differences, we characterized eight CUG clade species—C. albicans, C.dubliniensis, C. tropicalis, C. parapsilosis, Clavispora lusitaniae, M. guilliermondii, Debaryomyces hansenii, and Lodderomyceselongisporus—for host-relevant phenotypes, including nutrient utilization, stress tolerance, morphogenesis, interactions withphagocytes, and biofilm formation. Two species deviated from expectations based on animal studies and human incidence. C.dubliniensis was quite robust, grouping in nearly all assays with the most virulent species, C. albicans and C. tropicalis, whereasC. parapsilosis was substantially less fit than might be expected from its clinical importance. These findings confirm the utility ofin vitro measures of virulence and provide insight into the evolution of virulence in the CUG clade.

Candida species are the most important fungal pathogens ofhumans and are collectively responsible for a vast number of

infections. These range from superficial mucosal infections suchas vulvovaginal candidiasis, and oropharyngeal thrush, to life-threatening infections such as disseminated hematogenous andinvasive candidiasis. These latter infections have steadily increasedin incidence in the last 30 years and are associated with a stub-bornly high mortality rate as a result of the underlying immuno-deficiency of the patients and inadequate diagnostics and treat-ments (1).

Of the approximately 150 species in the genus, 95% of infec-tions are caused by just four species: C. albicans, C. tropicalis, C.parapsilosis, and C. glabrata (2–4). C. albicans is the dominantspecies, representing about half of disseminated disease and aneven greater percentage of mucosal infections. Other clinicallyrelevant species include Clavispora lusitaniae (anamorph: Can-dida lusitaniae), M. guilliermondii (anamorph: Candida guillier-mondii), C. krusei, and C. dubliniensis, while D. hansenii (syn-onym: Candida famata) and Lodderomyces elongisporus aresubjects of rare clinical reports. Because the genus Candida ispolyphyletic, a better sense of evolutionary relationships comes inthe “CUG clade,” a grouping of species that use an alternativegenetic code in which that codon specifies serine rather than leu-cine (5, 6). The CUG clade encompasses all Candida species com-monly isolated from patients other than C. glabrata and C. krusei.

Within the CUG clade there is great diversity in both genotypeand phenotype. M. guilliermondii, C. lusitaniae, and D. hanseniiare haploid, while the others are diploid. Originally classified asfungi imperfecti, sexual cycles are slowly being identified for mostof these species (for a review, see reference 7). Most importantly,clinical incidence of these species is correlated, albeit imperfectly,with virulence potential in animal assays. A series of experimentstesting several Candida species in mice grouped C. albicans and C.tropicalis as the most virulent (infections with high inoculums

were lethal), followed by C. glabrata and C. lusitaniae (not lethal,but organisms persisted in organs), with C. parapsilosis, C. krusei,and M. guilliermondii as the least virulent, with at least some of theanimals clearing even high inoculums from the kidneys (8). This isbroadly consistent with a variety of other studies using subsets ofthese species in mouse models of disseminated or mucosal infec-tions or gastrointestinal colonization (9–13). It is notable that inthese models, C. parapsilosis is consistently less virulent thanwould be predicted from its clinical incidence.

A variety of phenotypes have been correlated with virulence inCandida species, primarily C. albicans, including hyphal growth,adhesion and biofilm formation, resistance to reactive oxygen andnitrogen stresses, use of nonfermentable carbon sources, modula-tion of macrophage functions, tolerance of a range of extracellularpH, and secreted protease and lipase activity (for a review, seereference 14). Presumably, the ability to cause disease in the mam-malian host is the product of a combination of these phenotypesand others, but there are only a few instances in which virulence-related phenotypes were examined systematically across multipleCUG species; for instance, sensitivity to peroxide was assayed foreight species, finding differences that were imperfectly correlated

Received 3 April 2015 Accepted 25 June 2015

Accepted manuscript posted online 6 July 2015

Citation Priest SJ, Lorenz MC. 2015. Characterization of virulence-relatedphenotypes in Candida species of the CUG clade. Eukaryot Cell 14:931–940.doi:10.1128/EC.00062-15.

Address correspondence to Michael C. Lorenz, michael.lorenz@uth.tmc.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00062-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/EC.00062-15

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with virulence (15). The phenotypic diversity both in vitro and inanimal models comes from a combination of genomic (gene con-tent) and regulatory (expression) variations among these species.The substantial genomic differences between these species havebeen analyzed primarily in silico (16–18), with interspecies com-parisons at a molecular level only beginning to appear.

We assayed eight species of the CUG clade for a variety ofhost-relevant phenotypes, including interactions with phago-cytes, morphology in multiple conditions, nutritional flexibility,and stress resistance. Although there is not a perfect correlationbetween these in vitro phenotypes and virulence, we found in gen-eral that the most pathogenic species have the highest growthrates in a variety of conditions, are most resistant to relevantstresses, and are the most robust when confronted by phago-cytes. These findings are an important contribution to the dis-section of virulence within this genus and will inform future mo-lecular, genomic, and proteomic studies within this clade.

MATERIALS AND METHODSStrains and media. The fungal strains used are listed in Table 1. For eachspecies, the strain chosen was the one used for the respective genomesequencing project, while several additional strains of C. parapsilosis wereobtained from G. Butler. Strains were propagated on standard yeast media(19), including YPD (1% yeast extract, 2% peptone, 2% dextrose) andYNB (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% dextrose).Media were adjusted as indicated in the text with various stress-inducingagents. SLAD medium (0.17% yeast nitrogen base, 10 mM ammoniumsulfate, 2% dextrose, 2% agar) was prepared as described previously (20).Strains were grown at 37°C except for D. hansenii, which grows poorly atthat temperature and was propagated at room temperature (25°C) in-stead.

Cell culture experiments used the RAW264.7 murine macrophage-like cell line (American Type Culture Collection), which was propagatedin RPMI with glutamine and HEPES (Invitrogen) supplemented with10% fetal bovine serum (FBS; Fisher/HyClone) and grown in a 5% CO2

environment.Stress sensitivity assays. To assess growth on various carbon sources,

strains were grown overnight in YPD at 30°C and then diluted to anoptical density at 600 nm of 0.1 in 8 ml of YPD or in YNB with 2% glucose,ethanol, lactate or Casamino Acids (CAA), or 1% acetate, present as thesole carbon source. Each medium was set to pH 6, except for the CAA

media, which was adjusted to pH 4. Cultures were grown with aeration at37°C for 24 h and monitored by determining the optical density at theindicated times. Aliquots of the CAA cultures were also used for pH mea-surements to assess alkalinization of the media, as we have reported pre-viously (21, 22). Assays were done at least in triplicate. Doubling timeswere calculated from the exponential growth phase, between 2 and 6 hafter inoculation.

Sensitivities to other stresses were assayed using a BioTek SynergyMXautomated plate reader in a 96-well plate format. Strains were grownovernight in YPD at 30°C, washed with water, and resuspended in 200 �lof YPD medium containing the relevant stressor. The plate was incubatedat 30°C with periodic agitation, and the optical density was measuredevery 10 min for up to 16 h. Each experiment had three replicates percondition, and this was repeated at least three times. Doubling times werecalculated for rolling 200-min periods every 10 min from 1 to 12 h. Thetime at which the peak growth rate was achieved differed somewhat byspecies and condition; the maximal division rate is reported.

Morphological characterization. For determination of cellular mor-phology in liquid inducing conditions, strains were grown overnight inYPD at 30°C, washed with water, and diluted into control YPD medium,RPMI (pH 7.4), or 10% FBS in water. After 1 to 4 h at 37°C, the cultureswere centrifuged briefly to concentrate the cells before photographingthem at �400 with an Olympus IX-81 inverted microscope.

Cellular and colony morphologies were assessed on solid mediumunder nitrogen starvation and embedded conditions. Colony morphol-ogy was observed on solid SLAD medium in petri dishes. For imaging ofcellular morphology, agar pads of the same media were prepared on stan-dard microscope slides. Highly diluted cultures were spotted to thesepads, grown for 48 h at 37°C, and imaged using a Zeiss Axiostar micro-scope fitted with a trinocular camera mount. Cells were embedded in anagar matrix by mixing an average of 100 to 250 cells in YPD top agar (0.5%agar) at 42°C in petri dishes. After solidifying, they were grown 4 days at37°C and photographed at two resolutions using an Olympus IX-81 in-verted microscope.

Fungal-macrophage coculture experiments. (i) Morphology. RAW264.7macrophages were seeded on glass coverslips in 12-well plates at a densityof 106 cells/well in 1 ml of RPMI plus 10% FBS and allowed to adhere for2 h at 37°C. Fungal strains were grown overnight in YPD, washed withwater, resuspended in phosphate-buffered saline (PBS), and counted us-ing a hemocytometer. Fungal cells were added at ratios of 1:1 to 2.5:1 (withhigher ratios used for the weaker pathogens) incubated for 1 h. Cocultureswere washed twice with PBS and then treated with 350 ng of calcofluor

TABLE 1 Fungal strains used in this study

Candida designation Teleomorph Strain Origin of strain

Reference(s)

For origina For genome sequenceb

Candida albicans None SC5314 “Disseminated” 67 16, 68Candida dubliniensis None CD36 Oral 69 48Candida tropicalis None MYA-3404 Blood 70 17Candida lusitaniae Clavispora lusitaniae ATCC 42720 Blood 71 17Candida guilliermondii Meyerozyma guilliermondiic ATCC 6260 Lungs 72 17Lodderomyces elongisporusd None NRRL YB-4239 Orange juicee 73 17Candida famata Debaryomyces hansenii CBS767 Unknownf 18Candida parapsilosis None CDC317 Skin 47 17

CDC173 Invasive 47CDC177 Invasive 47CLIB214 Feces 74

a That is, the reference for the report of the isolation of the strain.b That is, the reference for the report(s) of the complete genome sequence.c In some older literature, the teleomorph is named Pichia guilliermondii.d L. elongisporus has never been classified as a Candida species.e Isolation source per Centraalbureau voor Schimmelcultures for the alias CBS 2065.f Deposited at Centraalbureau voor Schimmelcultures by Carlsberg Laboratory, origin unknown.

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white/ml for 10 s to stain nonphagocytosed cells. After two washes withPBS, the cells were fixed with paraformaldehyde and permeabilized with0.1% Triton X-100. A rabbit polyclonal anti-Candida-FITC antibody (LS-Bio) was then used to stain and visualize both intracellular and extracel-lular fungi. The proportion of filamentous cells (hyphal or pseudohyphal)was ascertained by scoring morphology in photomicrographs.

(ii) Cytotoxicity. The ability of the fungal species to kill macrophageswas assessed through detection of lactate dehydrogenase (LDH) in theculture supernatant using the CytoTox 96 kit (Promega) as describedpreviously (22). RAW264.7 cells were seeded in 96-well plates at 2.5 � 105

cells/well and allowed to adhere overnight. Fungal overnight cultures werewashed and diluted in PBS, added to the macrophages at a 3:1 ratio, andincubated for 5 h before the supernatant was removed and assayed forLDH activity according to the manufacturer’s protocol. The ability of eachspecies to induce macrophage damage was expressed as a percentage ofthe total LDH released from chemically lysed cells.

(iii) NO suppression assay. RAW264.7 macrophages were seeded at6 � 105 cells/well in 12-well plates. Fungal species were prepared as de-scribed above and added to the wells at fungal cell/macrophage ratios of1:10 or 1:100. Concurrently, 100 ng of lipopolysaccharide (LPS)/ml and100 U of gamma interferon (IFN-�)/ml were added, and the cocultureswere incubated at 37°C in 5% CO2 for 24 h. Cell-free culture supernatantswere assayed for the presence of nitrite, which spontaneously forms fromnitric oxide in aqueous solutions, using Griess reagent, as described pre-viously (23). Assays were performed in triplicate.

Biofilm formation. To assess biofilm formation, we used a modifica-tion of an established assay to measure adherence to polystyrene plates(24). Fungal strains were grown to log phase in YNB plus 2% glucose,washed with PBS, and inoculated to 96-well plates at 106 cells/well. Theplate was incubated for 1.5 h with gentle shaking at 37°C. Subsequently,nonadherent cells were removed by aspiration and washing twice withPBS. Biofilms were allowed to develop in YNB plus 2% glucose for 24 h at37°C. Then the wells were washed twice with PBS and air dried for 45 minbefore adding 0.4% crystal violet for 45 min. After extensive washing, thewells were destained with 95% ethanol for 45 min. The destain was trans-ferred to a fresh plate, and the absorbance at 595 nm was recorded.

RESULTS

The CUG clade contains highly virulent (and intensively studied)species such as C. albicans and much less virulent and virtuallyuncharacterized species such as M. guilliermondii and L. elongis-porus. The phylogenetic relationships between these species aredepicted in Fig. 1 in which the uneven link between phylogeny andvirulence is apparent: C. albicans and C. tropicalis are significantpathogens, while C. dubliniensis is not. C. parapsilosis is frequentlyisolated clinically, L. elongisporus is not. Among the haploid spe-cies, C. lusitaniae, although relatively rare clinically, is nonethelessmuch more common than M. guilliermondii, which in turn ismore common than D. hansenii (25). Thus, we sought to add to

the existing bioinformatics comparisons based on genome se-quence and predicted protein content (17, 26, 27) by understand-ing the phenotypic differences that may contribute to virulence inthese species.

Utilization of different carbon sources. We began the pheno-typic assessment of these species by measuring growth rates instandard media. In aerated cultures at 37°C in either rich YPD orminimal YNB medium, both with 2% glucose as the primary(YPD) or sole (YNB) carbon source, each species grew rapidly(Fig. 2A), with the exception of D. hansenii, which grows verypoorly at 37°C and was omitted from most of the remaining as-says. C. tropicalis had the fastest doubling times in both conditions(53.6 and 59.0 min in YPD and YNB-glucose, respectively), grow-ing slightly more quickly than C. albicans and C. lusitaniae. C.parapsilosis was consistently the slowest growing species, with acell division time ca. 50% longer than that for C. tropicalis underoptimal conditions.

The acquisition and utilization of alternative nonfermentablecarbon sources such as lactate or amino acids has been proposedto be important in some host niches (21, 22, 28–30); C. albicansmutants lacking the ability to metabolize nonsugar compoundsare attenuated in mouse models (22, 31–34). Thus, we assayedgrowth rates in minimal YNB medium with lactate, acetate, oramino acids (in the form of Casamino Acids) as the sole carbonsource. C. albicans, C. tropicalis, and C. lusitaniae utilized aminoacids effectively as the carbon source (doubling times of 78.8 to88.5 min), while the growth of the other species was much slower(Fig. 2B). Growth was uniformly slower in the presence of lactateand acetate (Fig. 2B), with significant variations between species;the doubling times for C. tropicalis, for instance, were far faster inacetate-containing media than other species, but among the slow-est in the presence of lactate. Although all species metabolizedacetate, there was significant variability between experiments inthe lag time before growth began, particularly for C. lusitaniae andM. guilliermondii. C. parapsilosis was the slowest growing speciesunder all of these conditions.

Tolerance to common stresses. To assess sensitivity to varioushost-relevant stresses, including reactive oxygen and nitrogen spe-cies (hydrogen peroxide and the nitric oxide donor Deta-NONO-ate), osmotic stress (sorbitol and sodium chloride), pH (pH 2 to9), and arsenate, we used a 96-well plate format with an automatedplate reader (Fig. 2C; see also Fig. S1 in the supplemental mate-rial). Growth was generally slower than in the broth cultures butC. tropicalis (111.5 min), C. albicans (117.3 min), and C. lusitaniae(129.4 min) were again the fastest-growing species, followed by C.dubliniensis (159.9 min). The other three species, in contrast, grewless well in the 96-well plates, perhaps due to the more limitedaeration in this format. The doubling time for M. guilliermondiiwas more than eight times longer in the plates compared to brothculture (572.9 versus 68.7 min).

When stressors were added to YPD in the 96-well plate assays,the growth patterns changed markedly. C. albicans and C. dublini-ensis were relatively resistant to hydrogen peroxide, NONOate,arsenate (which can induce oxidative stress), and pH extremes(Fig. 2C and D; additional concentrations of stress agents areshown in Fig. S1 in the supplemental material), with doublingtimes within 2-fold of the control. The addition of 1 M sodiumchloride greatly slowed the growth of all species (Fig. 2C and D),although this was not due to osmotic stress, because none of thespecies were sensitive to 1 M sorbitol (see Fig. S1 in the supple-

FIG 1 Phylogenetic relationships between CUG species. Phylogeny was deter-mined using the nucleotide and protein sequences of core set of conservedorthologous genes using the MrBayes 3.1.2 tool (66). (Adapted from Nature[17].)

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mental material). While C. albicans was fairly resilient under thesestress conditions, the other species were sensitive to specific stres-sors; C. tropicalis, for instance, failed to grow in 10 mM peroxideor 1 mM arsenate and grew slowly at pH 2. In contrast, C. lusita-niae and C. parapsilosis were quite resistant to peroxide but acutelysensitive to reactive nitrogen species. Growth patterns in stressconditions are summarized in the heat map in Fig. 2D.

Modulation of extracellular pH. We have previously de-scribed a phenomenon in which C. albicans neutralizes acidic me-dia through the excretion of ammonia derived from the catabo-

lism of amino acids as a carbon source, and we observed thisoccurring in other Candida species as well, including C. glabrata(21, 35). Alkalinization occurs optimally in minimal medium withCasamino Acids as the sole source of carbon, a condition in whichall species grew fairly well (Fig. 2B); thus, we tested their ability toneutralize the medium (see Fig. S2 in the supplemental material).All seven species rapidly raised the extracellular pH from 4 toabout 7.5 in about 12 h and, while there were some differences inthe kinetics, this largely correlated with growth rates in this media.

Morphology. A hallmark of C. albicans is its polymorphic na-

FIG 2 Growth rates and stress sensitivities for CUG clade species. (A) The exponential doubling time, calculated as described in Materials and Methods, is shownfor each species grown at 37°C in aerated (flask) cultures in YPD. An asterisk indicates a significantly different (P � 0.01) doubling time relative to C. albicans.(B) Doubling times for strains grown in aerated cultures in YNB medium containing 2% glucose, Casamino Acids (CAA) or lactate, or 1% acetate. The asterisksrepresent species with a significantly (P � 0.01) reduced growth rate relative to C. albicans in the same media. (C) The doubling times for each species in a varietyof stress conditions are plotted. The strains were grown in 96-well plates in an automated plate reader at 37°C with intermittent shaking in YPD mediumcontaining no additives, 10 mM hydrogen peroxide (H2O2), 8 mM Deta-NONOate (NONOate), 1 M sodium chloride (NaCl), 1 mM arsenate, or media adjustedto pH 2 or 9. Dashes next to the vertical axis indicate no growth (a calculated doubling time greater than 800 min), and asterisks indicate a significant (P � 0.01)reduction in growth rate compared to the same species in the absence of stress. (D) Heat map depiction of the data from panel B in which boxes are shadedaccording to growth rates in the indicated condition relative to the no-stress control. Darker blue colors indicate lower growth rates (greater growth inhibitionby that stressor).

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ture and ample evidence indicates that the transition betweenmorphological forms is required for virulence (36, 37). Of theother species, only C. tropicalis and C. dubliniensis form true hy-phae, and they do this far less readily than C. albicans (38, 39). Wegrew each species in standard hyphal-inducing conditions, in-cluding in serum or in RPMI (at pH 7.4). Cellular morphologywas consistent with previously published results (39), with abun-dant hyphae seen in C. albicans, some hyphae in C. dubliniensis,and a mix of pseudohyphae and rarer true hyphae in C. tropicalis(see Fig. S3 in the supplemental material). All other species re-mained in the yeast form.

Nitrogen limitation induces pseudohyphal formation in manyyeast species, including C. albicans, C. tropicalis and S. cerevisiae(20, 39–41), so we tested each of the CUG species on solid lowammonia SLAD medium. As previously reported, C. albicansforms pseudohyphae on this medium as observed using a low-magnification stereomicroscope (Fig. 3). The filamentous growthof C. tropicalis and C. lusitaniae was surprisingly robust on thismedium. To assess cellular morphology, cells were incubated onthin films of SLAD prepared on microscope slides for 24 to 48 hand then analyzed by higher-resolution differential interferencecontrast imaging. Although the clarity is compromised by the agarsubstrate, pseudohyphal cells were seen at least occasionally for allspecies except M. guilliermondii (Fig. 3). Interestingly, the floridfilamentous growth of C. tropicalis cells was largely composed oftrue hyphae, as evidenced by the parallel cell walls, branched hy-phae, and absence of constrictions at septae. It even appeared toform aerial hyphae on SLAD (see Video S1 in the supplementalmaterial). C. tropicalis hyphal forms were far more common un-der nitrogen limitation than in other reported hyphal-inducingconditions.

C. albicans cells embedded in an agar matrix also form abun-dant hyphae, and this is largely independent of other inducingstimuli, occurring in rich YPD medium even at 25 to 30°C. Theseconditions also bypass the Cph1p and Efg1p transcription factorsclassically associated with hyphal growth in vitro and in vivo incertain animal models (42–44). We examined the morphology ofeach CUG clade species when grown embedded in 0.5% agar inYPD (Fig. 3). Although the three-dimensional nature of thesestructures presents a challenge to clear photography, matrix em-bedding stimulated filamentous growth in most of the species,with M. guilliermondii again excepted. C. albicans exhibited theclassic “beads-on-a-string” morphology with yeast cells buddingfrom the septa of hyphae that could extend for hundreds of mi-crons. Hyphal growth was also florid in C. tropicalis, but yeast cellswere rarely seen budding from hyphae; rather, extensive angularbranches produced a dense hyphal network. Pseudohyphal pro-jections were observed at various frequencies in the other species.

Interactions with macrophages. Avid hyphal growth of C. al-bicans is also common after phagocytosis by macrophages. Weinvestigated morphology after phagocytosis under standard con-ditions using cells labeled with fluorescein isothiocyanate (FITC)-concanavalin A (Fig. 4). One hour after initiation of the coculture,germ tube formation was apparent in a majority of C. albicanscells, whereas they were more rarely seen (and were shorter) in C.dubliniensis. Pseudohyphae, but not true hyphae, were sometimesseen in phagocytosed C. tropicalis cells and swollen and/or elon-gated cells of C. parapsilosis were observed rarely. The remainingspecies remained exclusively in the yeast form.

We quantitated the cellular morphology in phagocytosed ver-

FIG 3 Morphology of CUG species. To assess colony morphology under ni-trogen limitation, strains were grown on SLAD medium in standard petridishes for 4 days at 37°C before imaging with a stereomicroscope at �20magnification. To determine cellular morphology, strains were grown at 37°Cfor 48 h on SLAD agar pads on microscope slides before imaging at �400. Toassess colony morphology under embedded conditions, cells were diluted inYPD-top agar (0.5% agar) at �100 cells/plate, followed by incubation at 37°Cfor 5 days before imaging on the stereomicroscope at �40. Cellular morphol-ogy was determined by imaging the same plates using an inverted microscope.The scale bar (10 �m) in the lower right applies to the insets with the cellularimages.

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sus nonphagocytosed cells, which were distinguished by stainingfixed, nonpermeabilized cells with calcofluor white, which bindsto the cell wall of only nonphagocytosed fungal cells. As seen inFig. 5, the proportion of hyphal or pseudohyphal cells were signif-icantly higher in cells that remained in the media than those thatwere phagocytosed for C. albicans, C. tropicalis, and C. parapsilosis,indicating that the phagolysosomal environment inhibited somefilamentation (the difference was not statistically significant for C.dubliniensis). Filamentous forms were not observed for the otherspecies.

The robust filamentous growth of C. albicans has been pro-posed to damage macrophages through physical disruption of themembrane, although recent reports ascribe some of the lysis tofungus-induced pyroptosis (45, 46). To assess the ability of theCUG species to lyse macrophages, we utilized a standard assay thatmeasures release of host LDH into the medium. The fungal specieswere incubated with macrophages at a 3:1 ratio for 5 h beforesupernatants were assayed for LDH activity (Fig. 6). C. albicans, C.dubliniensis, and C. tropicalis induced nearly as much LDH releaseas chemically lysed macrophages (the positive control), whilemacrophage integrity remained high in cocultures with the other

species. The species that induce lysis are the only species to fila-ment to a significant degree inside macrophages (Fig. 4 and 5),reinforcing a link between morphogenesis and macrophage dam-age. However, the substantial variation in the proportion ofphagocytosed cells in the hyphal or pseudohyphal form was notreflected in differences in LDH release, suggesting that nonmor-phogenetic factors also contribute to lysis.

We recently reported that C. albicans cells modulate macro-phage function by suppressing the production of reactive nitrogenspecies via an unknown soluble, secreted factor (23). We testedhow broadly this activity was conserved in the CUG clade by as-saying NO production in 24-h cocultures. Supernatants were an-alyzed using the Griess reagent, which detects nitrite, a spontane-ous breakdown product of NO in aqueous cultures at neutral pH.As seen in Fig. 7, even at a fungus/macrophage ratio of 1:100, C.albicans and C. dubliniensis effectively suppressed NO production.The other species inhibited NO release modestly at the higherfungus/macrophage ratio, with C. tropicalis and, to a lesser extent,C. lusitaniae being the most effective of this second group.

Biofilm formation. The formation of biofilms on medical im-plants, such as venous catheters, is an important risk factor in

FIG 4 Morphology of phagocytosed cells. RAW264.7 macrophages were allowed to adhere to glass coverslips in 12-well plates and then cocultured with eachCUG species for 1 h at 37°C in 5% CO2. After washing, fixing, and permeabilization, the fungal cells were incubated with an �-Candida polyclonal antibodyconjugated to FITC before imaging. Scale bar, 10 �m.

FIG 5 Quantitation of the morphology of phagocytosed cells. Photomicro-graphs of fungi cocultured with RAW264.7 cells (representative examples areshown in Fig. 4) were scored as filamentous (pseudohyphal or hyphal) or yeast.At least three replicate experiments were performed, from which an aggregatetotal of at least 100 cells were counted. Asterisks indicate a P value of �0.01relative to nonphagocytosed cells.

FIG 6 Macrophage damage induced by CUG species. Macrophage membraneintegrity was estimated by assaying release of LDH from the cells as describedin Materials and Methods and expressed relative to the maximum amount ofLDH activity released from chemically lysed cells.

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disseminated candidiasis and poses significant obstacles to effec-tive therapy. C. albicans biofilms are highly polymorphic, and hy-phal morphogenesis is required for optimal biofilm formation. Toexamine biofilms formed by CUG clade species, we used a stan-dard assay that measures adherence to a polystyrene substrate inwhich biomass is estimated by the binding of the dye crystal violet,using two medium conditions: YNB (Fig. 8A) or RPMI (Fig. 8B)There was a clear distinction between three species that robustlyadhered to the polystyrene in both conditions as measured by theretention of crystal violet (C. albicans, C. dubliniensis, and C. tropi-calis) and the others, which did not (Fig. 8). Curiously, C. lusita-niae adhered to the plastic surface well when grown in YNB butnot when grown in RPMI (Fig. 8).

Strain variation in C. parapsilosis. Our data clearly group C.parapsilosis with the less virulent species, a significant discrepancyrelative to its clinical incidence. The sequenced reference isolateused here, CDC317, was isolated from the skin of a health careworker and, despite the implication of this strain in a hospitaloutbreak of invasive infection (47), it is possible that it is lessrobust in these host-related assays than other clinical isolates. Toaddress this question, we examined two strains isolated from in-vasive infections: one isolated from feces and another CDC317strain obtained from another laboratory in several of our assays(see Fig. S4 in the supplemental material). Although some strainvariation was seen in growth rates and stress sensitivity, all of theC. parapsilosis strains were far less robust than the C. albicans(SC5314) control. One strain, CLIB214, a fecal isolate, was fre-quently seen in a pseudohyphal form, both in RPMI (see Fig. S4 inthe supplemental material) and in YPD (not shown), and formedslightly more robust biofilms, but this had little effect on macro-phage interactions or stress tolerance. Notably, in the macrophagelysis assay, our original CDC317 isolate outperformed the other C.parapsilosis strains (see Fig. S4 in the supplemental material).These data suggest that our conclusions based on a single strain aregenerally warranted.

DISCUSSION

In this study, we have assessed the fitness of eight species of theCUG clade in a variety of in vitro and ex vivo assays often used as

proxies for virulence, including assays for resistance to host-asso-ciated stresses, hyphal morphogenesis, and interactions withphagocytes. Given the clinical significance of these species and thepotential for them to be used to understand the evolution and/ormechanisms of fungal pathogenesis, the lack of comparative stud-ies has been a hindrance. Taken together, our results correlate wellwith expectations based on animal models of virulence (8, 10, 12,13), with C. albicans and C. tropicalis as the most robust species,though C. tropicalis was notably sensitive to oxidative stress. Al-though C. tropicalis is usually cited as the third or fourth mostcommon cause of invasive infections (2, 4, 25), this species per-forms about as well as C. albicans in animal models, includingthose of disseminated hematogenous infections, gastrointestinalcolonization, and dissemination from the gastrointestinal tract (8,10, 12, 13). At the other end of the spectrum, M. guilliermondii, L.elongisporus and D. hansenii (when we were able to test it) weregenerally less stress tolerant and less fit when confronted withmacrophages, an observation consistent with their low clinicalimpact.

The robustness in our assays of three species—C dubliniensis,C. lusitaniae, and C. parapsilosis— deviated from expectationsfrom clinical incidence and animal models. C. dubliniensis, al-though closely related to C. albicans is a substantially weakerpathogen: only 24 cases of invasive infections from C. dubliniensiswere identified among over 4,000 isolates from candidiasis pa-tients (3). The disparity between the general fitness of this speciesand its low incidence may come from the absence of several par-

FIG 7 Inhibition of macrophage NO production by CUG species. RAW264.7macrophages were seeded in 96-well plates and activated by the addition of 100ng of LPS/ml and 100 U of IFN-�/ml. At the same time, the fungal species wereadded at the indicated ratios and incubated for 24 h. NO production wasassayed by detecting nitrite in the media using Griess reagent as described inMaterials and Methods. Nitrite concentrations are expressed as a percentage ofthat produced by LPS/IFN-�-stimulated macrophages incubated in the ab-sence of fungi. Negative values indicate nitrite concentrations lower than thebackground produced by resting (unstimulated) macrophages.

FIG 8 Biofilm adherence in CUG species. The adherence stage of biofilmformation was assayed in a 96-well polystyrene plate format in which bio-mass is estimated by retention of crystal violet, as described in Materialsand Methods.

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ticularly important virulence factors found in C. albicans, includ-ing the Als3 adhesin/iron acquisition protein, the Sap4-6 pro-teases, and the invasin Iff4 (48). Thus, a plausible mechanism forthe difference in virulence and clinical incidence between thesespecies is a combination of loss of specific genes (in C. dublinien-sis) with expansion of gene families that mediate host interactions(in C. albicans).

C. lusitaniae clustered with the more virulent species in somemeasures and with the less virulent ones in others. In general, itperformed well in assays more reflective of in vitro, laboratoryconditions, including general growth rates and morphogenesisunder nitrogen starvation, but was more sensitive to stresses andmacrophage contact. The biofilm assay is illustrative: C. lusitaniaeadhered well when grown in the minimal yeast media YNB but notwhen grown in the tissue culture medium RPMI. Further workwill be needed to understand the genetic mechanisms by whichthis species has adapted to environmental but not host niches.

In contrast, C. parapsilosis performed poorly in nearly everyassay, a finding consistent with published reports indicating that itis much less virulent than C. albicans in animal models (8, 9, 49),despite its clinical incidence. Although intraspecies strain varia-tion can be significant (see, for instance, references 50, 51, 52, and53), several additional C. parapsilosis species fared no better thandid CDC317, the sequenced reference isolate used in most of ourstudies. Another recent study that looked at a larger set of C.parapsilosis clinical isolates also concluded that, while there werephenotypic differences in vitro, the effects on host interactionswere modest (54). The discrepancy between the lab and the clinicmight be explained if C. parapsilosis were more common as a com-mensal or more easily transmitted from person to person, suchthat patients were exposed to it more frequently. Indeed, there issome evidence for this: C. parapsilosis is more commonly isolatedfrom skin than C. albicans, with one study identifying it as themost commonly isolated yeast species on hands (55–57; see alsoreference 58). A few studies have linked cases in neonatal intensivecare units to transmission from health care workers (59–61), in-cluding the type strain CDC317 (47). The prevalence of C. parap-silosis fungemia in neonates suggests that this population is par-ticularly susceptible to this species (3, 61, 62) and that the riskfactors for these patients may not be modeled effectively in vitro orin mouse models.

The adaptations to the host environment that contribute topathogenesis come from the many changes in gene content be-tween species, which has been addressed in bioinformatic detail(17), but not at a mechanistic level for the most part. They alsoderive from changes in patterns of regulation, which are only be-ginning to be understood. A recent analysis of transcription fac-tors in C. parapsilosis using a small library of mutant strains dem-onstrated significant rewiring of regulatory networks in biofilmformation and the hypoxic response relative to C. albicans (63). C.dubliniensis differentiates into hyphae less readily than C. albicans,partly because it lacks a close homolog of the hyphal regulatorytranscription factor Sfl2 (64). These regulatory changes, and likelymany others, contribute to phenotypic differences relevant to vir-ulence, including hyphal and biofilm formation and responses tooxidative and hypoxic stresses. These might be more subtle thanour assays can detect; indeed, C. dubliniensis does poorly whenincubated competitively with C. albicans in several broth and bio-film assays (63–65).

As for any complex trait, the ability to cause pathology in a

mammalian host is a multifactorial phenomenon, and the analysisof closely related species with vastly different inherent virulence isa potentially powerful tool for understanding which genetic andregulatory adaptations are particularly important. Our study di-rectly compares a large number of species across this spectrum innumerous host-related assays for the first time, providing greaterinsight into the evolution of virulence in this species complex.Further study will be needed to understand the mechanistic basisfor the phenotypic differences we observe and to determine howto exploit these to improve outcomes for patients.

ACKNOWLEDGMENTS

We are grateful to D. Soll, A. Brown, J. Heitman, and G. Butler for the giftof strains. We thank other members of the Lorenz laboratory, especially S.Vylkova, H. Danhof, P. Miramón, and E. Vesely for advice and helpfuldiscussions.

This study was supported by Public Health Service grant R21AI105651to M.C.L.

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