INFECTION AND IMMUNITY, Apr. 2011, p. 1546–1558 0019-9567/11/$12.00 doi:10.1128/IAI.00650-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 4
Regulation of Innate Immune Response to Candida albicans Infections by ␣M2-Pra1p Interaction䌤 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 fungal diseases in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is one of the main tasks of cells of the innate immune system, and in vitro evidence suggests that integrin ␣M2 (CR3, Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using ␣M2-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate that neutrophils derived from mice deficient in ␣M2 have a reduced ability to kill C. albicans and that the deficient mice themselves exhibit increased susceptibility to fungal infection. Disruption of the PRA1 gene of C. albicans, the primary ligand for ␣M2, protects the fungus against leukocyte killing in vitro and in vivo, impedes the innate immune response to the infection, and increases fungal virulence and organ invasion in vivo. Thus, recognition of pH-regulated antigen 1 protein (Pra1p) by ␣M2 plays a pivotal role in determining fungal virulence and host response and protection against C. albicans infection. rare (that is, ca. 1 in 106 people) hereditary disease, leukocyte adhesion deficiency 1 (LAD-1), which is characterized by the low expression (mild LAD-1) or complete absence (severe) of all four of the beta-2 integrins due to mutations in the 2 gene (4, 5, 34), are highly susceptible to wide range of bacterial and fungal infections (7, 50, 80). Staphylococcus aureus and Streptococcus spp. are the most common bacterial pathogens, and Candida spp. are the primary fungi isolated from patients with LAD-1. The candidal skin infections described in older publications were reported to occur in ca. 16% of patients; Candida esophagitis has also been frequently reported (6, 7). In three more-recent reports, a total of 27 cases of LAD-1 patients were described, 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 patients or 46% of all cases of mortality) the cause of death was fungal septicemia (44, 65, 71). In most cases, the degree of severity of these symptoms may be correlated with the level of 2 expression on the patients’ leukocytes. Generally, patients with ⬍1% normal levels of 2 are the most susceptible to frequent and life-threatening systemic infections and usually do not survive to adulthood. Patients with higher levels of expression (up to 10% of normal) develop milder forms of LAD-1 and may survive to adulthood with proper medical care (10, 86). The fungal invasion usually starts in newborns and toddlers (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 peritonitis, and the formation of nodular and ulcerative lesions in later stages, which ultimately lead to sepsis and death (44, 53, 64). The principle beta-2 integrin involved in recognition of bacterial and fungal pathogens is ␣M2 (Mac-1, CD11b/CD18, and CR3) (37, 39). ␣M2 is a pivotal adhesion receptor on PMNs, cells of the monocytoid lineage, subsets of T lymphocytes, and NK cells (37, 38). The capacity of ␣M2 to support
Candida albicans is an opportunistic pathogen, a pleomorphic fungus existing in yeast or filamentous forms (19, 23). Although the yeast form can bind to gut mucosal membranes with further colonization (20, 49), it is thought that the filamentous morphology provides some advantage during interaction with the mammalian immune system as a part of fungal anti-host defense, and the ability of C. albicans to rapidly and reversibly switch between yeast and filamentous morphologies is crucial to its pathogenicity (16, 55, 65, 82). In recent years, Candida infections ranked as the fourth most common cause of bloodstream infections and are the leading cause of life-threatening nosocomial fungal infections (1, 87). The risk of developing opportunistic bloodstream infections is greatly increased in patients who are severely immunocompromised. Candida strains that are resistant to commonly used antimycotics have emerged rapidly (72, 98). Therefore, dissecting its pathogenic mechanisms and the host response to C. albicans infections is of great importance. The innate immune system provides the principle protection against disseminated candidiasis. Polymorphonuclear leukocytes (PMNs) have been shown to be the primary components of the host’s innate immune defenses against Candida infections (27, 59, 67). The most prominent receptors on leukocytes utilized in fungal or microbial recognition are the integrins of the beta-2 subfamily (62, 63). These cell surface receptors mediate migration of leukocytes to sites of infection and adhesion to microorganisms with subsequent phagocytosis and killing of many pathogens (12, 27, 62). Patients with defects in leukocyte phagocytic functions, such as individuals with the
* Corresponding author. Mailing address: Joseph J. Jacobs Center for Thrombosis and Vascular Biology and Department of Molecular Cardiology, Cleveland Clinic, Cleveland, OH 44195. Phone: (216) 4458211. Fax: (216) 445-8204. E-mail:
[email protected]. 䌤 Published ahead of print on 18 January 2011. 1546
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leukocyte adhesion, migration, and phagocytosis depends upon its ability to recognize and mediate responses to a diverse set of structurally unrelated ligands, including fibrinogen (104), complement fragment iC3b (11), and intracellular cell adhesion molecule-1 (ICAM-1, CD54), as well as numerous bacterial lipoproteins and fungal mannans and -glycans (101, 103). This cell surface receptor consists of two structurally distinct subunits, ␣M and 2, which associate noncovalently. The ␣M subunit is unique to this receptor, and the 2 subunit is shared with three other members of the beta-2 integrin subfamily: LFA-1 (␣L2), p150,95 (␣X2), and ␣D2 (reviewed in references 43 and 52). Of the beta-2 integrins, ␣M2 has been specifically implicated in the interaction of leukocytes with C. albicans: PMNs utilize ␣M2 to adhere only to the filamentous form of C. albicans and not to yeast cells (36, 37, 39). Although other leukocyte receptors, Dectin-1 and Toll-like receptor 2 (TLR2), which can bind fungal -glucan (13, 68, 99), and mannan-binding TLR4 (69) also participate in fungal recognition and are essential in leukocyte activation and eventually in activation of -2 integrins (42, 91), they do not directly mediate leukocyte migration and adhesion. Also Dectin-1 and TLR2 recognize yeast forms of C. albicans only (41). Recently, we identified C. albicans pH-regulated antigen 1 protein (Pra1p) (84), also known as fibrinogen binding protein 1 (Fbp1) (56) or C. albicans 58-kDa mannoprotein (mp58) (17), as the major ligand of ␣M2 among C. albicans proteins (88). Pra1p is expressed predominantly on the surface of the hyphal form and not the yeast form of C. albicans (25, 66, 85). The expression of Pra1p is strongly pH dependent, requiring a pH ⬎ 7, and is also regulated by nutrition and certain other fungal genes (25, 79, 84). It is a mannoprotein, with carbohydrate 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 small quantities of Pra1p may be present on the yeast form of C. albicans, it becomes highly glycosylated only on the filamentous forms (hyphal and pseudohyphal forms) (18, 19), and we found that sugar residues are important for recognition of C. albicans by ␣M2 (39, 88). However, the biological role and the significance of the ␣M2-Pra1p interaction in C. albicans virulence and host defense is unknown. The present study was undertaken to determine the role and significance of the ␣M2Pra1p interaction in fungal pathogenicity and its effects on host defense in vivo using ␣M2-deficient knockout (KO) mice and the ⌬pra1 strain of C. albicans in two distinct models of acute murine candidiasis.
MATERIALS AND METHODS Candida albicans strains. The C. albicans strains used were CAI-12 (control strain, iro1-ura3/IRO1-URA3), CAMB5-18 (⌬Pra1 strain, pra1::hisG/pra1::hisG iro1-ura3⌬/IRO1-URA3), and CAMB9 (“reintegrant” strain pra1::hisG/ PRA1-URA3-pra1::hisG iro1-ura3/iro1-ura3). Strains CAI-12 and CAMB9 were previously described (77, 84). Strain CAMB5-18 was derived from strain CAMB435 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 deletion on fungal cell morphology and germination, yeast cells of each strain were resuspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 0.1 M HEPES (pH 7.4) and 10% non-heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). Serial dilutions from 104 to 102 cells/ml in 50-l aliquots were grown in 96-well untreated plastic plates at 37°C in humid-
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ified air and 5% CO2. To detect changes in the cell morphology, the samples were examined microscopically at 60-min intervals. Animals. ␣M knockout (KO) mice (⌬␣M2) were kindly provided by Christy M. Ballantyne of Baylor College of Medicine, Houston, TX, and have been used in several studies (see, for example, references 28, 57, 76, and 81). These mice were backcrossed for more than 12 generations into a C57BL/J6 background, and all mice used were genotyped by PCR of blood DNA samples. Age-matched C57BL/J6 mice purchased from Jackson Laboratories (Bar Harbor, ME) were used as controls (WT mice). All protocols involving mice were approved by the Institutional Animal Care and Use Committee in accordance with Public Health Service policy, the Health Research Extension Act (PL99-158), and Cleveland Clinic policy. All murine experiments involving C. albicans infections were carried out in a BSL2 facility of the Cleveland Clinic Biological Resource Unit. The mice were maintained on a 12-h alternating light-dark cycle and supplied with food (Diet #2918; Harlan Teklad, Madison, WI) and sterilized water ad libitum. Murine PMN isolation. Peritoneal PMNs were isolated from murine peritoneum lavage of ⌬␣M and WT mice, both males and females, as described previously (75) using intraperitoneal thioglycolate as an inflammatory stimulus to recruit the cells. At 6 h after injection, the mice were euthanized by CO2 inhalation, the peritoneal cavity was opened, and leukocytes were harvested by washing 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 this manner were ⬎95% PMN. Peripheral blood PMNs were isolated from pooled heparinized murine blood taken from tail veins of WT or ⌬␣M mice, both males and females, by centrifugation through Ficoll-Hypaque, followed by 6% dextran sedimentation; the contaminating erythrocytes were removed by hypotonic lysis (102). The cells obtained by this technique were ⬎90% PMNs (21, 33, 106). For each experiment, PMNs from five to seven mice were pooled. Cell adhesion assays. To determine cell adhesion to the fungus, 48-well Costar tissue culture plates (Corning, Corning, NY) were precoated with 500 l of 0.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich) for 1 h at room temperature and washed 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 ml of RPMI 1640 medium, containing 1% FBS, were added, followed by incubation overnight at 37°C to germinate. The supernatant was removed, and adherent fungi were carefully washed with 0.5 ml of HBSS. In some experiments, wells were coated overnight with 200 l of 10 g/ml of purified iC3b, ICAM-1 (Calbiochem/EMD, Darmstadt, Germany), or Fc fragments of murine IgG and postcoated with 0.5 ml of 0.5% PVP without the addition of the fungus. Murine Fc fragments were prepared by digestion of mouse IgG (Sigma) with an immobilized papain-based Fab preparation kit (Pierce/Thermo, Rockford, IL) with further separation on protein A-agarose. A total of 105 peritoneal PMNs or human embryonic kidney cell line 293 expressing ␣M2 on their surfaces (HEK293/␣M2 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 were coated with PVP only. Each experimental point was tested in triplicate. Subsequently, plates were washed three times with PBS, and the number of adherent cells in each well was quantified by using a CyQuant cell proliferation assay kit (Molecular Probes, Eugene, OR) as previously described (88, 89). The data from cell adhesion and migration (see below) analyses are presented as percentages (mean ⫾ the standard error [SE]) of total cells (to which was assigned the value of 100%) and represent the results of three independent experiments. Cell migration assays. Peritoneal PMN or HEK293/␣M2 cell migration assays were performed in serum-free Dulbecco modified Eagle medium/F-12 medium (Invitrogen) using modified Boyden chambers (Costar transwell inserts in 24-well plate format; Corning) with tissue culture-treated polycarbonate filters with 3-m pores (14) as previously described (40, 74, 88, 89). Briefly, the lower chambers contained 600 l of medium with 5 ⫻ 105 C. albicans yeast of the selected strain, which were germinated overnight prior to beginning the analyses. Alternatively, purified iC3b, ICAM-1, or murine IgG Fc fragments at 10 g/ml were used as ligands. The upper chambers contained final volumes of 200 l of PMN or HEK293/␣M2 cell suspensions. The assays were initiated by addition of 50 l of cell suspension (105 cells/well) to the 150 l of medium in the upper chambers, 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 upper chamber of transwell inserts by using cotton swabs, and the migrated cells present 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 and previously (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 Monteil (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 RPMI 1640 medium per well). Then, 2 ⫻ 104 peripheral blood PMNs, WT or ⌬␣M, were added in 100 l of HBSS-HEPES buffer (pH 7.4) containing 2 mM CaCl2 and 2 mM MgCl2. In some experiments, PMNs, before addition to the assay wells, were preincubated 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 plates for 30 min at 37°C in 5% CO2, 2,7-dichlorodihydrofluorescein diacetate (DCFHDA; 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 fluorescence was 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. Background fluorescence from C. albicans was subtracted, and the data were normalized to the peroxide concentration, obtained from peripheral blood PMNs of WT mice after stimulation with GM-CSF/fMLP in the absence of C. albicans, which was assigned the value of 100%. PMN degranulation assay. To compare WT and ⌬␣M PMN degranulation in response to C. albicans, the level of intracellular -glucuronidase retained in PMNs granules upon activation was determined by modification of described methods (57, 93). C. albicans (107) of either CAI-12 or ⌬Pra1 strains were allowed to germinate overnight in 1 ml of RPMI 1640 medium; 106 peritoneal or peripheral blood PMNs, either WT or ⌬␣M derived, were then added in 0.5 ml of HBSS-HEPES (pH 7.4) containing 2 mM CaCl2 and 2 mM MgCl2, followed by incubation at 37°C in 5% CO2 for 30 min. After the incubation, cell-fungus mixtures were centrifuged, the pellets were resuspended in 0.5 ml of 0.2% Tween 20 to lyse leukocytes, and fungi, along with cell debris, were removed by centrifugation. The clarified lysates (100 l) were added to 100 l of 5 mg of phenolphthalein glucuronic acid (pH 4.6; Sigma-Aldrich)/ml. The mixtures were incubated at 37°C for 16 h, and the reaction was stopped by adding 1 ml of 1 M glycine (pH 10.4); the absorbance was read at 540 nm with a spectrophotometer. In some experiments, PMNs before addition to C. albicans were preincubated for 10 min with 1 M GM-CSF and 1 M fMLP (32, 51). As controls, PMNs without coincubation with C. albicans were used. The data obtained from nonstimulated WT peripheral blood PMNs in the absence of C. albicans or other activators were assigned a value of 100% granule retention. PMN-mediated C. albicans killing/phagocytosis ex vivo assays. C. albicans yeast forms (106) of the selected strains in 0.25 ml of high glucose RPMI 1640 containing 0.1 M HEPES (pH 7.8) were allowed to germinate in plastic tubes at 37°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) and mixed with 3 ⫻ 106 PMNs in 0.25 ml (1:3 ratio). The leukocyte-fungal mixtures were incubated at 37°C with slow shaking for 1 to 5 h. To determine the extent of killing and/or phagocytosis, aliquots of the peritoneal PMN-C. albicans suspension diluted with HBSS-HEPES were plated in serial dilutions on SDA plates, and the CFU were counted manually on day 2 using a Bel-Art Products colony counter. PMNs were not lysed before plating, and all fungal cells that remained ingested were recorded as “killed.” Obtained results were independently 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 added to the fungal-leukocyte mixture, lysed leukocytes were removed by centrifugation, and C. albicans cell pellets were resuspended in 0.25 ml of 2.5 mM methylene blue (Sigma) in HBSS-HEPES. The number of viable (nonstained) cells was counted in a hemacytometer using a microscope. Control samples contained C. albicans incubated without PMNs. The results obtained by both methods of quantitation of fungal viability showed good correlation with variance in the 5 to 10% range. Clearance of intraperitoneal C. albicans. To determine the clearance of intraperitoneal C. albicans, we used a modification of the method used previously to determine intraperitoneal clearance of S. aureus (35). To initiate acute peritoneal sepsis, 105 C. albicans of CAI-12, ⌬Pra1p, or reintegrant strains were allowed to germinate 1 h in RPMI 1640 medium at 37°C and injected intraperitoneally in 0.1 ml of RPMI 1640 into WT and ⌬␣M mice. After 6 h, mice were euthanized, and peritoneal lavage was collected by gently washing the peritoneal cavity with 4 ml of ice-cold PBS. In pilot experiments, we found that we were not able to recover total Candida cells from the peritoneum after 6 h of incubation, and a significant amount of fungi remained adherent to the peritoneal epithelium after lavage with PBS. To elute C. albicans adherent within the peritoneal cavities, we took advantage of the ability of detergents such as Tween to solubilize mammalian cell membranes and membrane proteins. After the initial lavage with PBS, the peritonea of some mice were lavaged a second time with 4 ml of PBS containing 0.5% Tween 80. All lavages were subsequently plated separately in serial of dilutions onto SDA plates to enumerate CFU. To ensure
INFECT. IMMUN. the complete recovery of adherent fungi from the peritonea by this lavage procedure, as much of the murine peritoneal epithelia as possible was removed from all mice with or without the various lavage protocols and then homogenized in PBS containing Tween 80; serial dilutions were plated onto SDA plates for CFU quantitation. Absence of the fungal CFU in Tween-lavaged peritonea was taken as evidence of complete fungal recovery by the PBS-Tween lavage. Leukocyte recruitment. Total PMNs recovered in a PBS intraperitoneal lavage after intraperitoneal C. albicans injection were quantified enzymatically as previously described (15, 94, 96). Briefly, 0.5 ml of the lavage fluid was diluted with 0.5 ml of PBS containing 2% Triton X-100 to lyse leukocytes. PMNs were quantified spectrophotometrically by measuring the myeloperoxidase activity using guaiacol (Sigma-Aldrich) as a substrate by determining the absorbance at 470 nm. As independent verification, total leukocytes (predominantly PMNs and macrophages) were also determined spectrophotometrically by intracellular esterase activity in the lavage lysates using p-nitrophenyl butyrate (Sigma-Aldrich) as a chromogenic substrate measuring the absorbance at 405 nm (73, 94). The amount of resident peritoneal leukocytes, determined from the activities of these enzymes in the absence of C. albicans administration, was assigned a value of 100%. Murine model of systemic disseminated candidiasis. The model of systematic candidiasis, described and applied to C57/BJ6 by Costantino et al. (24) and Zhao et al. (107), was used. Unless stated otherwise, for each experimental point seven to nine mice at 8 to 12 weeks of age and weights in the range of 18 to 20 g were used. Mice were injected with various dosages from 5 ⫻ 104 to 5 ⫻ 105 of the selected C. albicans strain: CAI-12 (control strain), CAMB5-18 (⌬Pra1p), or CAMB9 (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 of humane endpoints in a murine model of leukemia (2). The following symptoms were evaluated and scored as follows: coma, 15 points; weight loss of 20% (15 points), 15% (11 points), or 10% (8 points); abdominal swelling, 6 points; significant decrease in mobility, 4 points; and hunched posture, 3 points. Each group was monitored daily over the 30-day period, at approximately the same time each day, and the score was nonaccumulative and was recalculated for each mouse every 8 ⫾ 1.5 h. The primary endpoint was the number of mice surviving on day 30 within each experimental group. When mice scored 15 or more points before 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 additional scoring value, and 3% developed coma. After euthanasia for all causes, the mice were 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 of PBS, and serial dilutions of the homogenates were plated onto SDA plates for CFU 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 paired Student t test (for in vitro migration, adhesion degranulation, and superoxide ion production assays), analysis of variance (ANOVA; for in vitro killing and organ fungal burden assays), log-rank and Cox regression (for mice survival analysis)— all are from statistical package of SigmaPlot (version 10.0; Jandel Scientific Software, Chicago, IL). A Wilcoxon-Mann-Whitney rank-sum U-test (WMW U-test) for murine survival analysis was performed by using online calculation tools (www.psychol-ok.ru/statistics/mann-whitney/) and tables of critical values (www.statanalyse.org). The difference in the two groups was considered significant with P ⬍ 0.05. The data are expressed as means ⫾ the SD unless otherwise noted.
RESULTS To demonstrate the biological importance of the ␣M2 and Pra1p interaction in fungal recognition and as a prelude to subsequent in vivo studies, the interaction of PMNs derived from C57BL/J6 (WT mice) or ␣M2-deficient mice (⌬␣M mice) with C. albicans strains expressing or lacking Pra1p was examined. Previous studies had demonstrated that deletion of ␣M2 does not affect expression levels of the other 2 integrins on PMNs (28, 29, 57). Furthermore, it was shown that ⌬␣M PMNs were still able to emigrate through endothelium (28, 48, 57) and displayed similar levels of recruitment into the peritoneal cavity in thioglycolate-induced inflammation model to WT
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FIG. 1. Effects of Pra1p and ␣M2 on the adhesive and migratory functions of PMNs in vitro. In each panel, asterisks indicate P ⬍ 0.05, as calculated by an ANOVA test. (A) Effect of Pra1p and ␣M2 on PMN adhesion to C. albicans. PMNs (105) from WT mice (left group of bars), ⌬␣M mice (center group of bars), or HEK293/␣M2 cells (right group of bars) were added to the wells containing the indicated strains of germinated C. albicans of CAI-12 (black bars), ⌬Pra1p (white bars), or Pra1p-reintegrant (hatched bars) strains as adhesive substrates. After 30 min, nonadherent cells were removed by washing, and adherent cells were lysed and reacted with a DNA-binding fluorescent dye. The amount of adherent cell was found by using a standard curve obtained from known numbers of the cells stained under the same conditions. As a control, PMN adhesion to wells coated with 0.5% PVP was used. The results are presented as the percentile of adherent cells and are expressed as means ⫾ the SD of three independent experiments with triplicates in each experiment. (B) Adhesion of PMNs and HEK293/␣M2 cells to a panel of leukocyte ligands. PMNs or HEK293/␣M2 cells (all 105) were added to plates coated with 10 g of the indicated protein/ml. After 30 min, nonadherent cells were removed by washing, and adherent cells were stained with a DNA-binding fluorescent dye. As a control, PMN adhesion to wells coated with 0.5% PVP was used. The results are presented as the percentile of adherent cells, and expressed as mean ⫾ the SD of three independent experiments with triplicates in each experiment. (C) Effect of Pra1p and ␣M2 on PMN migration to C. albicans. PMNs or HEK293/␣M2 cells (105) were allowed to migrate overnight across a polycarbonate membrane with 3-m pores (5 m in the case of HEK23/␣M2) to the C. albicans strains indicated as in panel A. After incubation, migrated cells were lysed and stained with a DNA-binding fluorescent dye. The amount of adherent cell was found using standard curve obtained from known numbers of the cells stained under the same conditions. The results are presented as percentile of migrated cells and are expressed, after subtracting the background migration, as the mean of three independent experiments with triplicates in each experiment. (D) Migration of PMNs and HEK293/␣M2 cells to the panel of leukocyte ligands. PMNs or HEK293/␣M2 cells (105) were allowed to migrate overnight with 3-m pores (5 m in the case of HEK23/␣M2) to 5-g/ml leukocyte ligands indicated as in panel B. After incubation, migrated cells were lysed and stained with a DNA binding fluorescent dye. The results are presented as the percentile of migrated cells and, after subtracting background migration, expressed as means ⫾ the SD of three independent experiments with triplicates in each experiment.
mice (48, 57). However, some PMN functions associated with ␣M2, such as adherence to fibrinogen (95), iC3b-mediated phagocytosis (70), and degranulation, were impeded (28, 57). Therefore, in all ex vivo experiments we included additional controls to examine the impact of ␣M2 ablation on relevant functions of ⌬␣M leukocytes. Effect of ␣M2 and Pra1p elimination on PMN adhesion to C. albicans. In the first set of experiments, peritoneal PMNs from either WT or ⌬␣M mice were added to plates with germinated C. albicans cells of CAI-12 or ⌬Pra1 strains, and adhesion was quantified after 30 min of incubation. Both WT PMNs and HEK293/␣M2 cells adhered well to WT fungus. Disruption of the PRA1 gene ablated adhesion of the WT PMN (P ⬍ 0.05, Student t test), while reinsertion of PRA1 restored adhesion of the WT PMNs to the fungal substrate (P ⬎ 0.1). In contrast, PMNs derived from the ␣M2-deficient mice showed little adhesion to C. albicans (⬎90% reduction compared to the WT PMNs, P ⬍ 0.05), and the presence or absence of Pra1p had virtually no effect (Fig. 1A). In control experiments 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 ␣M2) (97), ICAM-1 (ligand for both ␣M2 and ␣L2) (26, 61), and purified murine IgG Fc fragments (IgG-Fc, ligand for leukocyte Fc receptors) (108); these results were also verified with ␣M2-expressing HEK293 cells (HEK293/ ␣M2 cells). As expected, PMNs from WT mice exhibited strong adhesion to all ligands tested. In contrast, ⌬␣M-PMNs showed adhesion to ICAM-1 and IgG-Fc at a level similar to WT PMNs (P ⬎ 0.05) but were not able to adhere to aM2 ligand iC3b, whereas HEK293/␣M2 cells adhered to all of the ␣M2 ligands, iC3b and ICAM-1, but not to murine IgG-Fc (Fig. 1B). In additional experiments, we microscopically examined ␣M2-Pra1p interaction by microphotography of coincubation of CAI-12 and ⌬Pra1 C. albicans strains, germinated and labeled on mannose with fluorescein isothiocyanate with purified ␣M2, labeled with phycoerythrin (PE). Existing literature indicates that leukocytes can bind to germ tubes and to hyphae of the filamentous form of C. albicans but generally not to the yeast form (36, 39) and that Pra1p is also predominantly expressed on the surfaces of these C. albicans forms (25, 85). Consistent with previous findings (39, 88),
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FIG. 2. Immunofluorescent staining of germinated CAI-12 and ⌬Pra1 C. albicans strains with isolated and PE-labeled ␣M2. C. albicans of WT (A) and ⌬Pra1 (B) strains were allowed to germinate in RPMI 1640 for 2 h. Purified and biotinylated ␣M2 (the method of ␣M2 isolation was described 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 were washed with D-PBS and incubated with R-phycoerythrin-streptavidin conjugate for 30 min at room temperature. Subsequently, the fungi were again washed with D-PBS, fluorescein isothiocyanate-GNL specific for the terminal ␣-D-mannosyl group was added, and the mixture was incubated for an additional 1 h. The cells were washed with D-PBS and observed by fluorescence microscopy (Leica Microsystems AG) at an optical magnification of ⫻800.
␣M2 binds only to germ tubes and hyphae but not to bud forms of C. albicans, and Pra1p elimination completely abrogates ␣M2 binding to the fungus (Fig. 2). These results are also consistent with previous reports that Pra1p is ex-
pressed predominantly on the surface of filamentous but not yeast forms of C. albicans (25, 66, 85). Thus, both ␣M2 and Pra1p are essential for the productive adhesion of murine PMNs to C. albicans.
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Effect of ␣M2 and Pra1p deletion on C. albicans-mediated PMN migration. Our prior studies showed that in addition to its adhesive properties, soluble Pra1p acts as a chemoattractant of PMNs (88). The need for ␣M2 engagement in this process was demonstrated by testing the migration of WT and ⌬␣M PMNs toward germinated cells of C. albicans. WT PMNs migrated well to the fungus (Fig. 1C), whereas the ⌬␣M PMNs showed little propensity to migrate (7% ⫾ 5% compared to WT PMN migration). In the presence of the pra1 deletion mutant, neither WT nor ⌬␣M PMNs demonstrated any migration over background (5% ⫾ 5% and 3% ⫾ 5%, (P ⬎ 0.05, Student t test), compared to WT PMN migration to control the CAI-12 C. albicans strain, respectively. PRA1 reinsertion into the C. albicans mutant restored the fungal attraction of WT PMNs, with migration occurring at the level observed with CAI-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/␣M2 cells (90). The ␣M2-expressing cells adhered and migrated to the control CAI-12 and reintegrant C. albicans strains but not to the ⌬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 toward ICAM-1 or IgG-Fc (P ⬎ 0.1) (Fig. 1D). Since ⌬␣M PMNs retain the ability to adhere and migrate to other ligands (see references 28 and 57), we conclude that adhesion and chemotaxis of PMNs toward C. albicans cells depends specifically on Pra1p-␣M2 interaction. Effect of ␣M2-Pra1 interaction on PMN oxidative burst. Previous studies had shown that, unlike bacterial and some other fungal pathogens, successful killing/phagocytosis of C. albicans by PMNs requires the oxidative burst, and mice deficient in myeloperoxidase demonstrated significantly impaired defense against C. albicans infections (8, 9). In a subsequent series of experiments, we compared the effects of Pra1 and/or ␣M2 elimination on superoxide ion production (oxidative burst) using peripheral blood PMNs (102). As a positive control, WT PMNs were activated with GM-CSF/fMLP, an ␣M2independent activation mechanism (32, 47, 83), and their level of superoxide production was assigned a value of 100%. As shown in Fig. 3A, WT and ⌬␣M PMNs upon activation with GM-CSF/fMLP demonstrated similar level of peroxide production (P ⬎ 0.05, Student t test). Incubation with control CAI-12 strain of C. albicans affected only WT PMNs; peroxide production increased from 8% ⫾ 5% to 78% ⫾ 18% (P ⬍ 0.01, Student t test). In contrast, incubation with ⌬Pra1 C. albicans had little effect on the oxidative burst: PMNs of both lines increased peroxide production by 10% only. The results indicate that despite the lack of ␣M2, the PMNs are still capable of peroxide production upon activation by ␣M2-independent agonists, and ␣M2-Pra1 interaction plays an essential role in oxidative burst in response to C. albicans. Effect of ␣M2-Pra1 interaction on neutrophil degranulation. Degranulation of granulocytes upon contact with a pathogen is an important mechanism of the innate immune response for destruction of pathogens. In our experiments, the extent of PMN degranulation in response to C. albicans was determined as the extent of intracellular retention of -glucuronidase (-GU) upon encounter with the fungus. The -GU level in unstimulated peripheral blood PMNs from WT mice was as-
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FIG. 3. Effect of ␣M2 or Pra1p elimination on peripheral blood PMN secretory functions. The values obtained with control murine WT PMNs are presented as black bars, and the values obtained with mutant ⌬␣M PMNs are presented as white bars. In each panel, asterisks indicate P ⬍ 0.05, as calculated by ANOVA. (A) Effect of ␣M2 and Pra1p elimination on peripheral blood PMN oxidative burst. Peripheral blood PMNs (2 ⫻ 104) were incubated with germinated CAI-12 or ⌬Pra1 C. albicans or with the non-␣M2-dependent activator GM-CSF/fMLP. Subsequently, peroxidase substrate 2,7-dichlorodihydrofluorescein diacetate was added, and the fluorescence of the samples was measured. For control, PMNs incubated 30 min without GM-CSF or C. albicans was used. Background fluorescence from C. albicans was subtracted, and the data were normalized to the peroxide concentration obtained from peripheral blood PMNs of WT mice after stimulation with GM-CSF/fMLP in the absence of C. albicans, which was assigned the value of 100%. The data are expressed as means of peroxide production ⫾ the SD of two independent experiments with triplicates in each experiment. (B) Effect of ␣M2 and Pra1p elimination 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, samples were pelleted, PMNs were lysed with 0.2% Tween, and the fungi and cell debris were removed by a second centrifugation. Subsequently, phenolphthalein glucuronic acid was added, and samples were incubated overnight. The reaction was stopped with glycine (pH 10.4), and absorbance was read at 540 nm. As controls, PMNs were used without C. albicans or GM-CSF/fMLP coincubation. The data obtained from nonstimulated WT peripheral blood PMNs in the absence of C. albicans or agonists were assigned a value of 100% -GU granule retention. The data are expressed as mean of -GU granule retention ⫾ the SD of two independent experiments with triplicates in each experiment.
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FIG. 4. Effect of Pra1p and ␣M2 on C. albicans killing/phagocytosis by murine PMNs. Peritoneal PMNs (3 ⫻ 106) were incubated with 6 10 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 dilutions of the PMN/C. albicans mixture were spread onto duplicate agar plates and the CFU were enumerated. The data are expressed as means (n ⫽ 5) ⫾ the SD. As controls, initial PMN-C. albicans suspensions were plated at time zero and assigned a value of 100%. Asterisks indicate P ⬍ 0.05, and double asterisks indicate P ⬎ 0.05 calculated by ANOVA.
signed the value of 100% -GU, corresponding to 0% degranulation. Upon activation with GM-CSF/fMLP, both WT and ⌬␣M blood PMNs demonstrated a substantial loss of intracellular -GU, to 22% ⫾ 8% and 27% ⫾ 6%, respectively (P ⬎ 0.05, Student t test) comparing to -GU levels in unstimulated PMNs (Fig. 3B). These results demonstrate that ⌬␣M PMNs can release their granule contents in response to agonists. After incubation with CAI-12 C. albicans, -GU in WT PMNs decreased to 39% ⫾ 11% (P ⬍ 0.05), whereas the response to CAI-12 by ⌬␣M PMNs was insignificant (98% ⫾ 16%, P ⬎ 0.05). In contrast, ⌬Pra1 fungus had no effect on -GU release by either WT or ⌬␣M PMNs: 94% ⫾ 18% and 97% ⫾ 19% (P ⬎ 0.05) for WT and ⌬␣M PMNs, respectively (Fig. 3B). Effect of ␣M2 and Pra1p on PMN-mediated killing of C. albicans. Fungal killing by PMNs paralleled their adhesive interactions. PMN from WT and ⌬␣M mice were incubated for 6 h with germinated C. albicans of all three strains, and the number of viable cells was quantified as CFU. Under these conditions, only 32% ⫾ 14% of CAI-12 C. albicans survived after incubation with WT PMNs. In contrast, 88% ⫾ 14% of the ⌬Pra1 C. albicans strain remained viable (P ⬍ 0.05, Student t test). Restoration of Pra1p in the reintegrant strain reimposed 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 PMNs derived from the ⌬␣M mice, fungal viability remained ⬃80% whether the C. albicans target strains did or did not express Pra1p. Thus, recognition of Pr1p by ␣M2 is pivotal for the efficient killing of C. albicans by murine PMNs. These differences were not attributable to an altered growth rate or germination of the mutant strain since these were comparable to the WT strain. To evaluate the impact of PRA1 deletion on growth rate, the WT and ⌬Pra1 C. albicans strains were resuspended at 105 cells/ml in RPMI 1640 medium containing 0.1 M HEPES (pH 7.8) and incubated in plastic tubes at 37°C with slow agitation. At 4, 8, and 16 h, the samples were diluted with PBS and spread on SDA plates, and the numbers of colonies
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were counted after 48 h of incubation at 30°C to determine the CFU. Only a 3 to 5% difference in the CFU content was found after 16 h of incubation, and the amounts of CFU after 4 and 8 h were indistinguishable and practically identical for both strains (results not shown). As can be seen on Fig. 2, PRA1 disruption also had no effect on the mutant fungus germination. Pra1p modulates ␣M2-mediated early response to C. albicans. An important element of the host response to pathogens is the recruitment of leukocytes to sites of infection. Among all leukocyte populations, cells recruited to sites of infection or inflammation within the first 6 h are predominantly PMNs (⬎95% of total leukocytes recruited) (73). The observed deficits in in vitro chemotaxis suggested that the interaction of Pra1p and ␣M2 may be critical for the attraction of PMNs and killing of C. albicans cells during the early stages of infection. To evaluate this prediction, we turned to an acute peritoneal sepsis model in mice. In this analysis, mice of both WT and ⌬␣M lines were challenged with 105 C. albicans intraperitoneally. After 6 h, mice were euthanized, and the peritoneal cavity was lavaged with PBS and subsequently with PBS containing 0.5% Tween 80 to solubilize and extract adherent invading fungi. These lavages were assayed separately (to avoid lysis of leukocytes from the first PBS lavage by Tween from second PBS-Tween lavage) for fungal CFU and normalized to values obtained by lavaging immediately after C. albicans injection. As shown in Fig. 5A, the total CFU recovered following the infection of WT mice was significantly altered by the presence or absence of Pra1p. In the absence of Pra1p, the number of recovered viable cells increased 4-fold from 16% ⫾ 10% for CAI-12 C. albicans to 65% ⫾ 12% for the pra1-null mutant (P ⬍ 0.05, WMW U-test). With restoration of PRA1 into the fungus, CFU levels again attained those observed with CAI-12 C. albicans (18% ⫾ 8%, P ⬎ 0.05). In the complementary experiment, mice lacking ␣M2 were infected. The recovery of viable fungal cells from these mice was insensitive to the presence or absence of Pra1p. Importantly, the recovery values, 65 to 75%, were essentially identical to the values obtained for the pra1 mutant in WT mice (P ⬎ 0.05). To test fungal invasion of organs, parts of murine peritoneal epithelia were removed from mice 6 h after fungal administration without prior PBS-Tween lavage, and the CFU within homogenates of these tissues were determined to assess differences in fungal adherence. The elimination of Pra1p caused significant increases in fungal cell adherence to all murine organs assayed in both control WT and KO mice. In Fig. 5B, we present the results of fungal adhesion to peritoneal epithelium (expressed as the total CFU found in the tissue homogenate). Pra1p elimination increased the number of CFU found in this organ from all mouse strains by 8- to 10-fold (P ⬍ 0.01). This pattern was consistent with the CFU distribution in PBS and Tween lavages; namely, with the loss of Pra1p, most recovered C. albicans CFU were adherent to the epithelium, requiring Triton to recover the fungal cells in the lavages (Fig. 5C). Role of Pra1 and ␣M2 on PMN recruitment to C. albicans. The differences in fungal survival could reflect either decreased killing of C. albicans by PMNs attracted to the infection site or a lack of PMN recruitment. Accordingly, we sought to determine the impact of Pra1p and ␣M2 deletion on leu-
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FIG. 5. Effect of Pra1p and ␣M2 on early stages of acute fungal peritonitis. A total of 105 C. albicans of CAI-12 (black bars), ⌬Pra1p (white bars), and reintegrant strains (hatched bars) were allowed to germinate for 1 h and injected into the peritoneal cavity of WT (left group of bars) or ⌬␣M (right group of bars) mice. After 6 h, mice were euthanized, and the peritoneal cavities were lavaged with 4 ml of D-PBS, followed by 4 ml of D-PBS containing 0.5% Tween 80 (PBSTween). Serial dilutions of each lavage were plated for CFU determination, and values were normalized to the injected suspension. The CFU recovered from each group of mice (n ⫽ 8) are expressed as mean percent ⫾ the SD. The single asterisk indicates P ⬍ 0.05, and the double asterisk indicates P ⬎ 0.05 calculated by ANOVA. In panel A is shown as a percentage of total inoculum the total number of recovered viable C. albicans in combined PBS ⫹ PBS-Tween lavages for each fungal-mice group (n ⫽ 8), and the fractional number of tissueadherent C. albicans from PBS-Tween lavage only is shown in panel C. In panel B are present results of separate control experiment, verifying that cells found in PBS-Tween lavages in fact were adherent to the peritoneal epithelia. Murine peritoneal epithelia were removed from the mice without previous lavage, weighed, homogenized, and plated at different dilutions onto agar plates. The number of C. albicans adherent to the epithelia was determined as CFU/g of the epithelial tissue. The data are presented as means of three independent experiments, three mice per group ⫾ the SD.
kocyte recruitment to C. albicans in the peritoneal cavity. Total leukocytes and PMNs were quantified in an intraperitoneal lavage of either WT or ⌬␣M mice 6 h after intraperitoneal administration of 105 nongerminated C. albicans. The PMNs in the lavage were quantified by myeloperoxidase activity. The number of resident peritoneal PMNs, determined by the activities of these enzymes in the absence of thioglycolate and/or C. albicans administration, was assigned a value of 100% the observed levels of resident PMNs and leukocytes in both mouse lines were similar (results not shown). By these criteria PMNs 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 recruit PMNs above background levels (P ⬎ 0.05, Fig. 6). Similarly, elimination of ␣M2 in the deficient mice almost completely abolished PMN recruitment. Similar results were obtained when total leukocytes were quantified in the lavage using a nonspecific intracellular esterase activity assay, indicating that PMNs constituted the majority of leukocytes attracted to the site of infection (Fig. 6). Thus, the absence of Pra1p or ␣M2 had equivalent effects, limiting PMN recruitment in vivo.
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FIG. 6. Leukocyte recruitment to C. albicans in WT and ⌬␣M mice. C. albicans (105) of CAI-12 or ⌬Pra1p strains were allowed to germinate for 1 h in RPMI 1640 media at 37°C and then injected into the peritoneal cavities of WT and ⌬␣M mice. After 5 h mice were euthanized and peritoneal lavages were collected by sequentially lavaging peritonea with PBS (first lavage) and then PBS plus 0.5% Tween 20 (second lavage). The lavages were plated separately in a series of dilutions onto SDA plates to enumerate surviving C. albicans as CFU. Leukocytes were determined in the first lavage (PBS) by enzymatic assay of neutrophil myeloperoxidase activity (PMNs only, white bars) or total leukocytes (predominantly PMNs, macrophages, and some monocytoid cells) by nonspecific intracellular esterase activity (black bars). Standard curves were developed from the lysates of a known number of leukocytes/PMNs for cell quantitation. Resident leukocytes were measured in the lavages obtained from noninfected mice and were assigned as 100%. The results are presented as means of three independent experiments, three mice per group, and are expressed as the fractional percent increase over control (uninfected) resident leukocytes/PMNs ⫾ the SD. Asterisks indicate P ⬍ 0.05 and double asterisks indicate P ⬎ 0.05 calculated by using the Student t test pared to the same murine line.
Elimination of Pra1p or ␣M2 increases C. albicans virulence in a murine model of systemic candidiasis. To examine the significance of the Pra1p-␣M2 interaction in the context of the total host-pathogen relationship, the mutants were studied in a murine model of disseminated candidiasis. When WT mice were challenged with 3 ⫻ 105 CAI-12 strain C. albicans cells 50% of the mice reached endpoint morbidity or mortality (see Materials and Methods for a complete list of criteria) in 152 ⫾ 14 h (Fig. 7A). Eliminating Pra1p from C. albicans dramatically decreased this time, since 50% of the mice introduced to 3 ⫻ 105 ⌬Pra1 C. albicans died in the range of 70 to 88 h. Genetic reintegration of the mutant restored to near WT values, 142 ⫾ 16 h (P ⫽ 0.096 compared to wild-type, log-rank test), confirming association of the virulence phenotype with PRA1. The complementary side of the Pra1p-␣M2 interaction was examined using ␣M2 KO mice. When challenged with CAI-12 C. albicans, 50% lethality occurred at 82 h in the ⌬␣M mice compared to 152 h in the WT mice (P ⬍ 0.05) (Fig. 7A). Mice lacking ␣M2 showed a similar enhanced susceptibility to each C. albicans strains tested. The median survival time was 70 h for mice given the ⌬Pra1p strain and 88 h for mice given the reintegrant Pra1p C. albicans. There was no statistically significant difference (P ⬎ 0.05, log-rank test) between these values, demonstrating that Pra1p was without influence in the absence of ␣M2. Importantly, these values were nearly identical to those of WT mice infected with the Pra1p mutant (P ⫽ 0.096), suggesting that the protective role of ␣M2 was nullified by the absence of Pra1p (Fig. 7A and B). The enhanced virulence of the pra1 mutant was confirmed by examining the dose response. Mice were challenged with a range of C. albicans doses from 5 ⫻ 104 to 5 ⫻ 106 cells/mouse. At all tested doses, the
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FIG. 7. Effect of leukocyte integrin ␣M2 on C. albicans virulence in a murine model of disseminated candidiasis. (A) Effect of Pra1p and ␣M2 elimination 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. After administration, the mice were inspected on an (8 ⫾ 2)-h basis and euthanized when they became moribund (e.g., at 20% weight loss). The average survivors in each group (n ⫽ 8) are presented as medians (25th and 75th). The P values were calculated by log-rank test. The complemented Kaplan-Meier graphic of this experiment is shown on the panel B. (C) Effect of Pra1p deletion on C. albicans virulence in WT mice. C. albicans of either WT (closed circles, solid line) or ⌬Pra1p (opened circles, dashed line) strains in the concentration range of 5 ⫻ 105 to 5 ⫻ 106 cells/mouse were administered by tail vein into WT mice. The mice were inspected every 8 h and euthanized immediately upon becoming moribund. The results are plotted as log10 of dose of yeast cells/g of mouse weight versus time to become moribund. Each point represents an individual mouse. The P values were calculated by log-rank test.
pra1 mutant produced more rapid killing than WT cells, with approximately half as many mutant as WT cells required to attain a similar time to endpoint in the mid-range of tested dosages (Fig. 7C). Elimination of Pra1p or ␣M2 increases C. albicans dissemination in the host. Because of the diverse roles of ␣M2 in containing microbial infections, the similarity in time course of ⌬pra1 and ⌬␣M2 infections may have different pathological reasons. Therefore, the extent of dissemination, organ fungal burden, and tissue targeting was assessed for all infected mice. Selected organs, kidney, lung, heart, spleen, and liver, were recovered from mice 16 and 40 h after infection, and the fungal burden was determined. Consistent with previous studies (92), the highest fungal burden was present in the kidneys and was comparable at 16 h in both lines of mice regardless of C. albicans challenge strain (P ⬎ 0.05, Student t test). Notable differences, however, occurred in other organs. In WT mice infected with the pra1 mutant, in all of infected animals notable fungal burden in the spleen, liver, and lungs appears as early as 16 h versus 40 h for the WT. At 40 h, the fungal burden in all organs 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 strain CAI-12. This same pattern of altered tissue burdens was ob-
served in ⌬␣M mice infected with either CAI-12 or pra1 mutant cells (P ⬎ 0.05 for kidney, P ⬎ 0.02 for other organs) (Table 1). These results showed that the absence of Pra1p from the pathogen or the absence of ␣M2 from the host altered dissemination of the infection to a similar extent and in a similar manner. DISCUSSION In the present study, we assessed the contributions of Pra1p and ␣M2 on the innate immune response to C. albicans infection and fungal pathogenesis. The major findings of our study are as follows. (i) The absence of ␣M2 significantly impairs the ability of murine PMNs to adhere to, migrate toward, and eliminate C. albicans by phagocytosis and/or oxidative burst in vitro but has no or has limited effect on the phagocytic functions of neutrophils, such as ␣M2-independent adhesive and migratory abilities, degranulation, and peroxide ion production. (ii) The absence of ␣M2 led to a major decrease in host resistance to C. albicans infection in both employed murine models of acute fungal peritonitis and hematogenously disseminated candidiasis. (iii) Deletion of Pra1p from C. albicans decreased PMN adhesion, migration, degranulation, oxidative burst, and fungal killing and increased viru-
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TABLE 1. Distribution of C. albicans within murine organs after the introduction of 3 ⫻ 105 C. albicans at 16 and 40 h of infection and at the endpointa C. albicans in murine organs (mean CFU/g of the indicated tissue ⫾ SE)b Time of examination and organ CAI-12
a b
⌬␣M mice
WT mice ⌬Pra1
CAI-12
⌬Pra1
16 h Kidney Spleen Liver Lungs Heart
(2.1 ⫻ 103) ⫾ 320 ND 100 ⫾ 80 80 (n ⫽ 4/6) ND
(2.2 ⫻ 103) ⫾ 300 260 ⫾ 120* 340 ⫾ 80* 360 ⫾ 80* ND
(2.4 ⫻ 103) ⫾ 420 320 (n ⫽ 4/6)* 220 (n ⫽ 3/6) 320 (n ⫽ 3/6)* ND
(2.3 ⫻ 103) ⫾ 600 400 ⫾ 120* 220 ⫾ 120 380 ⫾ 140* ND
40 h Kidney Spleen Liver Lungs Heart
(1.3 ⫻ 104) ⫾ (3 ⫻ 103) (3.2 ⫻ 103) ⫾ (1 ⫻ 103) (1.3 ⫻ 103) ⫾ 600 (2.1 ⫻ 103) ⫾ 800 ND
(6.9 ⫻ 104) ⫾ (1 ⫻ 104)* (3.3 ⫻ 104) ⫾ (1 ⫻ 104)* (1.4 ⫻ 104) ⫾ (2 ⫻ 103)* (5.2 ⫻ 104) ⫾ (1 ⫻ 104)* ND
(8.3 ⫻ 104) ⫾ (2 ⫻ 104)* (5 ⫻ 104) ⫾ (5 ⫻ 103)* (9 ⫻ 103) ⫾ (2 ⫻ 103)* (8.8 ⫻ 103) ⫾ (2 ⫻ 103)* 40 (n ⫽ 1/6)
(1.1 ⫻ 105) ⫾ (3 ⫻ 104)* (1.3 ⫻ 105) ⫾ (3 ⫻ 104)* (5.2 ⫻ 104) ⫾ (1 ⫻ 104)* (4.4 ⫻ 104) ⫾ (1 ⫻ 104)* ND
The endpoints were 180 ⫾ 60 h for WT CAI-12 and 84 ⫾ 36 h for all other strains. *, P ⬍ 0.05. ND, not determined. n, Number of animals.
lence, and these changes were all ␣M2 dependent. (iv) Removal of either ␣M2 or Pra1p significantly reduced leukocyte recruitment at the early stage of fungal infection. Together, these observations show the critical importance of recognition of fungal Pra1p by leukocyte integrin ␣M2 and that this interaction significantly contributes to the success of the host defense against C. albicans infection, especially on the initial stages of the infection. Two distinct murine models of disease, disseminated candidiasis and acute peritoneal sepsis, demonstrated increased C. albicans virulence upon deletion of the PRA1 gene. The PRA1null mutant induced lethality more rapidly (1.5- to 2-fold faster) than the same inoculum of WT C. albicans. The data of the fungal burden in the primary target organ of infection, the kidney (46, 78), support the virulence data. The early time point data (16 h) show similar kidney fungal burdens in all animals, indicating that the kidneys were seeded at similar levels, a finding consistent with an equivalent inoculum. At 40 h, there are statistically significant differences between WT mice infected with CAI-12 versus the ⌬Pra1 mutant and between WT mice infected with CAI-12 and ⌬␣M mice infected with either CAI-12 or the Pra1 mutant (P ⬍ 0.05 in all cases), and this correlates with a higher survival of WT mice infected with control C. albicans strain. The 5-fold difference in kidney was associated with increased 10- to 20-fold fungal burden in other organs (P ⬍ 0.02), this difference in dissemination appeared as early as the 16-h time point. Results from the acute peritonitis model of early stage infection suggest that this enhanced virulence may arise from suppressed fungal-leukocyte interaction. In mice that lack ␣M2, this difference in virulence between Pra1p-positive and -negative strains of C. albicans was no longer observed; disease progression and fungal burden in ⌬␣M mice was similar for both C. albicans strains. Since these strains have similar in vitro growth rates, these results suggest that the innate response fails to recognize and clear fungal cells lacking Pra1p. We cannot exclude the possibility that the in vivo growth rate of the mutant exceeds that of the WT, but a 4-fold enhancement
seems unlikely. These findings support our conclusion that both ␣M2 and Pra1p are involved in fungal recognition. The observed effect of ␣M2 elimination on C. albicans virulence is consistent with previous reports demonstrating that patients with leukocyte adhesion deficiency type 1 (LAD-1) syndrome showed increased susceptibility to C. albicans infection (7, 45, 50, 53, 86). LAD-1 syndrome is a rare hereditary disease characterized by increased susceptibility to infections due to the inability of leukocytes, in particular neutrophils, to migrate from the blood to sites of inflammation. The increased susceptibility of LAD-1 patients to C. albicans infection is ascribed to the significant impediment of leukocytes to form pus at the site of infection (7, 50) and to the reduced ability of LAD-1 neutrophils to kill the fungus. Lau et al. reported that neutrophils of a LAD-1 patient showed a 3-fold reduction in C. albicans killing in vitro, whether opsonizing or nonopsonizing killing was involved (53), and we recapitulated these results using ⌬␣M PMNs. LAD-1 arises from an absence of all beta-2 integrins on leukocytes caused by mutations in the 2 gene (4, 7). Unlike ⌬2 leukocytes, ⌬␣M leukocytes are still able to migrate to the sites of infection and inflammation using other 2 integrins, primarily ␣L2 (28, 57). The similarity in the migration rates of WT and ⌬␣M PMNs to thioglycolate-induced intraperitoneal inflammation, described in the literature (57) and observed by us, taken together with the failure of leukocytes to migrate to the peritoneal cavity of ␣M2-deficient mice 6 h after peritoneal infection with C. albicans, suggests that ␣M2 is the integrin responsible for leukocyte recruitment to C. albicans. The absence of neutrophil recruitment to ⌬Pra1 C. albicans in WT mice further indicates that Pra1p is a major ligand for leukocytes among C. albicans proteins and that ␣M2 is a major receptor for this protein (38, 39, 88). It is possible that PMNs can utilize ␣M2 for binding also to other ligands on the C. albicans surface. As seen on Fig. 2B, PE-labeled isolated ␣M2 slightly binds to the ends of some hyphae, probably by low-affinity interaction to fungal cell wall mannans through its lectin domain (100, 101, 105). Residual killing of ⌬Pra1 C. albicans by ⌬␣M PMNs also may be medi-
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ated by resident peritoneal macrophages and/or dendritic cells. Indeed, an ␣M2-independent mechanism for C. albicans recognition has been ascribed to macrophages and dendritic cells (30, 67), and macrophages have been shown to bind and engulf C. albicans in a Pra1p-independent fashion (60). Other leukocyte receptors that can bind fungal polysaccharides—Dectin-1, TLR2, (13, 68), and TLR4 (69)—also may be involved in fungal recognition, but they do not directly mediate leukocyte migration, adhesion, killing, and phagocytosis. Also, Dectin-1 and TLR2 recognize yeast forms of C. albicans only (41), and therefore they may play only minor roles in fungal elimination by PMNs, which can kill and/or phagocytose C. albicans filamentous forms only (36, 37, 39). Another notable finding in the presenting study is the observation of increased adhesion of Pra1 ⫺/⫺ C. albicans to peritoneal endothelia in the murine model of acute peritonitis (Fig. 5B). It indicates that the Pra1p may participate in the regulation of the fungal invasion and colonization by inhibition fungal adhesion to the host tissues. As noted above, the exact role of Pra1 in C. albicans physiology is still unclear, and the earlier observations that the Pra1 expression is environmentally and/or conditionally dependent and is regulated by nutrition (3, 19, 84) support this conclusion. The enhanced adhesiveness of the Pra1-depleted C. albicans may also contribute to increased virulence of the mutant fungus, but the difference in virulence between Pra1⫹/⫹ and Pra1⫺/⫺ C. albicans strains in ␣M2-KO mice and the CAI-12 strain in WT mice (Fig. 7A) were insignificant (P ⬎ 0.05 [ANOVA]), and the similar levels of organ fungal burdens at 16 h in these murine-fungal combinations (Table 1, P ⬎ 0.05 [ANOVA]) demonstrate that even if this contribution really takes a place, it plays minor role in increased fungal virulence of Pra1-KO fungus compared to the disruption of Pra1-␣M2 interaction. Thus, our results clearly demonstrate that recognition of Pra1p by ␣M2 plays a major role in determining fungal virulence and host protection against C. albicans infection. ACKNOWLEDGMENTS This study was supported by grant AIO 80596 from the National Institute of Allergy and Infectious Diseases and in part by grant P50 HL081011 from the National Institutes of Health. ⌬␣M-KO mice were kindly provided by Christy M. Ballantyne of Baylor College of Medicine, Houston, TX. We thank Edward Plow and Elzbieta Pluskota for productive discussions of results and Carla Drumm for help with mouse husbandry and organ collection. REFERENCES 1. Abelson, J. A., T. Moore, D. Bruckner, J. Deville, and K. Nielsen. 2005. Frequency of fungemia in hospitalized pediatric inpatients over 11 years at a tertiary care institution. Pediatrics 116:61–67. 2. Aldred, A. J., M. G. Cha, and K. A. Mechling-Gill. 2002. Determination of a humane endpoint in the L1210 model of murine leukemia. Contemp. Top. Lab. Anim. Sci. 41:24–27. 3. Alloush, H. M., J. L. Lopez-Ribot, and W. L. Chaffin. 1996. Dynamic expression of cell wall proteins of Candida albicans revealed by probes from cDNA clones. J. Med. Vet. Mycol. 34:91–97. 4. Anderson, D. C., et al. 1985. The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J. Infect. Dis. 152:668– 689. 5. Anderson, D. C., et al. 1985. Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies of three kindreds. Fed. Proc. 44:2671–2677. 6. Anderson, D. C., and T. A. Springer. 1987. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu. Rev. Med. 38:175–194.
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