Regulation of Innate Immune Response to Candida albicans ... · Candida albicans is a common opportunistic fungal pathogen and is the leading cause of ... MATERIALS AND METHODS Candida

INFECTION AND IMMUNITY, Apr. 2011, p. 1546–1558 Vol. 79, No. 40019-9567/11/$12.00 doi:10.1128/IAI.00650-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Regulation of Innate Immune Response to Candida albicansInfections by �M�2-Pra1p Interaction�

Dmitry A. Soloviev,1* Samir Jawhara,1 and William A. Fonzi2

Joseph J. Jacobs Center for Thrombosis and Vascular Biology and Department of Molecular Cardiology, Cleveland Clinic,Cleveland, Ohio 44195,1 and Department of Microbiology and Immunology, School of Medicine,

Georgetown University, Washington, DC 200572

Received 17 June 2010/Returned for modification 9 July 2010/Accepted 6 January 2011

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungaldiseases in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is one ofthe main tasks of cells of the innate immune system, and in vitro evidence suggests that integrin �M�2 (CR3,Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using�M�2-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate thatneutrophils derived from mice deficient in �M�2 have a reduced ability to kill C. albicans and that the deficientmice themselves exhibit increased susceptibility to fungal infection. Disruption of the PRA1 gene of C. albicans,the primary ligand for �M�2, protects the fungus against leukocyte killing in vitro and in vivo, impedes theinnate immune response to the infection, and increases fungal virulence and organ invasion in vivo. Thus,recognition of pH-regulated antigen 1 protein (Pra1p) by �M�2 plays a pivotal role in determining fungalvirulence and host response and protection against C. albicans infection.

Candida albicans is an opportunistic pathogen, a pleomor-phic fungus existing in yeast or filamentous forms (19, 23).Although the yeast form can bind to gut mucosal membraneswith further colonization (20, 49), it is thought that the fila-mentous morphology provides some advantage during interac-tion with the mammalian immune system as a part of fungalanti-host defense, and the ability of C. albicans to rapidly andreversibly switch between yeast and filamentous morphologiesis crucial to its pathogenicity (16, 55, 65, 82). In recent years,Candida infections ranked as the fourth most common cause ofbloodstream infections and are the leading cause of life-threat-ening nosocomial fungal infections (1, 87). The risk of devel-oping opportunistic bloodstream infections is greatly increasedin patients who are severely immunocompromised. Candidastrains that are resistant to commonly used antimycotics haveemerged rapidly (72, 98). Therefore, dissecting its pathogenicmechanisms and the host response to C. albicans infections isof great importance.

The innate immune system provides the principle protectionagainst disseminated candidiasis. Polymorphonuclear leuko-cytes (PMNs) have been shown to be the primary componentsof the host’s innate immune defenses against Candida infec-tions (27, 59, 67). The most prominent receptors on leukocytesutilized in fungal or microbial recognition are the integrins ofthe beta-2 subfamily (62, 63). These cell surface receptorsmediate migration of leukocytes to sites of infection and ad-hesion to microorganisms with subsequent phagocytosis andkilling of many pathogens (12, 27, 62). Patients with defects inleukocyte phagocytic functions, such as individuals with the

rare (that is, ca. 1 in 106 people) hereditary disease, leukocyteadhesion deficiency 1 (LAD-1), which is characterized by thelow expression (mild LAD-1) or complete absence (severe) ofall four of the beta-2 integrins due to mutations in the �2 gene(4, 5, 34), are highly susceptible to wide range of bacterial andfungal infections (7, 50, 80). Staphylococcus aureus and Strep-tococcus spp. are the most common bacterial pathogens, andCandida spp. are the primary fungi isolated from patients withLAD-1. The candidal skin infections described in older publi-cations were reported to occur in ca. 16% of patients; Candidaesophagitis has also been frequently reported (6, 7). In threemore-recent reports, a total of 27 cases of LAD-1 patients weredescribed, all of which had infections of different etiologies: C.albicans infections in 13 patients (48%), 17 patients (63%)dead from infections, and in 6 cases (22% of all LAD-1 pa-tients or 46% of all cases of mortality) the cause of death wasfungal septicemia (44, 65, 71). In most cases, the degree ofseverity of these symptoms may be correlated with the level of�2 expression on the patients’ leukocytes. Generally, patientswith �1% normal levels of �2 are the most susceptible tofrequent and life-threatening systemic infections and usuallydo not survive to adulthood. Patients with higher levels ofexpression (up to 10% of normal) develop milder forms ofLAD-1 and may survive to adulthood with proper medical care(10, 86). The fungal invasion usually starts in newborns andtoddlers (severe LAD-1) or in children (milder) as recurrent,severe skin and soft tissue infections that tend to be necrotic,leading to colitis, otitis, pneumonia with spontaneous perito-nitis, and the formation of nodular and ulcerative lesions inlater stages, which ultimately lead to sepsis and death (44, 53,64). The principle beta-2 integrin involved in recognition ofbacterial and fungal pathogens is �M�2 (Mac-1, CD11b/CD18,and CR3) (37, 39). �M�2 is a pivotal adhesion receptor onPMNs, cells of the monocytoid lineage, subsets of T lympho-cytes, and NK cells (37, 38). The capacity of �M�2 to support

* Corresponding author. Mailing address: Joseph J. Jacobs Centerfor Thrombosis and Vascular Biology and Department of MolecularCardiology, Cleveland Clinic, Cleveland, OH 44195. Phone: (216) 445-8211. Fax: (216) 445-8204. E-mail: [email protected].

� Published ahead of print on 18 January 2011.

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leukocyte adhesion, migration, and phagocytosis depends uponits ability to recognize and mediate responses to a diverse setof structurally unrelated ligands, including fibrinogen (104),complement fragment iC3b (11), and intracellular cell adhe-sion molecule-1 (ICAM-1, CD54), as well as numerous bacte-rial lipoproteins and fungal mannans and �-glycans (101, 103).This cell surface receptor consists of two structurally distinctsubunits, �M and �2, which associate noncovalently. The �M

subunit is unique to this receptor, and the �2 subunit is sharedwith three other members of the beta-2 integrin subfamily:LFA-1 (�L�2), p150,95 (�X�2), and �D�2 (reviewed in refer-ences 43 and 52). Of the beta-2 integrins, �M�2 has beenspecifically implicated in the interaction of leukocytes with C.albicans: PMNs utilize �M�2 to adhere only to the filamentousform of C. albicans and not to yeast cells (36, 37, 39). Althoughother leukocyte receptors, Dectin-1 and Toll-like receptor 2(TLR2), which can bind fungal �-glucan (13, 68, 99), andmannan-binding TLR4 (69) also participate in fungal recogni-tion and are essential in leukocyte activation and eventually inactivation of �-2 integrins (42, 91), they do not directly mediateleukocyte migration and adhesion. Also Dectin-1 and TLR2recognize yeast forms of C. albicans only (41).

Recently, we identified C. albicans pH-regulated antigen 1protein (Pra1p) (84), also known as fibrinogen binding protein1 (Fbp1) (56) or C. albicans 58-kDa mannoprotein (mp58)(17), as the major ligand of �M�2 among C. albicans proteins(88). Pra1p is expressed predominantly on the surface of thehyphal form and not the yeast form of C. albicans (25, 66, 85).The expression of Pra1p is strongly pH dependent, requiring apH � 7, and is also regulated by nutrition and certain otherfungal genes (25, 79, 84). It is a mannoprotein, with carbohy-drate moieties accounting for 18 to 30% of its molecular mass(19, 25). In addition to fibrinogen, Pra1p also binds factor H,factor H-like protein, and plasminogen (58). Although smallquantities of Pra1p may be present on the yeast form of C.albicans, it becomes highly glycosylated only on the filamen-tous forms (hyphal and pseudohyphal forms) (18, 19), and wefound that sugar residues are important for recognition of C.albicans by �M�2 (39, 88). However, the biological role and thesignificance of the �M�2-Pra1p interaction in C. albicans viru-lence and host defense is unknown. The present study wasundertaken to determine the role and significance of the �M�2-Pra1p interaction in fungal pathogenicity and its effects on hostdefense in vivo using �M�2-deficient knockout (KO) mice andthe �pra1 strain of C. albicans in two distinct models of acutemurine candidiasis.

MATERIALS AND METHODS

Candida albicans strains. The C. albicans strains used were CAI-12 (controlstrain, iro1-ura3/IRO1-URA3), CAMB5-18 (�Pra1 strain, pra1::hisG/pra1::hisGiro1-ura3�/IRO1-URA3), and CAMB9 (“reintegrant” strain pra1::hisG/PRA1-URA3-pra1::hisG iro1-ura3/iro1-ura3). Strains CAI-12 and CAMB9 werepreviously described (77, 84). Strain CAMB5-18 was derived from strainCAMB435 by reversion of the iro1-ura3 deletion as previously described (77).The strains were routinely maintained on Difco Sabouraud dextrose agar (SDA)plates (Becton Dickinson, Sparks, MD). To evaluate the effect of PRA1 deletionon fungal cell morphology and germination, yeast cells of each strain wereresuspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 0.1 MHEPES (pH 7.4) and 10% non-heat-inactivated fetal bovine serum (FBS; At-lanta Biologicals, Lawrenceville, GA). Serial dilutions from 104 to 102 cells/ml in50-�l aliquots were grown in 96-well untreated plastic plates at 37°C in humid-

ified air and 5% CO2. To detect changes in the cell morphology, the sampleswere examined microscopically at 60-min intervals.

Animals. �M knockout (KO) mice (��M�2) were kindly provided by ChristyM. Ballantyne of Baylor College of Medicine, Houston, TX, and have been usedin several studies (see, for example, references 28, 57, 76, and 81). These micewere backcrossed for more than 12 generations into a C57BL/J6 background,and all mice used were genotyped by PCR of blood DNA samples. Age-matchedC57BL/J6 mice purchased from Jackson Laboratories (Bar Harbor, ME) wereused as controls (WT mice). All protocols involving mice were approved by theInstitutional Animal Care and Use Committee in accordance with Public HealthService policy, the Health Research Extension Act (PL99-158), and ClevelandClinic policy. All murine experiments involving C. albicans infections were car-ried out in a BSL2 facility of the Cleveland Clinic Biological Resource Unit. Themice were maintained on a 12-h alternating light-dark cycle and supplied withfood (Diet #2918; Harlan Teklad, Madison, WI) and sterilized water ad libitum.

Murine PMN isolation. Peritoneal PMNs were isolated from murine perito-neum lavage of ��M and WT mice, both males and females, as describedpreviously (75) using intraperitoneal thioglycolate as an inflammatory stimulus torecruit the cells. At 6 h after injection, the mice were euthanized by CO2

inhalation, the peritoneal cavity was opened, and leukocytes were harvested bywashing the cavity with 4 ml of sterile ice-cold phosphate-buffered saline (PBS).The cells were centrifuged (10 min at 2,250 � g), counted with a hemacytometer,and suspended in RPMI 1640 medium (15, 94). The cells harvested in thismanner were �95% PMN. Peripheral blood PMNs were isolated from pooledheparinized murine blood taken from tail veins of WT or ��M mice, both malesand females, by centrifugation through Ficoll-Hypaque, followed by 6% dextransedimentation; the contaminating erythrocytes were removed by hypotonic lysis(102). The cells obtained by this technique were �90% PMNs (21, 33, 106). Foreach experiment, PMNs from five to seven mice were pooled.

Cell adhesion assays. To determine cell adhesion to the fungus, 48-well Costartissue culture plates (Corning, Corning, NY) were precoated with 500 �l of 0.5%polyvinylpyrrolidone (PVP; Sigma-Aldrich) for 1 h at room temperature andwashed with Hanks balanced salt solution (HBSS), and aliquots of 5 � 105 C.albicans yeast of either the CAI-12, the �Pra1, or the reintegrant strain in 0.5 mlof RPMI 1640 medium, containing 1% FBS, were added, followed by incubationovernight at 37°C to germinate. The supernatant was removed, and adherentfungi were carefully washed with 0.5 ml of HBSS. In some experiments, wellswere coated overnight with 200 �l of 10 �g/ml of purified iC3b, ICAM-1 (Cal-biochem/EMD, Darmstadt, Germany), or Fc fragments of murine IgG andpostcoated with 0.5 ml of 0.5% PVP without the addition of the fungus. MurineFc fragments were prepared by digestion of mouse IgG (Sigma) with an immo-bilized papain-based Fab preparation kit (Pierce/Thermo, Rockford, IL) withfurther separation on protein A-agarose. A total of 105 peritoneal PMNs orhuman embryonic kidney cell line 293 expressing �M�2 on their surfaces(HEK293/�M�2 cells [36, 39, 90]) were added in 200 �l of HBSS–20 mM HEPES(pH 7.4), and assay plates were incubated at 37°C for 30 min. Control wells werecoated with PVP only. Each experimental point was tested in triplicate. Subse-quently, plates were washed three times with PBS, and the number of adherentcells in each well was quantified by using a CyQuant cell proliferation assay kit(Molecular Probes, Eugene, OR) as previously described (88, 89). The data fromcell adhesion and migration (see below) analyses are presented as percentages(mean � the standard error [SE]) of total cells (to which was assigned the valueof 100%) and represent the results of three independent experiments.

Cell migration assays. Peritoneal PMN or HEK293/�M�2 cell migration assayswere performed in serum-free Dulbecco modified Eagle medium/F-12 medium(Invitrogen) using modified Boyden chambers (Costar transwell inserts in 24-wellplate format; Corning) with tissue culture-treated polycarbonate filters with3-�m pores (14) as previously described (40, 74, 88, 89). Briefly, the lowerchambers contained 600 �l of medium with 5 � 105 C. albicans yeast of theselected strain, which were germinated overnight prior to beginning the analyses.Alternatively, purified iC3b, ICAM-1, or murine IgG Fc fragments at 10 �g/mlwere used as ligands. The upper chambers contained final volumes of 200 �l ofPMN or HEK293/�M�2 cell suspensions. The assays were initiated by addition of50 �l of cell suspension (105 cells/well) to the 150 �l of medium in the upperchambers, and the plates were placed in a humidified incubator at 37°C in 5%CO2 for 16 h. After migration, nonmigrated cells were removed from upperchamber of transwell inserts by using cotton swabs, and the migrated cellspresent on the undersurface of the membrane, as well as in the lower chamber,were quantitated by using a CyQuant cell proliferation kit as described above andpreviously (74, 89).

PMN oxidative burst assay. Superoxide anion production by murine WT and��M PMNs were determined by modification of the method of Colin and Mon-teil (22). Briefly, CAI-12 or �Pra1 C. albicans samples were allowed to germinate

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overnight at 37°C in 96-well TC Costar plates (5 � 104 yeasts in 100 �l of RPMI1640 medium per well). Then, 2 � 104 peripheral blood PMNs, WT or ��M, wereadded in 100 �l of HBSS-HEPES buffer (pH 7.4) containing 2 mM CaCl2 and 2mM MgCl2. In some experiments, PMNs, before addition to the assay wells, werepreincubated with 1 �M granulocyte-macrophage colony-stimulating factor(GM-CSF; Sigma-Aldrich) in the presence of 1 �M formyl-Met-Leu-Phe (fMLP)for 15 min for complete activation (31, 32, 51, 102). After incubation of the platesfor 30 min at 37°C in 5% CO2, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) was added to a final concentration of 10 �M. Subsequently,the plates were incubated for an additional 20 min at 37°C, and the fluorescencewas read with a CytoFluor II fluorometer (Applied Biosystems, Carlsbad, CA),using wavelengths of 485 nm for excitation and 535 nm for emission. As controls,wells coated with 0.5% PVP instead of C. albicans were used. Backgroundfluorescence from C. albicans was subtracted, and the data were normalized tothe peroxide concentration, obtained from peripheral blood PMNs of WT miceafter stimulation with GM-CSF/fMLP in the absence of C. albicans, which wasassigned the value of 100%.

PMN degranulation assay. To compare WT and ��M PMN degranulation inresponse to C. albicans, the level of intracellular �-glucuronidase retained inPMNs granules upon activation was determined by modification of describedmethods (57, 93). C. albicans (107) of either CAI-12 or �Pra1 strains wereallowed to germinate overnight in 1 ml of RPMI 1640 medium; 106 peritoneal orperipheral blood PMNs, either WT or ��M derived, were then added in 0.5 mlof HBSS-HEPES (pH 7.4) containing 2 mM CaCl2 and 2 mM MgCl2, followedby incubation at 37°C in 5% CO2 for 30 min. After the incubation, cell-fungusmixtures were centrifuged, the pellets were resuspended in 0.5 ml of 0.2% Tween20 to lyse leukocytes, and fungi, along with cell debris, were removed by centrif-ugation. The clarified lysates (100 �l) were added to 100 �l of 5 mg of phenol-phthalein glucuronic acid (pH 4.6; Sigma-Aldrich)/ml. The mixtures were incu-bated at 37°C for 16 h, and the reaction was stopped by adding 1 ml of 1 Mglycine (pH 10.4); the absorbance was read at 540 nm with a spectrophotometer.In some experiments, PMNs before addition to C. albicans were preincubated for10 min with 1 �M GM-CSF and 1 �M fMLP (32, 51). As controls, PMNs withoutcoincubation with C. albicans were used. The data obtained from nonstimulatedWT peripheral blood PMNs in the absence of C. albicans or other activators wereassigned a value of 100% granule retention.

PMN-mediated C. albicans killing/phagocytosis ex vivo assays. C. albicansyeast forms (106) of the selected strains in 0.25 ml of high glucose RPMI 1640containing 0.1 M HEPES (pH 7.8) were allowed to germinate in plastic tubes at37°C for 1 h with slow agitation. The fungal cells were collected by centrifugation,washed twice with PBS, suspended in 0.25 ml of HBSS-HEPES (pH 7.4) andmixed with 3 � 106 PMNs in 0.25 ml (1:3 ratio). The leukocyte-fungal mixtureswere incubated at 37°C with slow shaking for 1 to 5 h. To determine the extentof killing and/or phagocytosis, aliquots of the peritoneal PMN-C. albicans sus-pension diluted with HBSS-HEPES were plated in serial dilutions on SDAplates, and the CFU were counted manually on day 2 using a Bel-Art Productscolony counter. PMNs were not lysed before plating, and all fungal cells thatremained ingested were recorded as “killed.” Obtained results were indepen-dently verified by a modification of the method of Lehrer et al. (54, 88).Briefly, at experiment endpoint the equal volume of 1% Tween 80 was addedto the fungal-leukocyte mixture, lysed leukocytes were removed by centrifu-gation, and C. albicans cell pellets were resuspended in 0.25 ml of 2.5 mMmethylene blue (Sigma) in HBSS-HEPES. The number of viable (nonstained)cells was counted in a hemacytometer using a microscope. Control samplescontained C. albicans incubated without PMNs. The results obtained by bothmethods of quantitation of fungal viability showed good correlation withvariance in the 5 to 10% range.

Clearance of intraperitoneal C. albicans. To determine the clearance of intra-peritoneal C. albicans, we used a modification of the method used previously todetermine intraperitoneal clearance of S. aureus (35). To initiate acute perito-neal sepsis, 105 C. albicans of CAI-12, �Pra1p, or reintegrant strains wereallowed to germinate 1 h in RPMI 1640 medium at 37°C and injected intraperi-toneally in 0.1 ml of RPMI 1640 into WT and ��M mice. After 6 h, mice wereeuthanized, and peritoneal lavage was collected by gently washing the peritonealcavity with 4 ml of ice-cold PBS. In pilot experiments, we found that we were notable to recover total Candida cells from the peritoneum after 6 h of incubation,and a significant amount of fungi remained adherent to the peritoneal epitheliumafter lavage with PBS. To elute C. albicans adherent within the peritonealcavities, we took advantage of the ability of detergents such as Tween to solu-bilize mammalian cell membranes and membrane proteins. After the initiallavage with PBS, the peritonea of some mice were lavaged a second time with 4ml of PBS containing 0.5% Tween 80. All lavages were subsequently platedseparately in serial of dilutions onto SDA plates to enumerate CFU. To ensure

the complete recovery of adherent fungi from the peritonea by this lavageprocedure, as much of the murine peritoneal epithelia as possible was removedfrom all mice with or without the various lavage protocols and then homogenizedin PBS containing Tween 80; serial dilutions were plated onto SDA plates forCFU quantitation. Absence of the fungal CFU in Tween-lavaged peritonea wastaken as evidence of complete fungal recovery by the PBS-Tween lavage.

Leukocyte recruitment. Total PMNs recovered in a PBS intraperitoneal lavageafter intraperitoneal C. albicans injection were quantified enzymatically as pre-viously described (15, 94, 96). Briefly, 0.5 ml of the lavage fluid was diluted with0.5 ml of PBS containing 2% Triton X-100 to lyse leukocytes. PMNs werequantified spectrophotometrically by measuring the myeloperoxidase activityusing guaiacol (Sigma-Aldrich) as a substrate by determining the absorbance at470 nm. As independent verification, total leukocytes (predominantly PMNs andmacrophages) were also determined spectrophotometrically by intracellular es-terase activity in the lavage lysates using p-nitrophenyl butyrate (Sigma-Aldrich)as a chromogenic substrate measuring the absorbance at 405 nm (73, 94). Theamount of resident peritoneal leukocytes, determined from the activities of theseenzymes in the absence of C. albicans administration, was assigned a value of100%.

Murine model of systemic disseminated candidiasis. The model of systematiccandidiasis, described and applied to C57/BJ6 by Costantino et al. (24) and Zhaoet al. (107), was used. Unless stated otherwise, for each experimental point sevento nine mice at 8 to 12 weeks of age and weights in the range of 18 to 20 g wereused. Mice were injected with various dosages from 5 � 104 to 5 � 105 of theselected C. albicans strain: CAI-12 (control strain), CAMB5-18 (�Pra1p), orCAMB9 (the reintegrant strain) in 0.1 ml of sterile saline through a tail vein.Mice were returned to cages and monitored. To determine the degree of distress,we developed a scoring system, similar to one described for the determination ofhumane endpoints in a murine model of leukemia (2). The following symptomswere evaluated and scored as follows: coma, 15 points; weight loss of 20% (15points), 15% (11 points), or 10% (8 points); abdominal swelling, 6 points; sig-nificant decrease in mobility, 4 points; and hunched posture, 3 points. Eachgroup was monitored daily over the 30-day period, at approximately the sametime each day, and the score was nonaccumulative and was recalculated for eachmouse every 8 � 1.5 h. The primary endpoint was the number of mice survivingon day 30 within each experimental group. When mice scored 15 or more pointsbefore 30 days, the animals were euthanized. In the 76 mice analyzed, 79%exhibited a 20% weight loss, 18% exhibited a 10% weight loss plus an additionalscoring value, and 3% developed coma. After euthanasia for all causes, the micewere subjected to pathological examination. To determine the extent of C.albicans invasion and fungal burden in individual organs, the livers, spleens,hearts, lungs, and kidney were harvested, weighed, and homogenized in 5 ml ofPBS, and serial dilutions of the homogenates were plated onto SDA plates forCFU quantitation. The variability (standard deviation [SD]) in the recovery of C.albicans from infected tissues within the same mice group was �30%.

Statistical analyses. Statistical significance was determined by using a pairedStudent t test (for in vitro migration, adhesion degranulation, and superoxide ionproduction assays), analysis of variance (ANOVA; for in vitro killing and organfungal burden assays), log-rank and Cox regression (for mice survival analysis)—all are from statistical package of SigmaPlot (version 10.0; Jandel ScientificSoftware, Chicago, IL). A Wilcoxon-Mann-Whitney rank-sum U-test (WMWU-test) for murine survival analysis was performed by using online calculationtools (www.psychol-ok.ru/statistics/mann-whitney/) and tables of critical values(www.statanalyse.org). The difference in the two groups was considered signifi-cant with P � 0.05. The data are expressed as means � the SD unless otherwisenoted.

RESULTS

To demonstrate the biological importance of the �M�2 andPra1p interaction in fungal recognition and as a prelude tosubsequent in vivo studies, the interaction of PMNs derivedfrom C57BL/J6 (WT mice) or �M�2-deficient mice (��M mice)with C. albicans strains expressing or lacking Pra1p was exam-ined. Previous studies had demonstrated that deletion of �M�2

does not affect expression levels of the other �2 integrins onPMNs (28, 29, 57). Furthermore, it was shown that ��M PMNswere still able to emigrate through endothelium (28, 48, 57)and displayed similar levels of recruitment into the peritonealcavity in thioglycolate-induced inflammation model to WT

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mice (48, 57). However, some PMN functions associated with�M�2, such as adherence to fibrinogen (95), iC3b-mediatedphagocytosis (70), and degranulation, were impeded (28, 57).Therefore, in all ex vivo experiments we included additionalcontrols to examine the impact of �M�2 ablation on relevantfunctions of ��M leukocytes.

Effect of �M�2 and Pra1p elimination on PMN adhesion toC. albicans. In the first set of experiments, peritoneal PMNsfrom either WT or ��M mice were added to plates with ger-minated C. albicans cells of CAI-12 or �Pra1 strains, andadhesion was quantified after 30 min of incubation. Both WTPMNs and HEK293/�M�2 cells adhered well to WT fungus.Disruption of the PRA1 gene ablated adhesion of the WTPMN (P � 0.05, Student t test), while reinsertion of PRA1restored adhesion of the WT PMNs to the fungal substrate(P � 0.1). In contrast, PMNs derived from the �M�2-deficientmice showed little adhesion to C. albicans (�90% reductioncompared to the WT PMNs, P � 0.05), and the presence orabsence of Pra1p had virtually no effect (Fig. 1A). In controlexperiments we compared adhesive properties of WT and ��M

PMNs to a panel of ligands for leukocyte receptors that play a

significant role in the immune response to infections: iC3b(ligand for �M�2) (97), ICAM-1 (ligand for both �M�2 and�L�2) (26, 61), and purified murine IgG Fc fragments (IgG-Fc,ligand for leukocyte Fc receptors) (108); these results werealso verified with �M�2-expressing HEK293 cells (HEK293/�M�2 cells). As expected, PMNs from WT mice exhibitedstrong adhesion to all ligands tested. In contrast, ��M-PMNsshowed adhesion to ICAM-1 and IgG-Fc at a level similar toWT PMNs (P � 0.05) but were not able to adhere to aM�2

ligand iC3b, whereas HEK293/�M�2 cells adhered to all of the�M�2 ligands, iC3b and ICAM-1, but not to murine IgG-Fc(Fig. 1B). In additional experiments, we microscopically exam-ined �M�2-Pra1p interaction by microphotography of coincu-bation of CAI-12 and �Pra1 C. albicans strains, germinatedand labeled on mannose with fluorescein isothiocyanate withpurified �M�2, labeled with phycoerythrin (PE). Existing lit-erature indicates that leukocytes can bind to germ tubes andto hyphae of the filamentous form of C. albicans but gener-ally not to the yeast form (36, 39) and that Pra1p is alsopredominantly expressed on the surfaces of these C. albicansforms (25, 85). Consistent with previous findings (39, 88),

FIG. 1. Effects of Pra1p and �M�2 on the adhesive and migratory functions of PMNs in vitro. In each panel, asterisks indicate P � 0.05, ascalculated by an ANOVA test. (A) Effect of Pra1p and �M�2 on PMN adhesion to C. albicans. PMNs (105) from WT mice (left group of bars),��M mice (center group of bars), or HEK293/�M�2 cells (right group of bars) were added to the wells containing the indicated strains ofgerminated C. albicans of CAI-12 (black bars), �Pra1p (white bars), or Pra1p-reintegrant (hatched bars) strains as adhesive substrates. After 30min, nonadherent cells were removed by washing, and adherent cells were lysed and reacted with a DNA-binding fluorescent dye. The amount ofadherent cell was found by using a standard curve obtained from known numbers of the cells stained under the same conditions. As a control, PMNadhesion to wells coated with 0.5% PVP was used. The results are presented as the percentile of adherent cells and are expressed as means � theSD of three independent experiments with triplicates in each experiment. (B) Adhesion of PMNs and HEK293/�M�2 cells to a panel of leukocyteligands. PMNs or HEK293/�M�2 cells (all 105) were added to plates coated with 10 �g of the indicated protein/ml. After 30 min, nonadherent cellswere removed by washing, and adherent cells were stained with a DNA-binding fluorescent dye. As a control, PMN adhesion to wells coated with0.5% PVP was used. The results are presented as the percentile of adherent cells, and expressed as mean � the SD of three independentexperiments with triplicates in each experiment. (C) Effect of Pra1p and �M�2 on PMN migration to C. albicans. PMNs or HEK293/�M�2 cells (105)were allowed to migrate overnight across a polycarbonate membrane with 3-�m pores (5 �m in the case of HEK23/�M�2) to the C. albicans strainsindicated as in panel A. After incubation, migrated cells were lysed and stained with a DNA-binding fluorescent dye. The amount of adherent cellwas found using standard curve obtained from known numbers of the cells stained under the same conditions. The results are presented aspercentile of migrated cells and are expressed, after subtracting the background migration, as the mean of three independent experiments withtriplicates in each experiment. (D) Migration of PMNs and HEK293/�M�2 cells to the panel of leukocyte ligands. PMNs or HEK293/�M�2 cells(105) were allowed to migrate overnight with 3-�m pores (5 �m in the case of HEK23/�M�2) to 5-�g/ml leukocyte ligands indicated as in panelB. After incubation, migrated cells were lysed and stained with a DNA binding fluorescent dye. The results are presented as the percentile ofmigrated cells and, after subtracting background migration, expressed as means � the SD of three independent experiments with triplicates in eachexperiment.


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�M�2 binds only to germ tubes and hyphae but not to budforms of C. albicans, and Pra1p elimination completely ab-rogates �M�2 binding to the fungus (Fig. 2). These resultsare also consistent with previous reports that Pra1p is ex-

pressed predominantly on the surface of filamentous but notyeast forms of C. albicans (25, 66, 85). Thus, both �M�2 andPra1p are essential for the productive adhesion of murinePMNs to C. albicans.

FIG. 2. Immunofluorescent staining of germinated CAI-12 and �Pra1 C. albicans strains with isolated and PE-labeled �M�2. C. albicans of WT(A) and �Pra1 (B) strains were allowed to germinate in RPMI 1640 for 2 h. Purified and biotinylated �M�2 (the method of �M�2 isolation wasdescribed in detail in Soloviev et al. [88]) was added at 1 �g/ml, and the samples were incubated for 1 h at 37°C. After incubation, the fungi werewashed with D-PBS and incubated with R-phycoerythrin-streptavidin conjugate for 30 min at room temperature. Subsequently, the fungi wereagain washed with D-PBS, fluorescein isothiocyanate-GNL specific for the terminal �-D-mannosyl group was added, and the mixture was incubatedfor an additional 1 h. The cells were washed with D-PBS and observed by fluorescence microscopy (Leica Microsystems AG) at an opticalmagnification of �800.


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Effect of �M�2 and Pra1p deletion on C. albicans-mediatedPMN migration. Our prior studies showed that in addition toits adhesive properties, soluble Pra1p acts as a chemoattractantof PMNs (88). The need for �M�2 engagement in this processwas demonstrated by testing the migration of WT and ��M

PMNs toward germinated cells of C. albicans. WT PMNs mi-grated well to the fungus (Fig. 1C), whereas the ��M PMNsshowed little propensity to migrate (7% � 5% compared toWT PMN migration). In the presence of the pra1 deletionmutant, neither WT nor ��M PMNs demonstrated any migra-tion over background (5% � 5% and 3% � 5%, (P � 0.05,Student t test), compared to WT PMN migration to control theCAI-12 C. albicans strain, respectively. PRA1 reinsertion intothe C. albicans mutant restored the fungal attraction of WTPMNs, with migration occurring at the level observed withCAI-12 C. albicans and WT PMNs (95% �12% of WT, P �0.5), but did not affect the migration of ��M PMNs (6% �5%). These results were reinforced using HEK293/�M�2 cells(90). The �M�2-expressing cells adhered and migrated to thecontrol CAI-12 and reintegrant C. albicans strains but not tothe �Pra1p strain (Fig. 1C). In control experiments, ��M

PMNs were not able to migrate to iC3b, whereas the ��M

PMNs demonstrated migration similar to WT PMNs towardICAM-1 or IgG-Fc (P � 0.1) (Fig. 1D). Since ��M PMNsretain the ability to adhere and migrate to other ligands (seereferences 28 and 57), we conclude that adhesion and che-motaxis of PMNs toward C. albicans cells depends specificallyon Pra1p-�M�2 interaction.

Effect of �M�2-Pra1 interaction on PMN oxidative burst.Previous studies had shown that, unlike bacterial and someother fungal pathogens, successful killing/phagocytosis of C.albicans by PMNs requires the oxidative burst, and mice defi-cient in myeloperoxidase demonstrated significantly impaireddefense against C. albicans infections (8, 9). In a subsequentseries of experiments, we compared the effects of Pra1 and/or�M�2 elimination on superoxide ion production (oxidativeburst) using peripheral blood PMNs (102). As a positive con-trol, WT PMNs were activated with GM-CSF/fMLP, an �M�2-independent activation mechanism (32, 47, 83), and their levelof superoxide production was assigned a value of 100%. Asshown in Fig. 3A, WT and ��M PMNs upon activation withGM-CSF/fMLP demonstrated similar level of peroxide pro-duction (P � 0.05, Student t test). Incubation with controlCAI-12 strain of C. albicans affected only WT PMNs; peroxideproduction increased from 8% � 5% to 78% � 18% (P � 0.01,Student t test). In contrast, incubation with �Pra1 C. albicanshad little effect on the oxidative burst: PMNs of both linesincreased peroxide production by 10% only. The results indi-cate that despite the lack of �M�2, the PMNs are still capableof peroxide production upon activation by �M�2-independentagonists, and �M�2-Pra1 interaction plays an essential role inoxidative burst in response to C. albicans.

Effect of �M�2-Pra1 interaction on neutrophil degranula-tion. Degranulation of granulocytes upon contact with a patho-gen is an important mechanism of the innate immune responsefor destruction of pathogens. In our experiments, the extent ofPMN degranulation in response to C. albicans was determinedas the extent of intracellular retention of �-glucuronidase(�-GU) upon encounter with the fungus. The �-GU level inunstimulated peripheral blood PMNs from WT mice was as-

FIG. 3. Effect of �M�2 or Pra1p elimination on peripheral bloodPMN secretory functions. The values obtained with control murineWT PMNs are presented as black bars, and the values obtained withmutant ��M PMNs are presented as white bars. In each panel, aster-isks indicate P � 0.05, as calculated by ANOVA. (A) Effect of �M�2and Pra1p elimination on peripheral blood PMN oxidative burst.Peripheral blood PMNs (2 � 104) were incubated with germinatedCAI-12 or �Pra1 C. albicans or with the non-�M�2-dependent activa-tor GM-CSF/fMLP. Subsequently, peroxidase substrate 2,7-dichloro-dihydrofluorescein diacetate was added, and the fluorescence of thesamples was measured. For control, PMNs incubated 30 min withoutGM-CSF or C. albicans was used. Background fluorescence from C.albicans was subtracted, and the data were normalized to the peroxideconcentration obtained from peripheral blood PMNs of WT mice afterstimulation with GM-CSF/fMLP in the absence of C. albicans, whichwas assigned the value of 100%. The data are expressed as means ofperoxide production � the SD of two independent experiments withtriplicates in each experiment. (B) Effect of �M�2 and Pra1p elimina-tion on peripheral blood PMN degranulation. Peripheral blood PMNs(107) were incubated 30 min with germinated CAI-12 and �Pra1 C.albicans strains or with GM-CSF/fMLP. After incubation, sampleswere pelleted, PMNs were lysed with 0.2% Tween, and the fungi andcell debris were removed by a second centrifugation. Subsequently,phenolphthalein glucuronic acid was added, and samples were incu-bated overnight. The reaction was stopped with glycine (pH 10.4), andabsorbance was read at 540 nm. As controls, PMNs were used withoutC. albicans or GM-CSF/fMLP coincubation. The data obtained fromnonstimulated WT peripheral blood PMNs in the absence of C. albi-cans or agonists were assigned a value of 100% �-GU granule reten-tion. The data are expressed as mean of �-GU granule retention � theSD of two independent experiments with triplicates in each experi-ment.


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signed the value of 100% �-GU, corresponding to 0% degran-ulation. Upon activation with GM-CSF/fMLP, both WT and��M blood PMNs demonstrated a substantial loss of intracel-lular �-GU, to 22% � 8% and 27% � 6%, respectively (P �0.05, Student t test) comparing to �-GU levels in unstimulatedPMNs (Fig. 3B). These results demonstrate that ��M PMNscan release their granule contents in response to agonists.After incubation with CAI-12 C. albicans, �-GU in WT PMNsdecreased to 39% � 11% (P � 0.05), whereas the response toCAI-12 by ��M PMNs was insignificant (98% � 16%, P �0.05). In contrast, �Pra1 fungus had no effect on �-GU releaseby either WT or ��M PMNs: 94% � 18% and 97% � 19%(P � 0.05) for WT and ��M PMNs, respectively (Fig. 3B).

Effect of �M�2 and Pra1p on PMN-mediated killing of C.albicans. Fungal killing by PMNs paralleled their adhesive in-teractions. PMN from WT and ��M mice were incubated for6 h with germinated C. albicans of all three strains, and thenumber of viable cells was quantified as CFU. Under theseconditions, only 32% � 14% of CAI-12 C. albicans survivedafter incubation with WT PMNs. In contrast, 88% � 14% ofthe �Pra1 C. albicans strain remained viable (P � 0.05, Stu-dent t test). Restoration of Pra1p in the reintegrant strainreimposed susceptibility to PMN killing, 42% � 16% viability,a finding similar to the level observed with the CAI-12 C.albicans control strain (P � 0.05, ANOVA, Fig. 4). With PMNsderived from the ��M mice, fungal viability remained 80%whether the C. albicans target strains did or did not expressPra1p. Thus, recognition of Pr1p by �M�2 is pivotal for theefficient killing of C. albicans by murine PMNs. These differ-ences were not attributable to an altered growth rate or ger-mination of the mutant strain since these were comparable tothe WT strain. To evaluate the impact of PRA1 deletion ongrowth rate, the WT and �Pra1 C. albicans strains were resus-pended at 105 cells/ml in RPMI 1640 medium containing 0.1 MHEPES (pH 7.8) and incubated in plastic tubes at 37°C withslow agitation. At 4, 8, and 16 h, the samples were diluted withPBS and spread on SDA plates, and the numbers of colonies

were counted after 48 h of incubation at 30°C to determine theCFU. Only a 3 to 5% difference in the CFU content was foundafter 16 h of incubation, and the amounts of CFU after 4 and8 h were indistinguishable and practically identical for bothstrains (results not shown). As can be seen on Fig. 2, PRA1disruption also had no effect on the mutant fungus germina-tion.

Pra1p modulates �M�2-mediated early response to C. albi-cans. An important element of the host response to pathogensis the recruitment of leukocytes to sites of infection. Among allleukocyte populations, cells recruited to sites of infection orinflammation within the first 6 h are predominantly PMNs(�95% of total leukocytes recruited) (73). The observed def-icits in in vitro chemotaxis suggested that the interaction ofPra1p and �M�2 may be critical for the attraction of PMNs andkilling of C. albicans cells during the early stages of infection.To evaluate this prediction, we turned to an acute peritonealsepsis model in mice. In this analysis, mice of both WT and��M lines were challenged with 105 C. albicans intraperitone-ally. After 6 h, mice were euthanized, and the peritoneal cavitywas lavaged with PBS and subsequently with PBS containing0.5% Tween 80 to solubilize and extract adherent invadingfungi. These lavages were assayed separately (to avoid lysis ofleukocytes from the first PBS lavage by Tween from secondPBS-Tween lavage) for fungal CFU and normalized to valuesobtained by lavaging immediately after C. albicans injection.As shown in Fig. 5A, the total CFU recovered following theinfection of WT mice was significantly altered by the presenceor absence of Pra1p. In the absence of Pra1p, the number ofrecovered viable cells increased 4-fold from 16% � 10% forCAI-12 C. albicans to 65% � 12% for the pra1-null mutant(P � 0.05, WMW U-test). With restoration of PRA1 into thefungus, CFU levels again attained those observed with CAI-12C. albicans (18% � 8%, P � 0.05). In the complementaryexperiment, mice lacking �M�2 were infected. The recovery ofviable fungal cells from these mice was insensitive to the pres-ence or absence of Pra1p. Importantly, the recovery values, 65to 75%, were essentially identical to the values obtained for thepra1 mutant in WT mice (P � 0.05).

To test fungal invasion of organs, parts of murine peritonealepithelia were removed from mice 6 h after fungal administra-tion without prior PBS-Tween lavage, and the CFU withinhomogenates of these tissues were determined to assess dif-ferences in fungal adherence. The elimination of Pra1p causedsignificant increases in fungal cell adherence to all murineorgans assayed in both control WT and KO mice. In Fig. 5B,we present the results of fungal adhesion to peritoneal epithe-lium (expressed as the total CFU found in the tissue homog-enate). Pra1p elimination increased the number of CFUfound in this organ from all mouse strains by 8- to 10-fold(P � 0.01). This pattern was consistent with the CFU dis-tribution in PBS and Tween lavages; namely, with the loss ofPra1p, most recovered C. albicans CFU were adherent tothe epithelium, requiring Triton to recover the fungal cellsin the lavages (Fig. 5C).

Role of Pra1 and �M�2 on PMN recruitment to C. albicans.The differences in fungal survival could reflect either de-creased killing of C. albicans by PMNs attracted to the infec-tion site or a lack of PMN recruitment. Accordingly, we soughtto determine the impact of Pra1p and �M�2 deletion on leu-

FIG. 4. Effect of Pra1p and �M�2 on C. albicans killing/phagocyto-sis by murine PMNs. Peritoneal PMNs (3 � 106) were incubated with106 C. albicans (3:1 ratio) of CAI-12 (black bars), �Pra1p (white bars),and reintegrant strains (hatched bars) at 37°C for 5 h. Serial dilutionsof the PMN/C. albicans mixture were spread onto duplicate agar platesand the CFU were enumerated. The data are expressed as means (n 5) � the SD. As controls, initial PMN-C. albicans suspensions wereplated at time zero and assigned a value of 100%. Asterisks indicateP � 0.05, and double asterisks indicate P � 0.05 calculated byANOVA.


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kocyte recruitment to C. albicans in the peritoneal cavity. Totalleukocytes and PMNs were quantified in an intraperitoneallavage of either WT or ��M mice 6 h after intraperitonealadministration of 105 nongerminated C. albicans. The PMNs inthe lavage were quantified by myeloperoxidase activity. Thenumber of resident peritoneal PMNs, determined by the ac-tivities of these enzymes in the absence of thioglycolate and/orC. albicans administration, was assigned a value of 100% theobserved levels of resident PMNs and leukocytes in bothmouse lines were similar (results not shown). By these criteriaPMNs in the peritoneal cavity increased 300% � 20% (P �0.02, Student t test) after infection of WT mice with CAI-12 C.albicans. In contrast, C. albicans lacking Pra1p failed to recruitPMNs above background levels (P � 0.05, Fig. 6). Similarly,elimination of �M�2 in the deficient mice almost completelyabolished PMN recruitment. Similar results were obtainedwhen total leukocytes were quantified in the lavage using anonspecific intracellular esterase activity assay, indicating thatPMNs constituted the majority of leukocytes attracted to thesite of infection (Fig. 6). Thus, the absence of Pra1p or �M�2

had equivalent effects, limiting PMN recruitment in vivo.

Elimination of Pra1p or �M�2 increases C. albicans viru-lence in a murine model of systemic candidiasis. To examinethe significance of the Pra1p-�M�2 interaction in the context ofthe total host-pathogen relationship, the mutants were studiedin a murine model of disseminated candidiasis. When WT micewere challenged with 3 � 105 CAI-12 strain C. albicans cells50% of the mice reached endpoint morbidity or mortality (seeMaterials and Methods for a complete list of criteria) in 152 �14 h (Fig. 7A). Eliminating Pra1p from C. albicans dramaticallydecreased this time, since 50% of the mice introduced to 3 �105 �Pra1 C. albicans died in the range of 70 to 88 h. Geneticreintegration of the mutant restored to near WT values, 142 �16 h (P 0.096 compared to wild-type, log-rank test), con-firming association of the virulence phenotype with PRA1. Thecomplementary side of the Pra1p-�M�2 interaction was exam-ined using �M�2 KO mice. When challenged with CAI-12 C.albicans, 50% lethality occurred at 82 h in the ��M micecompared to 152 h in the WT mice (P � 0.05) (Fig. 7A). Micelacking �M�2 showed a similar enhanced susceptibility to eachC. albicans strains tested. The median survival time was 70 hfor mice given the �Pra1p strain and 88 h for mice given thereintegrant Pra1p C. albicans. There was no statistically signif-icant difference (P � 0.05, log-rank test) between these values,demonstrating that Pra1p was without influence in the absenceof �M�2. Importantly, these values were nearly identical tothose of WT mice infected with the Pra1p mutant (P 0.096),suggesting that the protective role of �M�2 was nullified by theabsence of Pra1p (Fig. 7A and B). The enhanced virulence ofthe pra1 mutant was confirmed by examining the dose re-sponse. Mice were challenged with a range of C. albicans dosesfrom 5 � 104 to 5 � 106 cells/mouse. At all tested doses, the

FIG. 5. Effect of Pra1p and �M�2 on early stages of acute fungalperitonitis. A total of 105 C. albicans of CAI-12 (black bars), �Pra1p(white bars), and reintegrant strains (hatched bars) were allowed togerminate for 1 h and injected into the peritoneal cavity of WT (leftgroup of bars) or ��M (right group of bars) mice. After 6 h, mice wereeuthanized, and the peritoneal cavities were lavaged with 4 ml ofD-PBS, followed by 4 ml of D-PBS containing 0.5% Tween 80 (PBS-Tween). Serial dilutions of each lavage were plated for CFU determi-nation, and values were normalized to the injected suspension. TheCFU recovered from each group of mice (n 8) are expressed asmean percent � the SD. The single asterisk indicates P � 0.05, and thedouble asterisk indicates P � 0.05 calculated by ANOVA. In panel Ais shown as a percentage of total inoculum the total number of recov-ered viable C. albicans in combined PBS � PBS-Tween lavages foreach fungal-mice group (n 8), and the fractional number of tissue-adherent C. albicans from PBS-Tween lavage only is shown in panel C.In panel B are present results of separate control experiment, verifyingthat cells found in PBS-Tween lavages in fact were adherent to theperitoneal epithelia. Murine peritoneal epithelia were removed fromthe mice without previous lavage, weighed, homogenized, and platedat different dilutions onto agar plates. The number of C. albicansadherent to the epithelia was determined as CFU/g of the epithelialtissue. The data are presented as means of three independent exper-iments, three mice per group � the SD.

FIG. 6. Leukocyte recruitment to C. albicans in WT and ��M mice.C. albicans (105) of CAI-12 or �Pra1p strains were allowed to germi-nate for 1 h in RPMI 1640 media at 37°C and then injected into theperitoneal cavities of WT and ��M mice. After 5 h mice were eutha-nized and peritoneal lavages were collected by sequentially lavagingperitonea with PBS (first lavage) and then PBS plus 0.5% Tween 20(second lavage). The lavages were plated separately in a series ofdilutions onto SDA plates to enumerate surviving C. albicans as CFU.Leukocytes were determined in the first lavage (PBS) by enzymaticassay of neutrophil myeloperoxidase activity (PMNs only, white bars)or total leukocytes (predominantly PMNs, macrophages, and somemonocytoid cells) by nonspecific intracellular esterase activity (blackbars). Standard curves were developed from the lysates of a knownnumber of leukocytes/PMNs for cell quantitation. Resident leukocyteswere measured in the lavages obtained from noninfected mice andwere assigned as 100%. The results are presented as means of threeindependent experiments, three mice per group, and are expressed asthe fractional percent increase over control (uninfected) resident leu-kocytes/PMNs � the SD. Asterisks indicate P � 0.05 and doubleasterisks indicate P � 0.05 calculated by using the Student t test paredto the same murine line.


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pra1 mutant produced more rapid killing than WT cells, withapproximately half as many mutant as WT cells required toattain a similar time to endpoint in the mid-range of testeddosages (Fig. 7C).

Elimination of Pra1p or �M�2 increases C. albicans dissem-ination in the host. Because of the diverse roles of �M�2 incontaining microbial infections, the similarity in time course of�pra1 and ��M�2 infections may have different pathologicalreasons. Therefore, the extent of dissemination, organ fungalburden, and tissue targeting was assessed for all infected mice.Selected organs, kidney, lung, heart, spleen, and liver, wererecovered from mice 16 and 40 h after infection, and the fungalburden was determined. Consistent with previous studies (92),the highest fungal burden was present in the kidneys and wascomparable at 16 h in both lines of mice regardless of C.albicans challenge strain (P � 0.05, Student t test). Notabledifferences, however, occurred in other organs. In WT miceinfected with the pra1 mutant, in all of infected animals notablefungal burden in the spleen, liver, and lungs appears as early as16 h versus 40 h for the WT. At 40 h, the fungal burden in allorgans WT/�Pra1 mice was increased 4-fold (kidney, P � 0.05)to 10-fold (spleen and liver, P � 0.01) and 20-fold (lungs, P �0.01) compared to infection with the control C. albicans strainCAI-12. This same pattern of altered tissue burdens was ob-

served in ��M mice infected with either CAI-12 or pra1 mutantcells (P � 0.05 for kidney, P � 0.02 for other organs) (Table 1).

These results showed that the absence of Pra1p from thepathogen or the absence of �M�2 from the host altered dis-semination of the infection to a similar extent and in a similarmanner.

DISCUSSION

In the present study, we assessed the contributions of Pra1pand �M�2 on the innate immune response to C. albicans in-fection and fungal pathogenesis. The major findings of ourstudy are as follows. (i) The absence of �M�2 significantlyimpairs the ability of murine PMNs to adhere to, migratetoward, and eliminate C. albicans by phagocytosis and/or oxi-dative burst in vitro but has no or has limited effect on thephagocytic functions of neutrophils, such as �M�2-independentadhesive and migratory abilities, degranulation, and peroxideion production. (ii) The absence of �M�2 led to a major de-crease in host resistance to C. albicans infection in both em-ployed murine models of acute fungal peritonitis and hema-togenously disseminated candidiasis. (iii) Deletion of Pra1pfrom C. albicans decreased PMN adhesion, migration, degran-ulation, oxidative burst, and fungal killing and increased viru-

FIG. 7. Effect of leukocyte integrin �M�2 on C. albicans virulence in a murine model of disseminated candidiasis. (A) Effect of Pra1p and �M�2elimination on murine survival. C. albicans (3 � 105) of CAI-12 (black bars), �Pra1p (white bars), or reintegrant (hatched bars) strains of C.albicans were introduced in 100 �l of D-PBS via tail vein injection into WT (left group of bars) and ��M (right group of bars) mice. Afteradministration, the mice were inspected on an (8 � 2)-h basis and euthanized when they became moribund (e.g., at 20% weight loss). The averagesurvivors in each group (n 8) are presented as medians (25th and 75th). The P values were calculated by log-rank test. The complementedKaplan-Meier graphic of this experiment is shown on the panel B. (C) Effect of Pra1p deletion on C. albicans virulence in WT mice. C. albicansof either WT (closed circles, solid line) or �Pra1p (opened circles, dashed line) strains in the concentration range of 5 � 105 to 5 � 106 cells/mousewere administered by tail vein into WT mice. The mice were inspected every 8 h and euthanized immediately upon becoming moribund. The resultsare plotted as log10 of dose of yeast cells/g of mouse weight versus time to become moribund. Each point represents an individual mouse. The Pvalues were calculated by log-rank test.


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lence, and these changes were all �M�2 dependent. (iv) Re-moval of either �M�2 or Pra1p significantly reduced leukocyterecruitment at the early stage of fungal infection. Together,these observations show the critical importance of recognitionof fungal Pra1p by leukocyte integrin �M�2 and that this in-teraction significantly contributes to the success of the hostdefense against C. albicans infection, especially on the initialstages of the infection.

Two distinct murine models of disease, disseminated candi-diasis and acute peritoneal sepsis, demonstrated increased C.albicans virulence upon deletion of the PRA1 gene. The PRA1-null mutant induced lethality more rapidly (1.5- to 2-foldfaster) than the same inoculum of WT C. albicans. The data ofthe fungal burden in the primary target organ of infection, thekidney (46, 78), support the virulence data. The early timepoint data (16 h) show similar kidney fungal burdens in allanimals, indicating that the kidneys were seeded at similarlevels, a finding consistent with an equivalent inoculum. At40 h, there are statistically significant differences between WTmice infected with CAI-12 versus the �Pra1 mutant and be-tween WT mice infected with CAI-12 and ��M mice infectedwith either CAI-12 or the Pra1 mutant (P � 0.05 in all cases),and this correlates with a higher survival of WT mice infectedwith control C. albicans strain. The 5-fold difference in kidneywas associated with increased 10- to 20-fold fungal burden inother organs (P � 0.02), this difference in dissemination ap-peared as early as the 16-h time point.

Results from the acute peritonitis model of early stage in-fection suggest that this enhanced virulence may arise fromsuppressed fungal-leukocyte interaction. In mice that lack�M�2, this difference in virulence between Pra1p-positive and-negative strains of C. albicans was no longer observed; diseaseprogression and fungal burden in ��M mice was similar forboth C. albicans strains. Since these strains have similar in vitrogrowth rates, these results suggest that the innate responsefails to recognize and clear fungal cells lacking Pra1p. Wecannot exclude the possibility that the in vivo growth rate of themutant exceeds that of the WT, but a 4-fold enhancement

seems unlikely. These findings support our conclusion thatboth �M�2 and Pra1p are involved in fungal recognition.

The observed effect of �M�2 elimination on C. albicans vir-ulence is consistent with previous reports demonstrating thatpatients with leukocyte adhesion deficiency type 1 (LAD-1)syndrome showed increased susceptibility to C. albicans infec-tion (7, 45, 50, 53, 86). LAD-1 syndrome is a rare hereditarydisease characterized by increased susceptibility to infectionsdue to the inability of leukocytes, in particular neutrophils, tomigrate from the blood to sites of inflammation. The increasedsusceptibility of LAD-1 patients to C. albicans infection isascribed to the significant impediment of leukocytes to formpus at the site of infection (7, 50) and to the reduced ability ofLAD-1 neutrophils to kill the fungus. Lau et al. reported thatneutrophils of a LAD-1 patient showed a 3-fold reduction in C.albicans killing in vitro, whether opsonizing or nonopsonizingkilling was involved (53), and we recapitulated these resultsusing ��M PMNs. LAD-1 arises from an absence of all beta-2integrins on leukocytes caused by mutations in the �2 gene (4,7). Unlike ��2 leukocytes, ��M leukocytes are still able tomigrate to the sites of infection and inflammation using other�2 integrins, primarily �L�2 (28, 57). The similarity in themigration rates of WT and ��M PMNs to thioglycolate-in-duced intraperitoneal inflammation, described in the literature(57) and observed by us, taken together with the failure ofleukocytes to migrate to the peritoneal cavity of �M�2-deficientmice 6 h after peritoneal infection with C. albicans, suggeststhat �M�2 is the integrin responsible for leukocyte recruitmentto C. albicans. The absence of neutrophil recruitment to �Pra1C. albicans in WT mice further indicates that Pra1p is a majorligand for leukocytes among C. albicans proteins and that�M�2 is a major receptor for this protein (38, 39, 88).

It is possible that PMNs can utilize �M�2 for binding also toother ligands on the C. albicans surface. As seen on Fig. 2B,PE-labeled isolated �M�2 slightly binds to the ends of somehyphae, probably by low-affinity interaction to fungal cell wallmannans through its lectin domain (100, 101, 105). Residualkilling of �Pra1 C. albicans by ��M PMNs also may be medi-

TABLE 1. Distribution of C. albicans within murine organs after the introduction of 3 � 105 C. albicans at 16 and 40 hof infection and at the endpointa

Time of examinationand organ

C. albicans in murine organs (mean CFU/g of the indicated tissue � SE)b

WT mice ��M mice

CAI-12 �Pra1 CAI-12 �Pra1

16 hKidney (2.1 � 103) � 320 (2.2 � 103) � 300 (2.4 � 103) � 420 (2.3 � 103) � 600Spleen ND 260 � 120* 320 (n 4/6)* 400 � 120*Liver 100 � 80 340 � 80* 220 (n 3/6) 220 � 120Lungs 80 (n 4/6) 360 � 80* 320 (n 3/6)* 380 � 140*Heart ND ND ND ND

40 hKidney (1.3 � 104) � (3 � 103) (6.9 � 104) � (1 � 104)* (8.3 � 104) � (2 � 104)* (1.1 � 105) � (3 � 104)*Spleen (3.2 � 103) � (1 � 103) (3.3 � 104) � (1 � 104)* (5 � 104) � (5 � 103)* (1.3 � 105) � (3 � 104)*Liver (1.3 � 103) � 600 (1.4 � 104) � (2 � 103)* (9 � 103) � (2 � 103)* (5.2 � 104) � (1 � 104)*Lungs (2.1 � 103) � 800 (5.2 � 104) � (1 � 104)* (8.8 � 103) � (2 � 103)* (4.4 � 104) � (1 � 104)*Heart ND ND 40 (n 1/6) ND

a The endpoints were 180 � 60 h for WT CAI-12 and 84 � 36 h for all other strains.b *, P � 0.05. ND, not determined. n, Number of animals.


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ated by resident peritoneal macrophages and/or dendritic cells.Indeed, an �M�2-independent mechanism for C. albicans rec-ognition has been ascribed to macrophages and dendritic cells(30, 67), and macrophages have been shown to bind and engulfC. albicans in a Pra1p-independent fashion (60). Other leuko-cyte receptors that can bind fungal polysaccharides—Dectin-1,TLR2, (13, 68), and TLR4 (69)—also may be involved infungal recognition, but they do not directly mediate leukocytemigration, adhesion, killing, and phagocytosis. Also, Dectin-1and TLR2 recognize yeast forms of C. albicans only (41), andtherefore they may play only minor roles in fungal eliminationby PMNs, which can kill and/or phagocytose C. albicans fila-mentous forms only (36, 37, 39).

Another notable finding in the presenting study is the ob-servation of increased adhesion of Pra1 �/� C. albicans toperitoneal endothelia in the murine model of acute peritonitis(Fig. 5B). It indicates that the Pra1p may participate in theregulation of the fungal invasion and colonization by inhibitionfungal adhesion to the host tissues. As noted above, the exactrole of Pra1 in C. albicans physiology is still unclear, and theearlier observations that the Pra1 expression is environmen-tally and/or conditionally dependent and is regulated by nutri-tion (3, 19, 84) support this conclusion. The enhanced adhe-siveness of the Pra1-depleted C. albicans may also contributeto increased virulence of the mutant fungus, but the differencein virulence between Pra1�/� and Pra1�/� C. albicans strainsin �M�2-KO mice and the CAI-12 strain in WT mice (Fig. 7A)were insignificant (P � 0.05 [ANOVA]), and the similar levelsof organ fungal burdens at 16 h in these murine-fungal com-binations (Table 1, P � 0.05 [ANOVA]) demonstrate that evenif this contribution really takes a place, it plays minor role inincreased fungal virulence of Pra1-KO fungus compared to thedisruption of Pra1-�M�2 interaction. Thus, our results clearlydemonstrate that recognition of Pra1p by �M�2 plays a majorrole in determining fungal virulence and host protectionagainst C. albicans infection.

ACKNOWLEDGMENTS

This study was supported by grant AIO 80596 from the NationalInstitute of Allergy and Infectious Diseases and in part by grant P50HL081011 from the National Institutes of Health.

��M-KO mice were kindly provided by Christy M. Ballantyne ofBaylor College of Medicine, Houston, TX. We thank Edward Plowand Elzbieta Pluskota for productive discussions of results and CarlaDrumm for help with mouse husbandry and organ collection.

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