Isocitrate lyase and malate synthase activities of Coccidioides immitis

EXPERIMENTAL

MYCOLOGY

9, 318-325 (1985)

lsocitrate

Lyase and Malate Synthase Coccidioides immitis B. AGY*

MICHAEL Departments of *Microbiology

Activities

of

AND JOHN L. PAZNOKASV

and fBasic Medical Sciences, Washington State University, Pullman, Washington 99164-4340

Accepted for publication August 23, 1985 AGY, M. B., AND PAZNOKAS, J. L. 1985. Isocitrate lyase and malate synthase activities of Coccidioides immitis. Experimental Mycology 9, 318-325. The saprophytic phase of the fungal pathogen Coccidioides immitis contains both of the enzymes unique to the glyoxylate cycle (isocitrate lyase, EC 4.1.3.1, and malate synthase, EC 4.1.3.2) when grown in a variety of media regardless of whether or not glucose is present in those media. Significantly higher levels of each enzyme are found in cells grown in defined media. Enzyme activity varied as a function of growth, being higher during the germination phase than the exponential phase of growth. C. immitis arthroconidia do not germinate in medium containing glucose as the sole carbon source, but will germinate in this same medium with acetate as the carbon source. Glucose alone does not support vegetative growth and acetate is utilized preferentially over glucose when both are present in defined minimal medium. An inhibitor of isocitrate lyase, itaconic acid, inhibits germination and vegetative growth of C. immitis ‘in all growth supporting media tested. Itaconic acid acts synergistically with amphotericin B to inhibit arthroconidia germination and growth. o 1985 Academic Inc. INDEX DESCRIPTORS:

Press,

isocitrate lyase; glyoxylate cycle; Coccidioides immitis; itaconate growth

inhibition.

Coccidioides immitis is a dimorphic fungal pathogen of particular concern to people living in the desert regions of the Americas (Pappagianis, 1980). In its disseminated form, coccidioidomycosis is a devastating disease with only one effective chemotherapeutic agent, amphotericin B (amB)’ (Collins and Pappagianis, 1977; Hardenbroch and Barriere, 1982). Since amB regimens are invariably associated with toxic side effects (Kelly, 1980), alternative therapies are desirable. A number of effective antibacterial drugs act by interfering with essential metabolic functions unique to the bacterium. Since fungi are eucaryotic, the number of unique pathways available for attack are limited. i Abbreviations used: amB, amphotericin B; TCA, tricarboxylic acid; GY agar, 1% glucose-0.5% yeast extract-1.5% agar; S, 0.5% soytone; Y, 0.5% yeast extract; G, 1% glucose; A, 1% sodium acetate; MOPS, morpholinopropane sulfonic acid.

One pathway utilized by some fungi, but not by mammalian cells, is the glyoxylate cycle. Isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), the unique enzymes of the glyoxylate cycle, have been described in several procaryotic (Hillier and Charnetzky, 1981; McFadden and Howes, 1962; Reeves and Ajl, 1960) and eucaryotic (Huang, 1982; Kobr et al., 1969; O’Connell and Paznokas, 1980; Pate1 and McFadden, 1978; Roman0 and Kornberg, 1969) systems. These enzymes comprise the unique enzymatic component of the glyoxylate cycle first described in 1957 (Kornberg and Krebs, 1957). In these organisms, glyoxylate cycle enzyme activities are severely reduced in media containing glucose. An exception to this is Aspergillus nidulans (Roman0 and Kornberg, 1969) which preferentially utilizes acetate, even in media containing glucose. 318

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GLYOXYLATE

CYCLE

C. immitis reportedly has the enzymes of the Embden-Meyerhof pathway leading to the production of pyruvate, a functional pentose phosphate pathway, and all of the enzymes of the TCA cycle except 2-0x0gluturate dehydrogenase (EC 1.2.4.2) (Lones, 1967). In addition, Lones reported that C. immitis has isocitrate lyase activity, but no mention was made of malate synthase. Our results indicate that malate synthase is included in the enzymatic repertoire of C. immitis and its specific activity roughly parallels that of isocitrate lyase in crude extracts of cells grown in a variety of media whether or not glucose is present. Furthermore, we have begun to assess the importance of th,: glyoxylate cycle enzymes in the life cycle of C. immitis. Itaconic acid, a known inhibitor of isocitrate lyase (McFadden, 1969), inhibits the growth of a variety of microorganisms (Beliion and Kelley, 1979; Hillier and Charnetzky, 1981; O’Connell and Paznokas, 1980; Pate1 and McFadden, 1978). We have found that itaconate inhibits the isocitrate lyase activity of C. immitis in vitro and also inhibits the growth of C. immitis from arthroconidia. Itaconate also acts in synergism with the antifungal agent amphotericin B. MATERIALS

AND

METHODS

Fungal cultures. C. immitis strains Silveira and 95-271 were obtained from H. B. Levine and H. A. Walch, respectively. Silveira is a highly pathogenic strain with a reported LI& (mouse) of less than 50 arthroconidia (Brass et al., 1982). Strain 95 271, a diauxotrophic mutant of the virulent strain KS, is nonpathogenic in the guinea pig and will not germinate or grow at 37°C (Walch and Walch, 1965). Arthroconidia were obtained from agar slab cultures grown at 30°C on media containing 1% glucose, 0.5% yeast extract, and 1.5% agar (GY agar) in 25-cm* disposable tissue culture flasks (Corning Glass Words, Corning, N.Y.). hoculum. Arthroconidia were harvested aseptically in water by scraping the myce-

OF

Coccidioides

imrnitis

319

hum from the agar surface of Ii- to 2-m.onth GY cultures using a wire “rake” fashione from an inoculating loop. The resulting suspension was vortex mixed with glass beads (2- to 5-mm diameter) for 1 to 3 min and filtered over glass wool to remove large vegetative fragments. This treatment routinely produced suspensions of I to 5 x 107 arthroconidiaiml with few contaminati~8 hyphal fragments, Media. Complex media composed of various combinations of 0.5% Soytone (Difco Laboratories, Detroit, Mich.) (S), 0.5% yeast extract (Y), 1% glucose (G), a 1% sodium acetate (A) were used as cated. Strain 95-271 was cultured in t fined medium of Walch (Walch and Welch, 1965) supplemented with 100 mg/liter each of riboflavin and pam-aminobenzoic acid. All media were inoculated with sions of arthroconidia (volumes ceeding 1 ml120 ml medium) to produce a final concentration of lo5 to IO6 arthroconidia/ml medium. Liquid cultures, in cotton stoppered Erlenmeyer flasks, were incubated at room temperature (22-25°C) on a rotary shaker (New Brunswick Model G-2) set at 150 rpm. The ratio of culture ~01~~~ to nominal flask volume was between 0.3 and 0.4. Growth was monitored with a Klett-Summerson photoelectric colorimeter equipped with a No. 42 filter (400 to 465 nm). Klett units are related to optical density units (OD) in the formula OD = (Klett value x 2)ilOOO. In a GUS culture, a Klett value of 500 (OD I.0) c~~~esp~~ds to approximately 1 mg dry wt C. immitisiml. Cell-free extract preparation. ~ycelial cultures were collected on Whatma~ No. 1 filters and washed twice with equal volumes of deionized water. This material was disrupted by freeze-grinding with liquid nitrogen in a precooled mortar. grinding was repeated twice on eat producing a slurry that was reconstitut 0.5 to 2.0 ml with 0.1 M MOPS buffer pholinopropanesulfonic acid), taining 3.0 mM MgCl, and 0.5

320

AGY

AND

The resulting suspension was centrifuged at 17,000g for 15 min in a Beckman JA 21 rotor. The supernatant (crude extract) was decanted and frozen at - 20°C until assayed (within 10 days). Assays. Isocitrate lyase activity was determined by the method of McFadden and Howes (1960). Volumes of crude extract containing 5-10 pg protein were added to 3.5 ml of 0.1 M MOPS with 0.3 mM MgCI, and 1 .O mM EDTA (pH adjusted to 7.5 with NaOH) and placed at 30°C where the reaction was initiated by the addition of DL-isocitrate (final concentration 2.8 mM). The reaction was stopped with the addition of 0.1 ml of 5% phenylhydrazine * HCl and 0.4 ml 1 M oxalic acid, heated to boiling, and cooled on ice. The resulting glyoxylatephenylhydrazone mixture was acidified with 1.8 ml 12 N HCI and oxidized to the colored compound, 1,5-diphenylformazancarboxylic acid by 0.1 ml K,Fe(CN), (25% w/v) (Kramer et al., 1959). This final compound was measured spectrophotometritally at 520 nm. Malate synthase activity was determined by a modification of the citrate condensing enzyme assay of Srere (Srere et al., 1963). Volumes of crude extract containing 2.5 to 5 kg protein were added to a reaction mixture yielding 0.2 ml final volume. The final concentrations of reaction mixture components were 0.1 M MOPS, (pH 6.5) 1 mM MgCI, 0.5 mM glyoxylate, and 0.875 mM acetyl-CoA. Reaction mixtures were incubated for 15 min at 30°C and terminated by the addition of 0.8 ml of 1 mM 5’,5-dithiobis(2-nitrobenzoic acid) in 50 mM Tris-hydrochloride (pH 8). Isocitrate lyase and malate synthase activities are reported in micromoles of glyoxylate or micromoles of coenzyme A, respectively, formed per minute per milligram of protein. Protein was determined by the protein assay method of Bio-Rad Laboratories (Richmond, Calif.) using bovine serum albumin as a standard. Assays for acetate and glucose. Strain Silveira was grown in a modification of

PAZNOKAS

Walch’s defined medium containing 0.3% acetate and 0.5% glucose. Cells were removed by centrifugation and the supernatants filtered through 0.45~km Type HA nitrocellulose filters (Millipore Corp., Bedford, Mass.). Twenty microliters of filtrate was applied to an Aminex Hpx-87H column (Bio-Rad Laboratories) and eluted with 0.0001 N H,SO, at 800 psi. The eluate was monitored with a Beckman uv monitor at 214 nm. Glucose concentrations were assessed by gas-liquid chromatography of trimethylsilylated derivatives of the filtrate by a method described previously (Brobst and Lotl, 1966). Inhibition studies. The pH of itaconic acid solutions was adjusted with NaOH and the solutions were sterilized by filtration. Amphotericin B (E.R. Squibb and Sons, Inc., Princeton, N.J.) was reconstituted and diluted in accordance with manufacturer’s aliquoted, and stored at instructions, -20°C. Since amB potency is reportedly lost in culture systems (Cheung et al., 1975) 40% of the original concentration was added at 24-h intervals over the duration of these experiments. RESULTS

Zsocitrate lyase and malate synthase enzyme activity. The enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were found in extracts of cells of C. immitis grown on a variety of complex and defined media (Table 1). The specific activity of each enzyme, as determined with crude extracts of strain Silveira, varied with medium composition as well as stage of the growth cycle (Figs. 1 and 2). The data presented in Figs. 1 and 2 are representative of at least four repetitions of these experiments. Higher specific activities of both enzymes were observed with extracts of cells grown in media supplemented with acetate than in media without acetate and the highest activities were observed with extracts of cells grown in a defined medium containing acetate as the sole carbon and

GLYOXYLATE

CYCLE OF Cocci&ides

TABLE 1 Representative Specific Activities of the Enzymes Isocitrate Lyase and Malate Synthase in Extracts of C. imrnitis Strain Silveira Cells Harvested from Midexponential Growth in Several Complex and Defined Media

Growth medium” Y AY GY US AYS GYS AGY AGYS mA mAG

iminitis 9

Specific activityb

Age of culture (h)

Isocitrate lyase

Malate synthase

60 84 72 66 65 60 84 84 120 192

1.5 3.1 1.5 1.6 4.6 0.5 1.4 1.8 14 12

3.2 7.2 1.6 2.9 12.6 0.8 3.2 3.6 98 96

n Abbreviations for medium constituents: A, 1% sodium acetate; G, 1% glucose; Y, 0.5% yeast extract; and S, 0.5% Soytone. mAG, is a minimal medium described by Walch and Walch (1965); mA is a moditication of Walch’s medium lacking glucose. b Isocitrate lyase activity expressed as ymol glyoxylate formedlminimg protein x lo-‘. Malate synthase activity expressed as kmol free coenzyme A formediminimg protein x lo-*.

24

HO%S

120

FIG. 1. Isocitrate lyase (m) and malate synthase (C! activities in C. irnnzitis strain Silveira cells grown from arthroconidia in a complex medium containing acetate, yeast extract, and Soytone (AYS). Growth ( indicated in optical density units converted from Klett units described under Materials and Methods.

throconidia. Significant fluctuations in the specific activities of each enzyme were observed during the growth cycle (Figs. 1 and 2). In all media tested, isocitrate lyase and were highest energy source. Reduced levels of each en- malate synthase activities during germination of the arthroconi zyme were found in cells grown in complex media containing glucose although the ac- Samples removed prior to 48 hr containe tivity of each enzyme was no more than primarily ungerminated arthroco~idia 50% less than in cells not exposed to glu- which are difficult to disrupt. The specific cose. No significant differences were ob- activity of each enzyme decreased during served for extracts of cells grown in defined exponential growth (Figs. 1 and 2), then inmedia with or without glucose. creased as the cells entered the stationary Apparent K, values were determined for phase. This same general pattern was seen each substrate. For isocitrate lyase the K,n with a variety of different media (data not shown). for DL-isocitrate was 1.22 mM. For malate synthase the acetyl-CoA K, was 0.2 mM C. immitis arthroconidia germinated and and the glyoxylate K,, was 0.11 mM. No grew in complex media containing yeast exsignificant differences were observed when tract and/or Soytone at pH 5.0, 6.0, or 7.3. extracts from cells grown in complex or de- Addition of acetate (1%) to these media inat pH 5.0, but not at fined media were compared. Similar results hibited germination pH 6.0 or 7.3 The addition of glucose were obtained with strain 95-27 1. Growth and enzyme activities. In all no effect on germination. In Walch’s minimal medium (which congrowth experiments liquid media were inoculated with purified suspensions of ar- tains both acetate and glucose) arthroco-

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AGY AND PAZNOKAS

166

Y

24

HCZRS

HOURS

216

264

120

FIG. 2. Isocitrate lyase (m) and malate synthase (0) activities in C. immitis strain Silveira cells grown from arthroconidia in a complex medium containing glucose, yeast extract, and Soytone (GYS). Growth (0) is indicated in optical density units as described under Materials and Methods.

nidia germinated and grew at pH 6.0 and 7.3, but not at pH 5.0. Germination was delayed in this medium longer than was observed in a modified Walch’s medium containing acetate, but lacking glucose. No germination was observed in Walch’s minimal medium containing glucose, but lacking acetate (Fig. 3). Arthroconidia inoculated into Walch’s minimal medium containing acetate (0.3%) and glucose (0.5%) utilized the acetate in a markedly higher proportion (e.g., a filtrate from a stationary culture contained 19% of the original acetate and 94% of the original glucose concentrations). Effects of itaconate on isocitrate lyase activity. Isocitrate lyase activity in extracts of C. immitis was inhibited 70 to 80% by 0.05 mM itaconic acid. No significant differences were seen in the inhibition of isocitrate lyase activity from cells grown in defined media with acetate versus complex media without acetate but with glucose. Effects of itaconate on growth. If acetate

FIG. 3. Growth of C. imrnitis strain Silveira in defined medium containing 0.62% ammonium acetate (H), 1% glucose (O), and 0.62% ammonium acetate plus 1% glucose (0). Growth is indicated in optical density units converted from Klett units as described under Materials and Methods.

is metabolized by the glyoxylate bypass then one might expect itaconate to inhibit this pathway in vivo and thus inhibit growth on acetate. Such inhibition has been observed with Pseudomonas (McFadden, 1969) and Mucor (O’Connell and Paznokas, 1980) when acetate was the sole carbon and energy source, but itaconate did not inhibit these organisms when they were grown on glucose or complex media. Itaconate inhibition of growth of C. immitis (Silveira or 95-271) was observed not only in Walch’s minimal acetate media but also in complex media without acetate, i.e., GYS (Fig. 4). The inhibitory effect of itaconate on the growth of C. immitis was greater during germination than the later phases of growth. NaCl addition to minimal medium to a final concentration of 360 mM had little effect on the growth of C. immitis. At this concentration of NaCl the medium had a conductivity (pmhoicm) greater than that of medium with a final concentration of 200 mM itaconate. Amphotericin B has been used to syner-

GLYOXYLATE

CYCLE OF

Coccidioides

immitis

323

TABLE 2 Comparison of Synergistic Inhibitory Effects of Amphotericin 8” with Various Carboxylic Acids on the Growth of C. immitis Strain Silveira in GYS Medium

I 100 ITACONATE

200 hM)

300

FIG. 4. Effect of itaconic acid on the growth of C. irnrnitis strain Silveira from arthroconidia in complex media containing acetate (AYS) (U) or glucose (GYS) (0) at 72 h of growth. Control culture had no itaconate.

gistically enhance antimetabolic effects with several compounds in mammalian and fnngal cells (Medoff et al., 1972, 1973). Noninhibitory concentrations of itaconate (50 mJ4) and amB (0.0.5 &ml) used in combination completely blocked germination and growth of C. immitis brain Silveira arthroconidia in complex medium containing glucose (GYS) (Fig. 5). Other carboxylic acids were assayed in a similar manner and growth inhibition was normalized to acetate

Organic acidb

% Maximum growthC

Delay of growth initiation’ (h)

Itaconate Citrate Isocitrate Glyoxylate Oxaloacetate Fumarate Malonate Pyruvate o.-Ketoglutarate Succinate Aconitate Malate Acetate

0 0 0 0 0 7.5 93 95 95 100 100 100 100

72 48 12 12 24 24 12 0

a 0.05 pgiml culture-functional concentration was maintained by adding 0.02 &ml at 24-h intervals. b Each compound was added at 50 mM to a basal medium of glucose-yeast extract-Soytone. c Growth is based on Klett-Summerson calorimeter measurements.

(Table 2). These synergistic growth inhibitions, like the inhibition associated with higher concentrations of amB alone, are fungistatic. In all instances apparent normal germination and growth ensued within 72 h of the discontinuance of supplemental am additions (see Materials and Methods). DISCUSSION

0.1;

24

72 HOURS

120

168

FIG. 5. The synergistic effect of itaconate (ITA, 50 ~ and amphotericin B (amB) (0) on the germination of C. immitis in glucose-yeast extract-Soytone (GYS) medium compared to itaconate (0) or amphotericin B (0) added alone. A functional concentration of amB was maintained by adding 40% of the initial 0.05 p.g/ml to the cultures at 24-h intervals.

We report here on the activities of t glyoxylate bypass enzymes isocitrate lyase and malate synthase during germination and vegetative growth of Coccidioides immitis. These enzyme activities are present in germinating arthroconidia and cells of C. imrnitis even under conditions that would have repressed glyoxylate cycle enzymes in a variety of other organisms. ~~~11~0~~~~s indigophora (McFadden and Howes, 1962), Saccharomyces cerevisiae (GOnzZilezp 1977), and MLKO~ racemosus fQ’Conneli and Paznokas. 1980)I do not have measur-

324

AGY AND PAZNOKAS

able activities of either isocitrate lyase or malate synthase when grown in a medium containing glucose, but C. immitis cells have significant levels of both enzymes when grown in either complex or defined media containing glucose. Two indirect observtions support the argument that a functional glyoxylate cycle is important for the germination and growth of C. immitis. First, acetate but neither glucose nor a number of other organic acids or carbohydrates will support the germination of C. immitis arthroconidia. Second, even if glucose is present in the growth medium acetate is preferentially utilized by the organism. While C. immitis is unusual in this regard it is not unique since Aspergillus nidulans (Roman0 and Kornberg, 1969) also preferentially utilizes acetate over glucose from growth media. Itaconic acid, a known inhibitor of the isocitrate lyase activity of Pseudomonas (McFadden and Howes, 1962) and Mucor (O’Connell and Paznokas, 1980), also inhibits isocitrate lyase activity of C. immitis extracts in vitro. Additionally, itaconate inhibits the germination and growth of C. immitis arthroconidia; however, high concentrations of itaconate (100 to 300 mM) are required for maximal inhibition. These high concentrations are consistent with low membrane permeability for itaconate. Medoff et al. (1972) have shown that low concentrations of amB increase the entry of 5fluorocytosine into several different yeasts with enhanced antifungal effects. In a similar manner we have demonstrated synergism with a competitive inhibitor of isocitrate lyase, itaconic acid. While inhibitors can have well-defined effects on purified enzyme preparations, their effect(s) on an intact cell are extremely difficult to define. Consequently, we tested other carboxylic acids (Table 2) and found several of these to have growth inhibitory effects similar to itaconate. The precise mechanism of the observed inhibitors will remain unknown until the ac-

tion of amB and its degradative products are known. Presumably, itaconate and other carboxylic acid molecules have gained access to the cell interior via the putative pore formed by amB spanning the membrane (Andreoli, 1974; Holz, 1974). Once inside of the cell, itaconate could act as a specific competitive inhibitor of isocitrate lyase. It is unclear what the other organic acids might be doing under these conditions. Alternatively, the combination of the carboxylic acid and amB or a degradative product of amB have their antifungal effect outside of the cell, for example, at or in the cytoplasmic membrane. This has been proposed as the site of action for lysozyme (Collins and Pappagianis, 1974) and polymyxin B (Collins and Pappagianis, 1975) each in synergism with amB. Both lysozyme and polymyxin B are basic peptides whose binding sites could be competitively blocked by divalent cations. Although carboxylic acids are not positively charged at near neutral pH values, charge difference alone does not rule out interaction of the carboxylic acid with amB or a derivative that would enhance anticoccidiodal activity at the cytoplasmic membrane. ACKNOWLEDGMENTS This work was supported in part by a grant from the American Lung Association of Washington. We thank Dr. Wayne Loescher for his assistance in the gas chromatographic analysis of glucose. REFERENCES T. E. 1974. The structure and function of amphotericin B-cholesterol pores in lipid bilayer membranes. Ann. N.Y. Acad. Sci. 235: 448-468. BELLION, E., AND KELLEY, R. L. 1979. Inhibition by itaconate of growth of methylotrophic bacteria. J. Bacteviol. 138: 519-522. BRASS, C., LEVINE, H. B., AND STEVENS, D. A. 1982. Stimualtion and suppression of cell mediated immunity by endosporulation antigens of Coccidioides immitis. Infect. Immun. 35: 431-436. BROBST, K. M., AND LOTL, C. E. 1966. Determination of some components in corn syrup by gas-liquid chromatography of the trimethylsilyl derivatives. Cereal Chem. 43: 35-42. ANDREOLI,

GLYOXYLATE

CYCLE OF Cocci&ides

CHEUNG, S. C., MEDOFF, G., SCHLESSINGER,D., AND KOBAYSHIM, G. S. 1975. Response of yeast and myceiial phases of Histoplasma capsulatum to amphotericin B and actinomycin D. Antimicrob. Ag. Chemother. 8: 498-503. COLLINS,

M.

S.,

AND

PAPPAGIANIS,

D.

1974.

Lyso-

zyme-enhanced killing of Candida albicans and Coccidioides irnrnitis by amphotericin B. Sabouraadia 12: 329-340. COLLINS, M. S.. AND PAPPAGIANIS. D. 1975. Inhibition of Corcidioides immitis in vitro and enhancement of anticoccidiodal effects of amphotericin B by polymyxin B. Antimicrob. Ag. Chemother I: 781-787. COLLINS. M. S.. AND PAPPAGIANIs. D. 1977. Uniform susceptibility of various strains of Cpccidioides immitis to amphotericin B. Antimicrob. Ag. Chemother. 11: 1049-1055. GONZALEZ, E. 1977. Two carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae. J. Bucteriol. 129: 1343-1348. HARDENBROCH, M. H., AND BARRIERE, S. L. 1982. Coccidioidomycosis: Evaluation of parameters used to predict outcome with amphotericin B therapy. Mycopathologia 78: 65-71. HILLIER, S.. AND CHARNETZKY, W. T. 1981. Glyoxylate bypass enzymes in Yersinia species with multiple forms of isocitrate lyase in Yersinia pestis. J. Bacterial. 145: 452-458. HOLZ, R. W. 1974. The effects of polyene antibiotics nystatin and amphotericin B on thin lipid membranes. Ann. N. Y. Acad. Sci. 235: 460-479. HUANG, A. H. C. 1982. Metabolism in plant peroxisomes. In Recent Advances in Phytochemistry (L. L. Creasy and G. Hrazdina, Eds.). Plenum, New York/London. KELLY, P. C. 1980. Coccidioidal meningitis. In Coccidiodomycosis: A Text (D. A. Stevens, Ed.), pp. I63-193. Plenum Med. Book Co., New York/ London. KOBR? M. J., VANDERHAEGHE, F.: AND COMBEPINE, G. 1969. Particulate enzymes of the glyoxylate cycle in Neurospora crassa. Biochem. Biophys. Res. Commcm. 37: 460-465. KORNBERG. H. L., AND KREBS, H. A. 1957. Synthesis of cell constituents from C,-units by a modified tricarboxylic acid cycle. Nature (London) 197: 988991. KRAMER,

D.,

N.

KLEJN,

AND

R. A.

BASELICE.

1959.

Quantitative determination of glyoxylate acid. Anal. Chem. 31: 250-252.

immiris

325

6. W. 1967. Studies of intermediary metabolism in Coccidioides immitis. In Proceedings qf the Second Coccidioidomycosis Symposium (L. Ajello, Ed.), pp. 349-353. Univ. of Arizona Press. Tucson. MCFADDEN, B. A. 1969. In Methods of E~qm~!ogy (.I. M. Lowenstein, Ed.), Vol. XIII. pp. 163-170. Academic Press, New York. MCFADDEN, B. A., AND HOWES, W. V. 1960. The determination of glyoxylic acid in biological systems. Anal. Biochem. 1: 240-248. MCFADDEN, B. A., AND HOWE& W. V. 1962. Oxidative metabolism and the glyoxylate cycle in Pseudomonas irzdigofera. J. Bacterial. 79: 341-345. LONE%

MEDOFF, LIGER.

G., KOBAYASHL G. S., KWAN. D., AND VENKOV. P. 1972.

MEDOFF.

G.,

C. N..

SCHEL-

Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proc. Nail. Acad. Sci. USA 69: 196-199. KWAN.

C. N.,

SCHLESSINCER.

D..

AXD

6. S. 1973. Permeability control in animal ceils by polyenes: A possibility. Anrimicrob. Ag. Chemother. 3: 441-443. KOBAYASHI,

O’CONNELL, oxylare

B. T., AND PAZNOKAS, cycle in n/lucor racemosus.

J. L. 1980. G3yJ. Bactei-iol.

143:416-421.

D. 1980. Epidemiology of coccidioidomycosis. In Coccidioidomycosis: A Text 0. A. Stevens. Ed.), pp. 63-85. Pienum Med. Book Co.; New York/London. PATEL. T. R., AND MCFADDEN; B. M. 1978. Caenorhabditis elegulzs and Ascaris suuna: Inhibition of isocitrate lyase by itaconate. Exp. Par&to/. 44:

PAPPAGIANIS,

262-268. H. C.. AND AJL, S. 1960. Occurrence and function of isocitrate lyase and malate synthetase in bacteria. .I. Bacterial. 79: 341-345.

REEVES,

ROMANO,

A. W.,

AND

KORNBERG,

H.

1969. Regu-

L.

lation of sugar uptake by Aspergi//us nidulans. R. Sot. London. Ser. B 173: 475-490. SRERE:

P. A.,

BRAZIL,

H.,

AND

GONEN,

L.

A?oc.

1963. The

citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta Chem. Stand. 17: 5129-5134.

WALCH: II. A., AND WALCH, R. K. 1965. Studies with induced mutants of Coccidioides immitis. in Proceedings of the Second Coccidiodomycosis Symposium (L. Ajello, Ed.). Univ. of Arizona Press. Tucson.