Vol. 21, No.2
INFECTION AND IMMUNITY, Aug. 1978, p. 506-513 0019-9567/78/0021-0506$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Inhibition of Specific Amino Acid Uptake in Candida albicans by Lysosomal Extracts from Rabbit Alveolar Macrophages El L ENA M. PETERSON' AND R. A. CALDERONE'* Department of Pathology, School ofMedicine, University of California at Irvine, Irvine, California 92717,' and Department ofMicrobiology, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. 200072
Received for publication 17 April 1978
Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine, valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and RbV. Leakage of 'Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract. Phagocytic cells possess a number of mechanisms that contribute to the inactivation of microorganisms. In general, they fall into two main categories, oxidative and nonoxidative (4, 9-12, 15, 17, 19, 22-24). Numerous studies have focused on the components of these microbicidal systems, but relatively few studies have characterized the exact manner by which these systems ultimately cause inactivation of the invading organism.
Odeberg and Olsson (15) have investigated the mechanism by which cationic proteins from human granulocytes killed selected bacteria. They found that bacterial incorporation of radioactive precursors into protein, DNA, and RNA was inhibited when cationic proteins were present (15). Other studies have shown an increase in the permeability of the microbial envelope of Escherichia coli when incubated with cationic proteins from rabbit granulocytes (21). In previous studies, we demonstrated in vitro that rabbit alveolar macrophages (AM) phagocytized Candida albicans and inhibited its intracellular growth (16). The purpose of this study was to characterize the underlying mechanism(s) by which C. albicans was inhibited by rabbit alveolar macrophages. This was accomplished by examining the effect of lysosomal506
rich fractions obtained from rabbit alveolar macrophages on specific cellular functions of C. albicans. MATERIALS AND METHODS C. albicans The isolate of C. albicans used in this study was previously described (16). An 18-h culture of C. albicans, grown on brain heart infusion slants (370C), was collected in 0.9% saline, centrifuged, and suspended in citrate-phosphate buffer (0.02 M citric acid-0.003 M sodium phosphate [dibasic]), pH 5.6. Labeled materials. The following compounds were purchased from New England Nuclear Corp. (Boston, Mass.): L-[3H]valine, 5 Ci/mmol; L-[3H]lysine, 5 Ci/Mmol; L-[3H]leucine, 5 Ci/Mmol; L-[3H]proline, 6 Ci/mmol; L-[3H]glutamic acid, 45 Ci/Mmol; L[3H]phenylalanine, 60 Ci/mmol; L-[3S]methionine, 400 Ci/mmol; [3H]uridine, 48 Ci/mmol; 2-deoxy-D[3H]glucose, 8.3 Ci/mmol; T6rubidium as RbCl in 0.5 N HCI, 5 mCi/mg. Disruption and differential centrifugation of macrophages. AM were obtained as previously described (16), with the following modifications: rabbits were killed by the injection of air (15 ml) into the marginal ear vein, and 200 ml of sterile saline (0.9%) was used to lavage the lungs. The contents of a typical savage have been previously described (16). AM were washed and suspended in 0.25 M sucrose, and viability was determined by trypan blue exclusion. AM were then homogenized on ice in a hand-operated Potter-
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Elvehjem tissue grinder (5 ml) at 2-min intervals and checked for the percent breakage by phase-contrast microscopy. Cells were homogenized until 85 to 90% breakage was achieved as judged by the ratio of free nuclei to whole cells. The homogenate was diluted with 0.34 M sucrose to three times its volume and, after centrifugation (500 x g, 12 min), the resulting supernatant was removed and saved. The pellet was washed an additional three times, and the superna'tants were collected and pooled. The resulting pellet, as judged by phase-contrast microscopy, contained unbroken cells and nuclei. The pooled supernatant was then centrifuged at 15,000 x g, and the pellet obtained was the lysosomal-rich fraction, as determined by electron microscopy and enzymatic analysis, and referred to as the "15-g" pellet. Extraction of lysosomal-rich fraction. The 15g pellet was suspended in 1 ml of 0.34 M sucrose and subjected to six cycles of freeze-thawing. The freezethawed material was centrifuged at 15,000 x g for 12 min, and the supernatant was collected. This supernatant was dialyzed overnight in citrate-phosphate buffer (40C, 2,000 x vol). All extracts referred to as freeze-thawed (FT) were treated in this manner and stored at -200C. Enzymatic and chemical determinations. (i) Acid phosphatase. Acid phosphatase was measured by a colorimetric assay usingp-itrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) as the substrate by the method of Andersch and Szczypinski (1). A standard curve was constructed by measuring the optical density readings at 400 nm of an alkaline solution of various concentrations of p-nitrophenol (Sigma). (ii) Lysozyme. Lysozyme was measured by following the lysis of a standardized, UV-killed culture of Micrococcus lysodeikticus (Difco Laboratories, Detroit, Mich.) by the procedure of Smolelis and Hartsel (20). A standard curve was prepared by measuring the change in turbidity of the bacterial suspension when incubated with known amounts of lysozyme (Difco). Turbidity was measured at 540 nm in a Bausch and Lomb.Spectronic 20 spectrophotometer. (iii) Protein. Protein determinations were carried out by the method of Lowry et al. (13) using bovine serum albumin as a standard. Effect of FT extracts on incorporation by C. albicams. An 18-h culture of C. albicans grown on brain heart infusion slants (370C) was washed, suspended in citrate-phosphate buffer (pH 5.6), and added to tissue culture tubes (13 by 100 mm) (105 cells per tube). Varying concentrations of extract (5 to 100 pg of protein) were added in a total volume of 0.1 ml per tube. Controls received an equal volume of buffer. Subsequently, [3H]leucine (5 pCi per tube) was added to each tube. The total volume of all mixtures was 0.205 ml. All tubes were incubated (370C, 150 rpm) for 45 min, and all reactions were stopped by precipitating cultures on ice with cold 15% trichloroacetic acid. Nonspecific trapping of label was determined by labeling cultures on ice and precipitating immediately with cold 15% trichloroacetic acid. Subsequently, all precipitated cultures were filtered onto glass fiber filters (Whatman GF/A) and washed with 5% trichloroacetic acid followed by 95% ethanol. All radioactive measurements were made in an In-
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tertechnique liquid scintillation counter which had approximately a 30% efficiency for 3H on GF/A filters in a scintillation liquid of the following composition: 0.1 g of p-bis-[5-phenyloxazolyl)]benzene and 5 g of 2,5-diphenyloxazole dissolved in 1.0 liter of scintillation grade toluene. All cultures were run in duplicate, and each experiment was repeated at least once. Similar data were obtained for each experiment. Effect of FT extract on the uptake of amino acids, uridine, and 2-deoxy-n-glucose by C. albicans. Equal volumes of prewarmed citrate-phosphate buffer (pH 5.6) and FT extract (1 mg/ml) were added to 25-ml flasks, respectively. The labeled compound (amino acid, sugar, or uridine) was added, followed by C. albicans (106/ml) to give a 1:1 (vol/vol) ratio of cells to buffer or cells to extract. At appropriate intervals, in duplicate, 0.1 ml of the mixture was added to 5 ml of cold citrate-phosphate buffer containing a 100x concentration of the unlabeled compound. Dialyzed bovine serum albumin (50 to 100 pLg/0.1 ml of citrate-phosphate buffer) was also used as a control to measure lysosomal-specific inhibition of amino acid uptake. Cells were then collected on glass fiber filters (Whatman GF/A) which had been soaked in the unlabeled compound. Subsequently, filters were covered with NCS tissue solubilizer (Amersham Corp., Arlington Heights, Ill.) for 30 min, at which time toluenebased fluor (described previously) was added and the vials were counted. Viability of C. albcanm treated with extract. FT extract (50 pg of protein per 0.1 ml) in citratephosphate buffer (pH 5.6) was added to one-half of the tissue culture tubes. The remaining tubes, which served as controls, contained an equal volume of citrate-phosphate buffer. C. albicans suspended in citrate-phosphate buffer was added to all tubes (105 cells per tube). Incubations were carried out in a heating block (370C, 150 rpm). At designated times, the incubation mixtures were diluted with sterile distilled water and plated on Sabouraud-dextrose agar. Plates were incubated for 24 h (370C) and counted for colonyforming units. Effect of FT extract on "rubidium uptake and release by C. albicans. (i) Labeling of C. albians with "rubidium. A modification of the procedure of Drazin and Lehrer (3) was used. A 16-h culture of C. albicans (370C, 150 rpm) grown in Sabouraud-dextrose broth was centrifuged (3,000 x g, 10 min), washed twice, and suspended in sterile distilled water (107 cells per ml). Cells were distributed in test tubes (106 cells per tube) which contained 0.1 ml of one-eighthstrength citrate-phosphate buffer (pH 5.6). "Rubidium was diluted with distilled water, and 0.24 appropriate intervals, ACi was added to each tube. At with cold distilled waduplicate cultures were diluted ter, filtered over membrane filters (0.45-pm pore size, Millipore Corp., Bedford, Mass.), washed twice with cold distilled water, and counted in a Beckman Biogamma Counter. (ii) "Rubidium release. C. albicans was grown and washed as described above. Washed cells were resuspended in sterile distilled water (3 x 107 cells per ml). Equal volumes of cells and isotope, diluted in sterile distilled water (100 pCi/ml), were incubated at 370C (150 rpm, 2 h). Labeled cells were centrifuged
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(500 x g), washed three times with sterile distilled water to remove unincorporated isotope, and resuspended in sterile distilled water (107 cells per ml). The 86Rb release assay was carried out in heavy-walled glass test tubes (10 by 75 mm), containing either 0.1 ml of citrate-phosphate buffer (pH 5.6) or varying concentrations of FT extract in a total volume of 0.1 ml. The release assay was initiated by the addition of 0.1 ml of TMRb-labeled C. albicans (106 cells). At designated time intervals, the cell suspensions were centrifuged at 15,000 x g (10 min, 400). Supernatants were removed and 0.1 ml was counted. All samples were done in duplicate. Zero time readings were carried out by the addition of labeled cells to extract or buffer on ice, followed by immediate centrifugation and supernatant removal. Total input counts per tube were determined by counting 0.1 ml (106 C. albicans) of prelabeled cells in duplicate. 86Rb release was calculated, after all zero time counts had been subtracted, as follows: ([(counts per minute of extract-treated cells) - (counts per minute of control cells)]/[input counts per minute]) x 100. (iii) "Rb uptake. FT extract (100 pg/0.1 ml) in one-eighth-strength citrate-phosphate buffer (pH 5.6) was added to sterile tissue culture tubes. Controls received an equal volume (0.1 ml) of one-eighthstrength citrate-phosphate buffer. C. albicans, grown in Sabouraud-dextrose broth, was washed and suspended as previously described and added to all tubes (106 cells per tube), followed by 0.24 jCi of MRb. At various times, ice-cold distilled water (5 ml) was added to duplicate cultures. Immediately, cells were collected on membrane filters (Millipore Corp.), washed twice with distilled water, and counted. Zero time readings were done on ice and treated as above.
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80
e~
60 60
C
-
C 40
20
10
50 100 Extract (pg protein) RESULTS FIG. 1. Dose effect of FT extract on [3HJleucine Effect of alveolar macrophage lysosomal incorporation by C. albicans. Cells (105 per tube) were extracts on C. albicans. Lysosomal-rich frac- labeled with [3Hjleucine (5 ,uCi per tube) for 45 min tions were obtained from AM to evaluate their at 370C.
role in the inhibition of Candida. To obtain such fractions, acid phosphatase and lysozyme were used as lysosomal markers in order to evaluate the enrichment procedure. Although these enzymes are distributed throughout the cell, their greatest concentration seems to be within lysosomes. AM were disrupted until 85 to 90% breakage had been achieved. At this percent breakage, some lysosomes were still contained in unbroken cells and these appeared in the nuclear pellet. In addition, some of the released lysosomes were themselves disrupted and their contents appeared in the fraction termed "supernatant." Nevertheless, preliminary experiments indicated that this percent breakage was most favorable for obtaining lysosomes. The 15-g fraction was extracted by freezethawing. To investigate the effects of the extract on C. albicans metabolism, C. albicans [3H]leucine incorporation was measured in the presence and absence of the extract. Figure 1 illustrates the effect of various concentrations of
the extract (micrograms of protein) on [3H]leucine incorporation by C. albicans. The extract had a marked effect on leucine incorporation by Candida up to 50 yg of extract protein per 105 Candida, above which the inhibition leveled off. FT extracts (100 jg of protein per 105 cells) were also tested for their ability to affect incorporation ofother amino acids by Candida. Table 1 shows that the incorporation of the majority of amino acids tested was inhibited when compared to incorporation by control cells. There was also variation in the percent inhibition depending on the amino acid tested. However, the incorporation of proline and glutamic acid was always above the incorporation of control cells. Since the inhibition of amino acid incorporation could be a reflection of decreased uptake of the labeled amino acids by Candida, it was decided to investigate the effect of the FT extract on amino acid uptake. Uptake of methionine, valine, lysine, phenylalanine, and leucine
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AMINO ACID UPTAKE INHIBITION IN C. ALBICANS
a
were all drastically reduced compared to controls (Fig. 2a through d, Fig. 3a). However, proline uptake was comparable to the control (Fig. 3b), and glutamic acid uptake was only slightly affected (Fig. 3c). These data (expressed as percent of control) are summarized in Table 2. The effect of FT extract on uptake by Candida appears to be specific for amino acids since the uptake of 2-deoxy-D-glucose, uridine, and Rb+ TABLE 1. Incorporation of amino acids by C. albicans in the presence of FT extract % Controla Compound incorporated 6.6 ..... ...... [3S]methionine ........ 10.0 [3H]leucine ... 13.5 [3H]phenylalanine ............ ........ 35.5 [3H]valine ... .......... 59.0 [3H]lysine ............ 121.0 ..... [3H]glutamic acid ...... 125.0 ........... [3H]proline a Cells were labeled in buffer (control) or in FT extract (100 pg) for 45 min at 37"C.
E
b
E
Minutes a
20
[[35SI
[3H] L-Va line
L-Methionine
I
E
C
c
C7 40
IW
ED
509Q
Minutes
FIG. 3. (a) Uptake of i[4HJleucine by C. albicans in the presence (0) and absence (0) ofFT extract. (b) Uptake of I_[3Hlproline by C. albicans in the presence (0) and absence (0) of FT extract. (c) Uptake of z-[3HJglutamic acid by C. albicans in the presence (0) and absence (0) of FT extract.
TABLE
2. Uptake by C. albicans in the presence of FT extract % Controla Compound 4.3 ......... ......... L-[3H]phenylalanine L-[3H]lysine .......................... 9.8 10.1 ............ L-[3S]methioine ........ 12.0 L-[3H]valine ......................... L-[3Hlleucine ........................ 20.3 L-[3H]glutamic acid ........... ........ 74.8 L-[3H]proline ........................ 82.0 91.3 86Rubidium ..... 95.1 2-Deoxy-D-[3H]glucose . 112.2 [14C]Unidine aPercent control is calculated from the 10-min readings on the uptake curves.
was not affected (Table 2). As an additional control, uptake of leucine and proline was measured in the presence of bovine serum albumin. Uptake of both amino acids was similar to that observed with buffer alone. Minutes Minutes Viability of C. albicans when treated with FT FIG. 2. (a) Uptake of L-[3Slmethionine by C. alof protein per 105 cells) was deterextract bicans in the presence (0) and absence (0) of FT mined (50 ,yg counts by plate (colony-forming units per extract. (b) Uptake of z-[3Hlvaline by C. albicans in the presence (0) and absence (0) of FT extract. (c) milliliter) (Fig. 4). A significant decrease in viaUptake of z43NHlysine by C. albicans in the presence bility was not detected until 16 h after the initial (0) and absence (0) of FT extract. (d) Uptake of L- exposure of Candida to the extract. After this [3HJphenylalantne by C. albicans in the presence time, there was a steady decline in viability of (0) and absence (0) of FT extract. the cells incubated with extract. Control colony-
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PETERSON AND CALDERONE lx
25
106
0)
Lob
lx
0)
10 5
a) -
0 00
co u
0) 0-
lxI
Hours
FIG. 4. Viability of C. albicans (colony-forming units per milliliter) measured after incubation in citrate phosphate buffer (0) or FT extract (50 pg of protein per culture (0).
forming units remained constant during their prolonged incubation in buffer. The influx and efflux of TMRb was determined in the presence and absence of FT extract in order to evaluate the effect of FT extract on the ability of the cytoplasmic membrane to transport Rb+ and to exert a permeability barrier
Extract (pg protein) FIG. 5. Percent 'rubidium release by C. albicans incubated with FT extract. Percent 'rubidium release = {[(counts per minute of extract-treated cells) - (counts per minute of control cells))/[input counts per minutes) X 100.
TABLE 3. Heat stability of 15-g extract as measured by [3H]leucine incorporation by C. albicansc Sample
Counts/ min
% Control
Control 31,013 Unheated extract 13.9 4,328 Extract, 560C, 1 h 3,389 10.9 Extract, 1000C, 10 min 5,500 17.7 Extract, 1000C, 30 min 4,801 15.5 a Freeze-thawed extract, 50 jig per assay tube. bEach assay tube was labeled for 45 min at 370C with 5,UCi of [3H]leucine (60 Ci/mmol). c 105 cells per tube.
has been demonstrated in Candida that Rb+ can replace K+ in studies of membranebound ion transport (3). Preliminary studies showed that 'Rb influx in the strain of C. albicans used throughout these experiments is competitively inhibited by K+. Uptake of 'Rb+ by dida. The purpose of this study was to characCandida in the presence of the extract was terize the mechanism(s) of growth inhibition. equivalent to the controls (Table 2). However, Since the phagocytosis process is followed by leakage of 'Rb from C. albicans, preloaded with lysosomal fusion with the phagocytic vacuole isotope, was found to be increased in the pres- and the release of its contents into the phagolyence of FT extract. Figure 5 represents the dose sosome, we decided to investigate the role, if effect of FT extract on MRb leakage. any, of lysosomal contents in the inactivation of The heat stability of FT extract was assessed Candida. This approach has been used by many by utilizing the [3H]leucine incorporation assay investigators (15, 22-24) in establishing microb(Table 3). The FT extract appeared to be heat icidal mechanisms of phagocytic cells. stable, in that it maintained its ability to inhibit In retrospect, one of the major disadvantages [3H]leucine incorporation by C. albicans after of working with normal rabbit AM is the yield being boiled for 30 min. of cells obtained from one rabbit (10' cells per rabbit). This problem is further compounded DISCUSSION when one attempts not only to enrich for lysoIn a previous study, we demonstrated that the somes but also to extract them. The end result AM restricts the intracellular growth of Can- is approximately 1 mg (protein) of lysosomal function. It
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AMINO ACID UPTAKE INHIBITION IN C. ALBICANS
extract per rabbit. This was a major consideration in designing experiments to explore the mechanism(s) of inactivation. Since intact AMs affected leucine incorporation by C. albicans (16), the AM lysosomal extracts were also examined for similar effects. The extracts did have an inhibitory effect on [3H]leucine incorporation by C. albicans. Since the decrease in incorporation could be a reflection of an effect on uptake, the uptake of several amino acids was measured with and without the FT extract. We found that lysine, methionine, phenylalanine, and valine uptake was significantly inhibited by FT extract, but glutamic acid and proline uptake was unaffected. Similarly, in the presence of FT extract, incorporation of proline and glutamic acid in trichloroacetic acidprecipitable material was unaffected, whereas the other amino acids mentioned above were inhibited. These studies indicated that protein synthesis by C. albicans was not affected by FT extract but rather appeared that way due to an inhibition in the uptake of the labeled amino acid under question. If protein synthesis were truly affected, one would not expect any labeled amino acid to give incorporation data equal to controls. Since this was observed with proline and glutamic acid, protein synthesis, at least initially (45 min), is not affected by FT extract but rather some amino acids are hampered in their entry into the cell. As mentioned above, methionine, lysine, valine, phenylalanine, and leucine uptake was lower in extract-treated cells when compared to controls. However, uptake of glutamic acid and proline was only slightly or not at all affected. Grenson et al. (7) presented evidence for the existence of a general amino acid permease in Saccharomyces cerevisiae. They found that all amino acids examined, except proline and glutamic acid, were transported by this general amino acid permease. They reasoned that proline was not transported by the general permease due to its configuration, and that the second carboxyl group of the dicarboxylic amino acids seemed to hinder transport. In addition to this general amino acid permease, specific permeases for arginine (8), lysine (6), methionine (5), and proline (14) have been described in yeast. Grenson et al., in attempting to distinguish between specific and general amino acid permeases, were able to depress the general permease with a high concentration of ammonium ions (7). They measured uptake of amino acids with and without the ammonium ions and found that, for all amino acids tested except glutamic acid, uptake increased approximately 10-fold when ammonium ions were depleted from the culture media. Proline was not
511
tested for it has been shown in Saccharomyces chevalieri (14) that the proline permease is also affected by ammonium ions. Grenson et al. also isolated a mutant (gap) which had lost the general amino acid permease. Uptake in the gap mutant was not greater in the absence of ammonium ions, as was seen in the wild type. However, in the absence of ammonium ions, there was no difference in the uptake rate of proline between the wild-type strain and the gap mutant. This, coupled with the fact that proline, even at high concentrations, did not inhibit the uptake of amino acids by the general amino acid permease, suggested that it was not a substrate of the general permease transport system. Therefore, it can be postulated that FT extract interferes with a general amino acid permease of C. albicans. This interference ofuptake appeared to be unique to amino acids for 2deoxy-D-glucose, uridine, and Rb+ showed no decrease in uptake. The low level of uptake present for valine, lysine, leucine, methionine, and phenylalanine probably represents uptake by their specific permeases. These specific amino acid transport systems have been shown to be low-velocity constitutive systems (18). Uptake by FT extract-treated cells is depressed about 10-fold in comparison to control cells, and this number correlates well with the findings of Grenson et al. that, without the general amino acid permease, uptake was decreased 10-fold (7). Uptake studies with C. albicans are sparse. In preliminary studies, we have found that phenylalanine inhibits uptake of leucine whereas proline does not, indicating the existence of such a gap transport system. The dimorphic fungus Histoplasma capsulatum appears to differ from Saccharomyces sp. and C. albicans in that leucine and proline seem to share a common transport system (2). Our data for H. capsulatum indicate that both proline and leucine uptake are inhibited by the AM extracts. Odeberg and Olsson (15) studied the effect of the cationic components of a lysosomal-rich fraction obtained from human granulocytes on leucine incorporation by E. coli and Staphylococcus aureus. They found that the cationic proteins inhibited leucine incorporation in these organisms. They did not do uptake studies but postulated that the inhibition in the incorporation of radioactive precursors could be due to inhibition of the transport of the radioactive precursors or inhibition of macromolecular synthesis. Therefore, components which contribute to the bactericidal mechanism of human granulocytes might be similar to the AM in which uptake of the phagocytized microorganism is drastically affected. In the studies done with intact AM (16), it
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PETERSON AND CALDERONE
was found that the growth of Candida was retarded since the cells did not germinate, and that [3H]leucine incorporation in the phagocytized yeast was dramatically decreased. Although it has not been established whether the contents of the FT extract are active in the phagolysosome, one can postulate that, by restricting uptake of amino acids by the phagocytized yeast, the AM could effectively restrict the growth of the fungus. In other studies, the viability of Candida incubated with FT extracts was decreased. However, this decrease took a relatively long time (16 h) to manifest itself, so that the process responsible for killing is a slow one. Rubidium has been used in bacteria (15) and Candida (3) as an analog of K+ in studying membrane transport and damage. Since a very slow rate of killing of Candida with FT extract was observed, any membrane damage that might occur early after exposure to the extract would be slight. Therefore, a sensitive seRb release assay was used to detect any membrane damage. Cells treated with FT extract gave evidence of seRb leakage. This release of seRb increased with increasing amounts of extract used. However, FT extract had no effect on seRb uptake of C. albicans. Therefore, although not affecting entry of seRb into the cell, FT extract affected the capacity of the cytoplasmic membrane to retain intracellular TMRb. Likewise, Odeberg and Olsson (15) found that E. coli treated with cationic protein from neutrophils behaved in a similar fashion. This indicated that FT extracts damage the membrane, and this could be a major factor affecting the viability of C. albicans. If the leakage were slow but steady, then this could account for the lag before detecting a decrease in viability by plate counts. The inhibitory component(s) in the AM FT extract was heat stable. Heat-stable, microbicidal lysosomal components, obtained from polymorphonuclear leukocytes, have been reported by others (11, 15). Azurophil granules, obtained from human polymorphonuclear leukocytes, contained a heat-stable, bacteriostatic component and a heat-labile bactericidal component
(17).
Many components within the AM can contribute to the stasis or even death of C. albicans. Certainly, our findings concerning the uptake of amino acids by Candida treated with extract reveal a static or even a cidal mechanism within the AM. Stasis of the invading organism is critical if the AM is to resist overgrowth by Candida or other fungi. If the AM does not kill the ingested organism, it must restrict its growth until the AM can be cleared from the alveoli or
INFECT. IMMUN.
until other cells of the immune system can be recruited. The ability of the FT extract to affect the cytoplasmic membrane of C. albicans is yet another candidate for a candidacidal mechanism in vitro. It is reasonable to assume that multiple mechanisms of resistance have evolved within the AM. ACKNOWLEDGMENTh E. P. was supported by Public Health Service training grant A10028-11 from the National Institute of Allergy and Infectious Diseases. This investigation was supported by grants from the Brown-Hazen Fund of the Research Corporation of America and the Washington Heart Association. We thank T. Sreevalsan for his suggestions and Stephanie Coleman for typing the Manuscript.
LITERATURE CITED 1. Andersch, M. A., and A. J. Szczypinski. 1947. Use of p-nitrophenyl phosphate as the substrate in the determination of serum acid phosphatase. Am. J. Clin. Pathol. 17:571-574. 2. Dabrowa, N., and D. H. Howard. 1976. Uptake of Lproline by Histoplasma capsulatum. Can. J. Microbiol. 22:1188-1190. 3. Drazin, R. E., and R. I. Lehrer. 1976. TMRubidium release: a rapid and sensitive assay for amphotericin B. J. Infect. Dis. 134:238-244. 4. Drazin, R. E., and R. L. Lehrer. 1977. Fungicidal properties of a chymotrypsin-like cationic protein from human neutrophils: adsorption to Candida parapsilosis. Infect. Immun. 17:382-388. 5. Gits, J., and M. Grenson. 1967. Multiplicity of the amino acid permeates in Saccharomyces cerevisiae. Inl. Evidence for a specific methionine-transporting system. Biochim. Biophys. Acta 135:507-516. 6. Grenson, M. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. II. Evidence for a specific lysine-transporting system. Biochim. Biophys.
Acta 127:339-346. 7. Grenson, M., C. Hou, and M. Crabell. 1970. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease.
J. Bacteriol. 103:770-777. 8. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of the amino acid permeates in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127:325-338. 9. Hirsch, J. G. 1960. Further studies on the preparation and properties of phagocytin. J. Exp. Med. 111:323-337. 10. Lehrer, R. I. 1975. The fungicidal mechanism of human monocytes. I. Evidence for myeloperoxidase-independent candidacidal mechanisms J. Clin. Invest.
55:338-346.
11. Lehrer, R. I., K. M. Ladra, and R. B. Hake. 1975. Non-
oxidative fungicidal mechanisms of mammalian granulocytes: demonstration of components with candidaci-
dal activity in human, rabbit, and guinea pig leukocytes. Infect. Immun. 11:1226-1234. 12. Leigh, P. C. M., M. T. Van den Barsellar, and P. VanFurth. 1977. Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes. Infect. Immun. 17:313-318. 13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
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14. Magana-Schwencke, N., and J. Schwencke. 1969. A proline transport system in Saccharomyces chevalieri. Biochim. Biophys. Acta 173:313-323. 15. Odeberg, HE, and I. Olson. 1976. Mechanic for the microbicidal activity of cationic proteins ofhuman granulocytes. Infect. Immun. 14:1269-1275. 16. Peterson, E. Me, and R. A. Calderone. 1977. Growth inhibition of Candida albicans by rabbit alveolar macrophages. Infect. Immun. 15:910-915. 17. Rest, R. F., M. H. Cooney, and J. K. SpitznageL 1978. Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of SabnoneUa typhimurium LT-2. Infect. Immun. 19:131-137. 18. Roon, J., F. Larimore, and J. S. Levy. 1975. Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae. J. Bacteriol. 12:325-331. 19. Selvaraj, R. J., B. B. Paul, R. R. Strauss, A. Jacobs, and A. J. Sbarra. 1974. Oxidative peptide cleavage
20.
21.
22.
23. 24.
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and decarboxylation by MPO-H20rCl antimicrobial system. Infect. Immune. 9:255-260. Smolelis, A. N., and S. E. Hartel. 1949. The determination of lysozyme. J. Bacteriol. 58:731-736. Weiss, J., R. C. Franson, S. Beckerdite, K. Schmeidler, and P. Elabach. 1975. Partial characterization and purification of a rabbit granulocyte factor that increases permeability in Escherichia coli. J. Clin. Invest. 55:3342. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteins of polymorphonuclear leukocyte lysosomes. I. Resolution of antibacterial and enzymatic activities. J. Bacteriol. 91:750-754. Zeya, H. I., and J. K. Spitznagel. 1968. Arginine-rich proteins of polymorphonuclear leukocyte lysosomes. J. Exp. Med. 127:927-941. Zeya, H. I., and J. K. Spitznagel. 1971. Characterization of cationic bearing granules of polymorphonuclear leukocytes. Lab. Invest. 24:229-236.
INFECTION AND IMMUNITY, Aug. 1978, p. 506-513 0019-9567/78/0021-0506$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Inhibition of Specific Amino Acid Uptake in Candida albicans by Lysosomal Extracts from Rabbit Alveolar Macrophages El L ENA M. PETERSON' AND R. A. CALDERONE'* Department of Pathology, School ofMedicine, University of California at Irvine, Irvine, California 92717,' and Department ofMicrobiology, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. 200072
Received for publication 17 April 1978
Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine, valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and RbV. Leakage of 'Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract. Phagocytic cells possess a number of mechanisms that contribute to the inactivation of microorganisms. In general, they fall into two main categories, oxidative and nonoxidative (4, 9-12, 15, 17, 19, 22-24). Numerous studies have focused on the components of these microbicidal systems, but relatively few studies have characterized the exact manner by which these systems ultimately cause inactivation of the invading organism.
Odeberg and Olsson (15) have investigated the mechanism by which cationic proteins from human granulocytes killed selected bacteria. They found that bacterial incorporation of radioactive precursors into protein, DNA, and RNA was inhibited when cationic proteins were present (15). Other studies have shown an increase in the permeability of the microbial envelope of Escherichia coli when incubated with cationic proteins from rabbit granulocytes (21). In previous studies, we demonstrated in vitro that rabbit alveolar macrophages (AM) phagocytized Candida albicans and inhibited its intracellular growth (16). The purpose of this study was to characterize the underlying mechanism(s) by which C. albicans was inhibited by rabbit alveolar macrophages. This was accomplished by examining the effect of lysosomal506
rich fractions obtained from rabbit alveolar macrophages on specific cellular functions of C. albicans. MATERIALS AND METHODS C. albicans The isolate of C. albicans used in this study was previously described (16). An 18-h culture of C. albicans, grown on brain heart infusion slants (370C), was collected in 0.9% saline, centrifuged, and suspended in citrate-phosphate buffer (0.02 M citric acid-0.003 M sodium phosphate [dibasic]), pH 5.6. Labeled materials. The following compounds were purchased from New England Nuclear Corp. (Boston, Mass.): L-[3H]valine, 5 Ci/mmol; L-[3H]lysine, 5 Ci/Mmol; L-[3H]leucine, 5 Ci/Mmol; L-[3H]proline, 6 Ci/mmol; L-[3H]glutamic acid, 45 Ci/Mmol; L[3H]phenylalanine, 60 Ci/mmol; L-[3S]methionine, 400 Ci/mmol; [3H]uridine, 48 Ci/mmol; 2-deoxy-D[3H]glucose, 8.3 Ci/mmol; T6rubidium as RbCl in 0.5 N HCI, 5 mCi/mg. Disruption and differential centrifugation of macrophages. AM were obtained as previously described (16), with the following modifications: rabbits were killed by the injection of air (15 ml) into the marginal ear vein, and 200 ml of sterile saline (0.9%) was used to lavage the lungs. The contents of a typical savage have been previously described (16). AM were washed and suspended in 0.25 M sucrose, and viability was determined by trypan blue exclusion. AM were then homogenized on ice in a hand-operated Potter-
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AMINO ACID UPTAKE INHIBITION IN C. ALBICANS
Elvehjem tissue grinder (5 ml) at 2-min intervals and checked for the percent breakage by phase-contrast microscopy. Cells were homogenized until 85 to 90% breakage was achieved as judged by the ratio of free nuclei to whole cells. The homogenate was diluted with 0.34 M sucrose to three times its volume and, after centrifugation (500 x g, 12 min), the resulting supernatant was removed and saved. The pellet was washed an additional three times, and the superna'tants were collected and pooled. The resulting pellet, as judged by phase-contrast microscopy, contained unbroken cells and nuclei. The pooled supernatant was then centrifuged at 15,000 x g, and the pellet obtained was the lysosomal-rich fraction, as determined by electron microscopy and enzymatic analysis, and referred to as the "15-g" pellet. Extraction of lysosomal-rich fraction. The 15g pellet was suspended in 1 ml of 0.34 M sucrose and subjected to six cycles of freeze-thawing. The freezethawed material was centrifuged at 15,000 x g for 12 min, and the supernatant was collected. This supernatant was dialyzed overnight in citrate-phosphate buffer (40C, 2,000 x vol). All extracts referred to as freeze-thawed (FT) were treated in this manner and stored at -200C. Enzymatic and chemical determinations. (i) Acid phosphatase. Acid phosphatase was measured by a colorimetric assay usingp-itrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) as the substrate by the method of Andersch and Szczypinski (1). A standard curve was constructed by measuring the optical density readings at 400 nm of an alkaline solution of various concentrations of p-nitrophenol (Sigma). (ii) Lysozyme. Lysozyme was measured by following the lysis of a standardized, UV-killed culture of Micrococcus lysodeikticus (Difco Laboratories, Detroit, Mich.) by the procedure of Smolelis and Hartsel (20). A standard curve was prepared by measuring the change in turbidity of the bacterial suspension when incubated with known amounts of lysozyme (Difco). Turbidity was measured at 540 nm in a Bausch and Lomb.Spectronic 20 spectrophotometer. (iii) Protein. Protein determinations were carried out by the method of Lowry et al. (13) using bovine serum albumin as a standard. Effect of FT extracts on incorporation by C. albicams. An 18-h culture of C. albicans grown on brain heart infusion slants (370C) was washed, suspended in citrate-phosphate buffer (pH 5.6), and added to tissue culture tubes (13 by 100 mm) (105 cells per tube). Varying concentrations of extract (5 to 100 pg of protein) were added in a total volume of 0.1 ml per tube. Controls received an equal volume of buffer. Subsequently, [3H]leucine (5 pCi per tube) was added to each tube. The total volume of all mixtures was 0.205 ml. All tubes were incubated (370C, 150 rpm) for 45 min, and all reactions were stopped by precipitating cultures on ice with cold 15% trichloroacetic acid. Nonspecific trapping of label was determined by labeling cultures on ice and precipitating immediately with cold 15% trichloroacetic acid. Subsequently, all precipitated cultures were filtered onto glass fiber filters (Whatman GF/A) and washed with 5% trichloroacetic acid followed by 95% ethanol. All radioactive measurements were made in an In-
507
tertechnique liquid scintillation counter which had approximately a 30% efficiency for 3H on GF/A filters in a scintillation liquid of the following composition: 0.1 g of p-bis-[5-phenyloxazolyl)]benzene and 5 g of 2,5-diphenyloxazole dissolved in 1.0 liter of scintillation grade toluene. All cultures were run in duplicate, and each experiment was repeated at least once. Similar data were obtained for each experiment. Effect of FT extract on the uptake of amino acids, uridine, and 2-deoxy-n-glucose by C. albicans. Equal volumes of prewarmed citrate-phosphate buffer (pH 5.6) and FT extract (1 mg/ml) were added to 25-ml flasks, respectively. The labeled compound (amino acid, sugar, or uridine) was added, followed by C. albicans (106/ml) to give a 1:1 (vol/vol) ratio of cells to buffer or cells to extract. At appropriate intervals, in duplicate, 0.1 ml of the mixture was added to 5 ml of cold citrate-phosphate buffer containing a 100x concentration of the unlabeled compound. Dialyzed bovine serum albumin (50 to 100 pLg/0.1 ml of citrate-phosphate buffer) was also used as a control to measure lysosomal-specific inhibition of amino acid uptake. Cells were then collected on glass fiber filters (Whatman GF/A) which had been soaked in the unlabeled compound. Subsequently, filters were covered with NCS tissue solubilizer (Amersham Corp., Arlington Heights, Ill.) for 30 min, at which time toluenebased fluor (described previously) was added and the vials were counted. Viability of C. albcanm treated with extract. FT extract (50 pg of protein per 0.1 ml) in citratephosphate buffer (pH 5.6) was added to one-half of the tissue culture tubes. The remaining tubes, which served as controls, contained an equal volume of citrate-phosphate buffer. C. albicans suspended in citrate-phosphate buffer was added to all tubes (105 cells per tube). Incubations were carried out in a heating block (370C, 150 rpm). At designated times, the incubation mixtures were diluted with sterile distilled water and plated on Sabouraud-dextrose agar. Plates were incubated for 24 h (370C) and counted for colonyforming units. Effect of FT extract on "rubidium uptake and release by C. albicans. (i) Labeling of C. albians with "rubidium. A modification of the procedure of Drazin and Lehrer (3) was used. A 16-h culture of C. albicans (370C, 150 rpm) grown in Sabouraud-dextrose broth was centrifuged (3,000 x g, 10 min), washed twice, and suspended in sterile distilled water (107 cells per ml). Cells were distributed in test tubes (106 cells per tube) which contained 0.1 ml of one-eighthstrength citrate-phosphate buffer (pH 5.6). "Rubidium was diluted with distilled water, and 0.24 appropriate intervals, ACi was added to each tube. At with cold distilled waduplicate cultures were diluted ter, filtered over membrane filters (0.45-pm pore size, Millipore Corp., Bedford, Mass.), washed twice with cold distilled water, and counted in a Beckman Biogamma Counter. (ii) "Rubidium release. C. albicans was grown and washed as described above. Washed cells were resuspended in sterile distilled water (3 x 107 cells per ml). Equal volumes of cells and isotope, diluted in sterile distilled water (100 pCi/ml), were incubated at 370C (150 rpm, 2 h). Labeled cells were centrifuged
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PETERSON AND CALDERONE
(500 x g), washed three times with sterile distilled water to remove unincorporated isotope, and resuspended in sterile distilled water (107 cells per ml). The 86Rb release assay was carried out in heavy-walled glass test tubes (10 by 75 mm), containing either 0.1 ml of citrate-phosphate buffer (pH 5.6) or varying concentrations of FT extract in a total volume of 0.1 ml. The release assay was initiated by the addition of 0.1 ml of TMRb-labeled C. albicans (106 cells). At designated time intervals, the cell suspensions were centrifuged at 15,000 x g (10 min, 400). Supernatants were removed and 0.1 ml was counted. All samples were done in duplicate. Zero time readings were carried out by the addition of labeled cells to extract or buffer on ice, followed by immediate centrifugation and supernatant removal. Total input counts per tube were determined by counting 0.1 ml (106 C. albicans) of prelabeled cells in duplicate. 86Rb release was calculated, after all zero time counts had been subtracted, as follows: ([(counts per minute of extract-treated cells) - (counts per minute of control cells)]/[input counts per minute]) x 100. (iii) "Rb uptake. FT extract (100 pg/0.1 ml) in one-eighth-strength citrate-phosphate buffer (pH 5.6) was added to sterile tissue culture tubes. Controls received an equal volume (0.1 ml) of one-eighthstrength citrate-phosphate buffer. C. albicans, grown in Sabouraud-dextrose broth, was washed and suspended as previously described and added to all tubes (106 cells per tube), followed by 0.24 jCi of MRb. At various times, ice-cold distilled water (5 ml) was added to duplicate cultures. Immediately, cells were collected on membrane filters (Millipore Corp.), washed twice with distilled water, and counted. Zero time readings were done on ice and treated as above.
INFECT. IMMUN.
80
e~
60 60
C
-
C 40
20
10
50 100 Extract (pg protein) RESULTS FIG. 1. Dose effect of FT extract on [3HJleucine Effect of alveolar macrophage lysosomal incorporation by C. albicans. Cells (105 per tube) were extracts on C. albicans. Lysosomal-rich frac- labeled with [3Hjleucine (5 ,uCi per tube) for 45 min tions were obtained from AM to evaluate their at 370C.
role in the inhibition of Candida. To obtain such fractions, acid phosphatase and lysozyme were used as lysosomal markers in order to evaluate the enrichment procedure. Although these enzymes are distributed throughout the cell, their greatest concentration seems to be within lysosomes. AM were disrupted until 85 to 90% breakage had been achieved. At this percent breakage, some lysosomes were still contained in unbroken cells and these appeared in the nuclear pellet. In addition, some of the released lysosomes were themselves disrupted and their contents appeared in the fraction termed "supernatant." Nevertheless, preliminary experiments indicated that this percent breakage was most favorable for obtaining lysosomes. The 15-g fraction was extracted by freezethawing. To investigate the effects of the extract on C. albicans metabolism, C. albicans [3H]leucine incorporation was measured in the presence and absence of the extract. Figure 1 illustrates the effect of various concentrations of
the extract (micrograms of protein) on [3H]leucine incorporation by C. albicans. The extract had a marked effect on leucine incorporation by Candida up to 50 yg of extract protein per 105 Candida, above which the inhibition leveled off. FT extracts (100 jg of protein per 105 cells) were also tested for their ability to affect incorporation ofother amino acids by Candida. Table 1 shows that the incorporation of the majority of amino acids tested was inhibited when compared to incorporation by control cells. There was also variation in the percent inhibition depending on the amino acid tested. However, the incorporation of proline and glutamic acid was always above the incorporation of control cells. Since the inhibition of amino acid incorporation could be a reflection of decreased uptake of the labeled amino acids by Candida, it was decided to investigate the effect of the FT extract on amino acid uptake. Uptake of methionine, valine, lysine, phenylalanine, and leucine
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AMINO ACID UPTAKE INHIBITION IN C. ALBICANS
a
were all drastically reduced compared to controls (Fig. 2a through d, Fig. 3a). However, proline uptake was comparable to the control (Fig. 3b), and glutamic acid uptake was only slightly affected (Fig. 3c). These data (expressed as percent of control) are summarized in Table 2. The effect of FT extract on uptake by Candida appears to be specific for amino acids since the uptake of 2-deoxy-D-glucose, uridine, and Rb+ TABLE 1. Incorporation of amino acids by C. albicans in the presence of FT extract % Controla Compound incorporated 6.6 ..... ...... [3S]methionine ........ 10.0 [3H]leucine ... 13.5 [3H]phenylalanine ............ ........ 35.5 [3H]valine ... .......... 59.0 [3H]lysine ............ 121.0 ..... [3H]glutamic acid ...... 125.0 ........... [3H]proline a Cells were labeled in buffer (control) or in FT extract (100 pg) for 45 min at 37"C.
E
b
E
Minutes a
20
[[35SI
[3H] L-Va line
L-Methionine
I
E
C
c
C7 40
IW
ED
509Q
Minutes
FIG. 3. (a) Uptake of i[4HJleucine by C. albicans in the presence (0) and absence (0) ofFT extract. (b) Uptake of I_[3Hlproline by C. albicans in the presence (0) and absence (0) of FT extract. (c) Uptake of z-[3HJglutamic acid by C. albicans in the presence (0) and absence (0) of FT extract.
TABLE
2. Uptake by C. albicans in the presence of FT extract % Controla Compound 4.3 ......... ......... L-[3H]phenylalanine L-[3H]lysine .......................... 9.8 10.1 ............ L-[3S]methioine ........ 12.0 L-[3H]valine ......................... L-[3Hlleucine ........................ 20.3 L-[3H]glutamic acid ........... ........ 74.8 L-[3H]proline ........................ 82.0 91.3 86Rubidium ..... 95.1 2-Deoxy-D-[3H]glucose . 112.2 [14C]Unidine aPercent control is calculated from the 10-min readings on the uptake curves.
was not affected (Table 2). As an additional control, uptake of leucine and proline was measured in the presence of bovine serum albumin. Uptake of both amino acids was similar to that observed with buffer alone. Minutes Minutes Viability of C. albicans when treated with FT FIG. 2. (a) Uptake of L-[3Slmethionine by C. alof protein per 105 cells) was deterextract bicans in the presence (0) and absence (0) of FT mined (50 ,yg counts by plate (colony-forming units per extract. (b) Uptake of z-[3Hlvaline by C. albicans in the presence (0) and absence (0) of FT extract. (c) milliliter) (Fig. 4). A significant decrease in viaUptake of z43NHlysine by C. albicans in the presence bility was not detected until 16 h after the initial (0) and absence (0) of FT extract. (d) Uptake of L- exposure of Candida to the extract. After this [3HJphenylalantne by C. albicans in the presence time, there was a steady decline in viability of (0) and absence (0) of FT extract. the cells incubated with extract. Control colony-
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INFECT. IMMUN.
PETERSON AND CALDERONE lx
25
106
0)
Lob
lx
0)
10 5
a) -
0 00
co u
0) 0-
lxI
Hours
FIG. 4. Viability of C. albicans (colony-forming units per milliliter) measured after incubation in citrate phosphate buffer (0) or FT extract (50 pg of protein per culture (0).
forming units remained constant during their prolonged incubation in buffer. The influx and efflux of TMRb was determined in the presence and absence of FT extract in order to evaluate the effect of FT extract on the ability of the cytoplasmic membrane to transport Rb+ and to exert a permeability barrier
Extract (pg protein) FIG. 5. Percent 'rubidium release by C. albicans incubated with FT extract. Percent 'rubidium release = {[(counts per minute of extract-treated cells) - (counts per minute of control cells))/[input counts per minutes) X 100.
TABLE 3. Heat stability of 15-g extract as measured by [3H]leucine incorporation by C. albicansc Sample
Counts/ min
% Control
Control 31,013 Unheated extract 13.9 4,328 Extract, 560C, 1 h 3,389 10.9 Extract, 1000C, 10 min 5,500 17.7 Extract, 1000C, 30 min 4,801 15.5 a Freeze-thawed extract, 50 jig per assay tube. bEach assay tube was labeled for 45 min at 370C with 5,UCi of [3H]leucine (60 Ci/mmol). c 105 cells per tube.
has been demonstrated in Candida that Rb+ can replace K+ in studies of membranebound ion transport (3). Preliminary studies showed that 'Rb influx in the strain of C. albicans used throughout these experiments is competitively inhibited by K+. Uptake of 'Rb+ by dida. The purpose of this study was to characCandida in the presence of the extract was terize the mechanism(s) of growth inhibition. equivalent to the controls (Table 2). However, Since the phagocytosis process is followed by leakage of 'Rb from C. albicans, preloaded with lysosomal fusion with the phagocytic vacuole isotope, was found to be increased in the pres- and the release of its contents into the phagolyence of FT extract. Figure 5 represents the dose sosome, we decided to investigate the role, if effect of FT extract on MRb leakage. any, of lysosomal contents in the inactivation of The heat stability of FT extract was assessed Candida. This approach has been used by many by utilizing the [3H]leucine incorporation assay investigators (15, 22-24) in establishing microb(Table 3). The FT extract appeared to be heat icidal mechanisms of phagocytic cells. stable, in that it maintained its ability to inhibit In retrospect, one of the major disadvantages [3H]leucine incorporation by C. albicans after of working with normal rabbit AM is the yield being boiled for 30 min. of cells obtained from one rabbit (10' cells per rabbit). This problem is further compounded DISCUSSION when one attempts not only to enrich for lysoIn a previous study, we demonstrated that the somes but also to extract them. The end result AM restricts the intracellular growth of Can- is approximately 1 mg (protein) of lysosomal function. It
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AMINO ACID UPTAKE INHIBITION IN C. ALBICANS
extract per rabbit. This was a major consideration in designing experiments to explore the mechanism(s) of inactivation. Since intact AMs affected leucine incorporation by C. albicans (16), the AM lysosomal extracts were also examined for similar effects. The extracts did have an inhibitory effect on [3H]leucine incorporation by C. albicans. Since the decrease in incorporation could be a reflection of an effect on uptake, the uptake of several amino acids was measured with and without the FT extract. We found that lysine, methionine, phenylalanine, and valine uptake was significantly inhibited by FT extract, but glutamic acid and proline uptake was unaffected. Similarly, in the presence of FT extract, incorporation of proline and glutamic acid in trichloroacetic acidprecipitable material was unaffected, whereas the other amino acids mentioned above were inhibited. These studies indicated that protein synthesis by C. albicans was not affected by FT extract but rather appeared that way due to an inhibition in the uptake of the labeled amino acid under question. If protein synthesis were truly affected, one would not expect any labeled amino acid to give incorporation data equal to controls. Since this was observed with proline and glutamic acid, protein synthesis, at least initially (45 min), is not affected by FT extract but rather some amino acids are hampered in their entry into the cell. As mentioned above, methionine, lysine, valine, phenylalanine, and leucine uptake was lower in extract-treated cells when compared to controls. However, uptake of glutamic acid and proline was only slightly or not at all affected. Grenson et al. (7) presented evidence for the existence of a general amino acid permease in Saccharomyces cerevisiae. They found that all amino acids examined, except proline and glutamic acid, were transported by this general amino acid permease. They reasoned that proline was not transported by the general permease due to its configuration, and that the second carboxyl group of the dicarboxylic amino acids seemed to hinder transport. In addition to this general amino acid permease, specific permeases for arginine (8), lysine (6), methionine (5), and proline (14) have been described in yeast. Grenson et al., in attempting to distinguish between specific and general amino acid permeases, were able to depress the general permease with a high concentration of ammonium ions (7). They measured uptake of amino acids with and without the ammonium ions and found that, for all amino acids tested except glutamic acid, uptake increased approximately 10-fold when ammonium ions were depleted from the culture media. Proline was not
511
tested for it has been shown in Saccharomyces chevalieri (14) that the proline permease is also affected by ammonium ions. Grenson et al. also isolated a mutant (gap) which had lost the general amino acid permease. Uptake in the gap mutant was not greater in the absence of ammonium ions, as was seen in the wild type. However, in the absence of ammonium ions, there was no difference in the uptake rate of proline between the wild-type strain and the gap mutant. This, coupled with the fact that proline, even at high concentrations, did not inhibit the uptake of amino acids by the general amino acid permease, suggested that it was not a substrate of the general permease transport system. Therefore, it can be postulated that FT extract interferes with a general amino acid permease of C. albicans. This interference ofuptake appeared to be unique to amino acids for 2deoxy-D-glucose, uridine, and Rb+ showed no decrease in uptake. The low level of uptake present for valine, lysine, leucine, methionine, and phenylalanine probably represents uptake by their specific permeases. These specific amino acid transport systems have been shown to be low-velocity constitutive systems (18). Uptake by FT extract-treated cells is depressed about 10-fold in comparison to control cells, and this number correlates well with the findings of Grenson et al. that, without the general amino acid permease, uptake was decreased 10-fold (7). Uptake studies with C. albicans are sparse. In preliminary studies, we have found that phenylalanine inhibits uptake of leucine whereas proline does not, indicating the existence of such a gap transport system. The dimorphic fungus Histoplasma capsulatum appears to differ from Saccharomyces sp. and C. albicans in that leucine and proline seem to share a common transport system (2). Our data for H. capsulatum indicate that both proline and leucine uptake are inhibited by the AM extracts. Odeberg and Olsson (15) studied the effect of the cationic components of a lysosomal-rich fraction obtained from human granulocytes on leucine incorporation by E. coli and Staphylococcus aureus. They found that the cationic proteins inhibited leucine incorporation in these organisms. They did not do uptake studies but postulated that the inhibition in the incorporation of radioactive precursors could be due to inhibition of the transport of the radioactive precursors or inhibition of macromolecular synthesis. Therefore, components which contribute to the bactericidal mechanism of human granulocytes might be similar to the AM in which uptake of the phagocytized microorganism is drastically affected. In the studies done with intact AM (16), it
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PETERSON AND CALDERONE
was found that the growth of Candida was retarded since the cells did not germinate, and that [3H]leucine incorporation in the phagocytized yeast was dramatically decreased. Although it has not been established whether the contents of the FT extract are active in the phagolysosome, one can postulate that, by restricting uptake of amino acids by the phagocytized yeast, the AM could effectively restrict the growth of the fungus. In other studies, the viability of Candida incubated with FT extracts was decreased. However, this decrease took a relatively long time (16 h) to manifest itself, so that the process responsible for killing is a slow one. Rubidium has been used in bacteria (15) and Candida (3) as an analog of K+ in studying membrane transport and damage. Since a very slow rate of killing of Candida with FT extract was observed, any membrane damage that might occur early after exposure to the extract would be slight. Therefore, a sensitive seRb release assay was used to detect any membrane damage. Cells treated with FT extract gave evidence of seRb leakage. This release of seRb increased with increasing amounts of extract used. However, FT extract had no effect on seRb uptake of C. albicans. Therefore, although not affecting entry of seRb into the cell, FT extract affected the capacity of the cytoplasmic membrane to retain intracellular TMRb. Likewise, Odeberg and Olsson (15) found that E. coli treated with cationic protein from neutrophils behaved in a similar fashion. This indicated that FT extracts damage the membrane, and this could be a major factor affecting the viability of C. albicans. If the leakage were slow but steady, then this could account for the lag before detecting a decrease in viability by plate counts. The inhibitory component(s) in the AM FT extract was heat stable. Heat-stable, microbicidal lysosomal components, obtained from polymorphonuclear leukocytes, have been reported by others (11, 15). Azurophil granules, obtained from human polymorphonuclear leukocytes, contained a heat-stable, bacteriostatic component and a heat-labile bactericidal component
(17).
Many components within the AM can contribute to the stasis or even death of C. albicans. Certainly, our findings concerning the uptake of amino acids by Candida treated with extract reveal a static or even a cidal mechanism within the AM. Stasis of the invading organism is critical if the AM is to resist overgrowth by Candida or other fungi. If the AM does not kill the ingested organism, it must restrict its growth until the AM can be cleared from the alveoli or
INFECT. IMMUN.
until other cells of the immune system can be recruited. The ability of the FT extract to affect the cytoplasmic membrane of C. albicans is yet another candidate for a candidacidal mechanism in vitro. It is reasonable to assume that multiple mechanisms of resistance have evolved within the AM. ACKNOWLEDGMENTh E. P. was supported by Public Health Service training grant A10028-11 from the National Institute of Allergy and Infectious Diseases. This investigation was supported by grants from the Brown-Hazen Fund of the Research Corporation of America and the Washington Heart Association. We thank T. Sreevalsan for his suggestions and Stephanie Coleman for typing the Manuscript.
LITERATURE CITED 1. Andersch, M. A., and A. J. Szczypinski. 1947. Use of p-nitrophenyl phosphate as the substrate in the determination of serum acid phosphatase. Am. J. Clin. Pathol. 17:571-574. 2. Dabrowa, N., and D. H. Howard. 1976. Uptake of Lproline by Histoplasma capsulatum. Can. J. Microbiol. 22:1188-1190. 3. Drazin, R. E., and R. I. Lehrer. 1976. TMRubidium release: a rapid and sensitive assay for amphotericin B. J. Infect. Dis. 134:238-244. 4. Drazin, R. E., and R. L. Lehrer. 1977. Fungicidal properties of a chymotrypsin-like cationic protein from human neutrophils: adsorption to Candida parapsilosis. Infect. Immun. 17:382-388. 5. Gits, J., and M. Grenson. 1967. Multiplicity of the amino acid permeates in Saccharomyces cerevisiae. Inl. Evidence for a specific methionine-transporting system. Biochim. Biophys. Acta 135:507-516. 6. Grenson, M. 1966. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. II. Evidence for a specific lysine-transporting system. Biochim. Biophys.
Acta 127:339-346. 7. Grenson, M., C. Hou, and M. Crabell. 1970. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease.
J. Bacteriol. 103:770-777. 8. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of the amino acid permeates in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127:325-338. 9. Hirsch, J. G. 1960. Further studies on the preparation and properties of phagocytin. J. Exp. Med. 111:323-337. 10. Lehrer, R. I. 1975. The fungicidal mechanism of human monocytes. I. Evidence for myeloperoxidase-independent candidacidal mechanisms J. Clin. Invest.
55:338-346.
11. Lehrer, R. I., K. M. Ladra, and R. B. Hake. 1975. Non-
oxidative fungicidal mechanisms of mammalian granulocytes: demonstration of components with candidaci-
dal activity in human, rabbit, and guinea pig leukocytes. Infect. Immun. 11:1226-1234. 12. Leigh, P. C. M., M. T. Van den Barsellar, and P. VanFurth. 1977. Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes. Infect. Immun. 17:313-318. 13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
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14. Magana-Schwencke, N., and J. Schwencke. 1969. A proline transport system in Saccharomyces chevalieri. Biochim. Biophys. Acta 173:313-323. 15. Odeberg, HE, and I. Olson. 1976. Mechanic for the microbicidal activity of cationic proteins ofhuman granulocytes. Infect. Immun. 14:1269-1275. 16. Peterson, E. Me, and R. A. Calderone. 1977. Growth inhibition of Candida albicans by rabbit alveolar macrophages. Infect. Immun. 15:910-915. 17. Rest, R. F., M. H. Cooney, and J. K. SpitznageL 1978. Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of SabnoneUa typhimurium LT-2. Infect. Immun. 19:131-137. 18. Roon, J., F. Larimore, and J. S. Levy. 1975. Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae. J. Bacteriol. 12:325-331. 19. Selvaraj, R. J., B. B. Paul, R. R. Strauss, A. Jacobs, and A. J. Sbarra. 1974. Oxidative peptide cleavage
20.
21.
22.
23. 24.
513
and decarboxylation by MPO-H20rCl antimicrobial system. Infect. Immune. 9:255-260. Smolelis, A. N., and S. E. Hartel. 1949. The determination of lysozyme. J. Bacteriol. 58:731-736. Weiss, J., R. C. Franson, S. Beckerdite, K. Schmeidler, and P. Elabach. 1975. Partial characterization and purification of a rabbit granulocyte factor that increases permeability in Escherichia coli. J. Clin. Invest. 55:3342. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteins of polymorphonuclear leukocyte lysosomes. I. Resolution of antibacterial and enzymatic activities. J. Bacteriol. 91:750-754. Zeya, H. I., and J. K. Spitznagel. 1968. Arginine-rich proteins of polymorphonuclear leukocyte lysosomes. J. Exp. Med. 127:927-941. Zeya, H. I., and J. K. Spitznagel. 1971. Characterization of cationic bearing granules of polymorphonuclear leukocytes. Lab. Invest. 24:229-236.
