Arch. Microbiol. 116, 61- 67 (1978)

Archives of

Hicrobiolow 9 by Springer-Verlag 1978

The Alternate Respiratory Pathway of Candida albicans MAXWELL G. SHEPHERD, CHIN MOI CHIN, and PATRICK A. SULLIVAN Department of Biochemistry, University of Otago, Dunedin, New Zealand

Abstract. Candida albicans c o n t a i n s a cryptic cyanide a n d a n t i m y c i n A insensitive respiratory system. This alternate oxidase was f o u n d (i) at all growth rates f r o m # = 0.05 to 0.26 in a c h e m o s t a t culture a n d (ii) in b o t h mycelial a n d yeast forms of the organism. N e i t h e r c h l o r a m p h e n i c o l n o r cycloheximide p r e v e n t e d the expression of the alternate oxidase. Salicylh y d r o x a m i c acid was a p o t e n t i n h i b i t o r of the cyanide insensitive respiration. T h e r e s p i r a t i o n o f m i t o c h o n dria g r o w n in the presence of a n t i m y c i n A was n o t i n h i b i t e d by cyanide or a n t i m y c i n A b u t was i n h i b i t e d by s a l i c y l h y d r o x a m i c acid. Key words: R e s p i r a t i o n - A l t e r n a t e oxidase - Cytoc h r o m e c oxidase - Candida albicans - C y a n i d e a n d a n t i m y c i n A insensitive respiration.

The early studies o n the respiratory system of Candida albicans p r o d u c e d some u n u s u a l results. W a r d a n d N i c k e r s o n (1958) reported t h a t r e s p i r a t i o n in C. albicans is n o t m e d i a t e d by the classical c y t o c h r o m e c oxidase a n d the r e s p i r a t i o n of resting cells is resistant to a n d even s t i m u l a t e d by cyanide, c a r b o n m o n o x i d e a n d s o d i u m azide. Chiew et al. (1972) confirmed the s t i m u l a t i o n of r e s p i r a t i o n by c y a n i d e b u t f o u n d the classical c y t o c h r o m e c oxidase in respiratory particles. M o r e recently, C h i n et al. (1975), K o t et al. (1975, 1976) a n d H e r r m a n n (1967, 1970) have r e p o r t e d that there is a n alternate oxidase in C. albicans which appears to be similar to the alternate cyanide-insensitive p a t h w a y f o u n d in a n u m b e r of p l a n t s a n d yeast ( D o w n i e a n d G a r l a n d , 1973; S c h o n b a u m et al., 1971 ; H e n r y et al., 1974; L a m b o w i t z a n d S l a y m a n , 1971). Abbreviations9 KCN = potassium hydroxamic acid

cyanide;

SHAM = salicyl

This p a p e r describes the n a t u r e a n d the expression of the alternate respiratory p a t h w a y of C. albicans u n d e r a variety of growth c o n d i t i o n s .

MATERIALS AND METHODS Reagents. All chemicals were analytical reagent grade. Enzyme substrates and other biological substances were obtained from Sigma. Thioglycollic acid was obtained from May & Baker Ltd, Dagenham, England. Helicase was from Industrie Biologique Fran~aise, Genevillers, France, and crude B glucuronidase (snail gut extract) was from the Sigma Chemical Co., U.S.A. Sorbitol and mannitol were from Sigma and BDH respectively and were purified by batchwise treatment with Dowex 50 H + prior to use. Organism and Culture Conditions. Candida albicans CMI 45348 was obtained from the Commonwealth Mycological Institute, Surrey. C. albicans ATCC 10261 and ATCC 10259 were obtained from the American Type Culture Collection, Maryland, U.S.A. All other strains were obtained from patients at Dunedin Public Hospital and were kindly provided by Mr. H. Sfiott of the Diagnostic Laboratory. All strains were propagated on either Sabouraud dextrose agar or malt extract agar at 28~C. For submerged cultures two basic media were used. N-limiting medium contained (per litre): carbon source, 15 g; (NH4)2SOr 0.5 g; KH2PO~, 2.0 g; CaCI2 . 2 HzO, 0.05 g; ZnSO4 . 7 H20, 1 rag; CuSO4 95 H20, I rag; FeSO4 - 7 H20, 0.01 g; biotin, 25 ~tg; final pH 5.2. C-limiting medium contained (per litre): carbon source, 10g; (NH4)2SO4, 2g; KH2PO4, 2.0g; C a C I 2 . 2 H 2 0 , 0.05 g; MgSO4 - 7 H20, 0.05 g; ZnSO4 . 7 H20, 1 mg; CuSO, 95 H20, I rag; FeSO4 - 7 H20, 0.01 g; biotin, 25 ~tg; final pH 5.2. Slants (8 g vials) of C. albieans were shaken with 5 ml of Tween 80 solution (500 ppm). This produced a suspension containing approximately 5 x 108 cells which was used to inoculate the fermenter. Shake Culture Experiments. A 2 1 Erlenmeyer flask containing I 1 C-limiting medium with glucose as carbon source, was inoculated with 5 x 108 cells and shaken on a gyratory shaker at 250 rev./min at 28~ this flask was modified with three vertical flutes to improve aeration. Chernostat Studies. The chemostat and the conditions of growth were as described previously (Shepherd and Sullivan, 1976). The temperature of growth, unless otherwise specified, was 30 _+ 0.1~C. The air flow and the stirrer were adjusted to provide a Po2 (determined by a Johnson-type steam-sterilizab!eoxygen electrode; John-

0302-8933/78/0116/0061/$ O1.40

62 son et al., 1964) between 5 and 90~ saturation of oxygen. The culture was regarded as having attained a steady state following a change in operating conditions when it had grown for at least seven doubling times, or for at least two doubling times during which there was no change in the monitored parameters of growth, i.e. E540, Po2 of the culture, Pco~ of the effluent gas (determined in a Lira model 303 infrared analyzer; Mine Safety Appliances Co. Ltd, Glasgow), or concentration of one of the substrates. The fungal cell dry matter was determined from the E54oof a sample by using a calibration curve of E54o versus dry weight. Separate calibration curves for mycelial, pseudo-mycelial and yeast cells of C. albicans CM1 45 348 were required. Oxygen Uptake Measurements. The oxygen uptake rates of cells were measured in a Clark-type oxygen electrode (Yellow Springs Instrument Co., Ohio, U.S.A.; electrode 5331). Where necessary the sample was diluted with spent medium obtained just before the oxygen uptake determination. The reaction chamber was enclosed in a constant-temperature water jacket and the reaction mixture was stirred by means of a magnetic bar. Metabolic Quotients. The specific growth, #, of the steady-state continuous culture is equal to the dilution rate, D, and is expressed in units of reciprocal time (h-i). The specific oxygen uptake (qo2) or respiration rate is expressed as IJl 02. mg dry wt. 1 . min-L In a typical assay 0.3 ml of the cell suspension was added to the oxygen electrode chamber which contained 2.7 ml of spent medium. The qo2 of this solution was determined and then 30 pl of 40 mM potassium cyanide was added. The qo~ values were then measured at the time indicated in the figures. Addition of a further 30 pl after 60 min caused no change in the qo2. In some instances the qo~ was determined in 0.1 M KHzPO4-NaHPO4 buffer, pH 5.6. Cells from the chemostat were washed three times (by centrifuging) and finally suspended in the buffer. The qo~ of an appropriate dilution was determined as described above. Preparation of Spheroplasts and a Mitochondrial Fraction. Cells were collected for 48 h from chemostat cultures of C. albicans ATCC 10261 and stored at 4~C. The yield was 4 0 - 45 g wet weight. Spheroplasts and a mitochondrial fraction were prepared essentially as described by Kovac et al. (1972) with modifications as described below. Cells were initially suspended in 0.5 M thioglycollate buffered with 0.1 M Tris-HC1, pH 9.3 (0.75 ml/g wet weight) and incubated at 25~C for 30 rain. For the cell wall digestion either helicase, 4.4 mg protein/g wet weight ceils or crude B-glucuronidase, 16.0 mg protein/g wet weight cells were used under conditions described by Kovac et al. (1972). About 7 0 - 9 0 ~ of the cells were converted to spheroplasts after 1.5-2 h. The spheroplasts v~erewashed three times in 0.44 M mannitol containing 50 mM KH2PO4-NazHPO4 buffer pH 6.5, 1 mM EDTA and 0A ~ w/v bovine serum albumin and finally suspended in the same solution at 1 ml/g wet weight. Treatment with a motor-driven Potter-Elvehjem homogenizer for 15 s was used to disrupt the spheroplasts. The mitochondrial fraction isolated by the procedure of Kovac et al. (1972) was finally suspended in 2.4 ml of the 0.44 M mannitol solution from which the bovine serum albumin had been omitted. Yields of the mitochondrial fraction were in the range 2 - 4 mg protein/g wet weight of cells.

RESULTS Expression o f the Alternate Oxidase T y p i c a l results for the expression the alternate oxidase of Candida albicans are s h o w n in F i g u r e 1. T h e cryptic n a t u r e of the alternate oxidase is a p p a r e n t ; there is

Arch. Microbiol., Vol. 116 (1978)

~-

w ......... . ......... -...

g ~ 1-5 ,'v

0

5

10

15

20

25

30

35

40

45

50

time offer oddilion of KeN(minutes)

Fig. 1. The effect of the specific growth rate (/2) on the expression of the alternate respiration in steady state cultures of Candida albicans ATCC 10261. The culture conditions are as described in "Materials and Methods". A N-limiting medium with glucose as a carbon source was used. qo2 values for these cells were determined in an appropriate dilution of spent medium. Potassium cyanide was added to a final concentration of 0.4 mM after the constant qo2-values were determined. 9 /1= 0.26; 9 # = 0.15; 9 # = 0.08; [] tt= 0.05

a lag o f some 10 m i n before the alternate oxidase is expressed, Initially 9 0 - 9 5 ~ o f the total respiration is cyanide sensitive. After 25 rain the respiration had recovered to a level of b e t w e e n 95 a n d 120~o of the original. As the growth rate decreased f r o m / 2 = 0.26 to /2 = 0.05 the qo2 for b o t h the n o r m a l a n d the alternate r e s p i r a t i o n decreased by 3 6 ~ a n d 4 6 ~ respectively. It should be n o t e d that at all the steady states for these c h e m o s t a t cultures o f C. albicans A T C C 10261 the cultures were c o m p o s e d entirely: of yeast cells. W i t h this strain, altering the growth rate c h a n g i n g the c a r b o n or n i t r o g e n source did n o t affect the m o r p h o l o g y (cf. C. albicans C M I 45346; Shepherd a n d Sullivan, 1976). A similar change in the qo~ for b o t h the n o r m a l a n d alternate respiration was observed for cells harvested at different times d u r i n g a b a t c h c u l t u r e ; the qo2 values were m u c h higher for cells harvested d u r i n g the early log phase t h a n for s t a t i o n a r y phase cells. T h e alternate r e s p i r a t i o n was almost completely i n h i b i t e d by salicyl h y d r o x a m i c acid at final concent r a t i o n s of 3 m M (Figs. 2 a n d 3). Salicyl h y d r o x a m i c acid did not, however, i n h i b i t the respiration of u n treated cells or cells which h a d been treated sequentially with salicyl h y d r o x a m i c acid a n d p o t a s s i u m c y a n i d e ; the p r e i n c u b a t i o n of cells with cyanide or a n t i m y c i n A was essential for salicyl h y d r o x a m i c acid i n h i b i t i o n . As d e m o n s t r a t e d in Figures 2 a n d 3, c h l o r a m p h e n i col did n o t affect the expression of the alternate oxidase activity. Cells g r o w n in the presence of chlora m p h e n i c o l showed the same p a t t e r n of expression for

M. G. Shepherd et al. : The Alternate Respiratory Pathway of Candida albicans

KCN

63

Table 1. Growth of Candida albicans in the presence of respiratory chain inhibitors

SHAM

Conditions of growth 1-5

Final cancantration

Dry weight at 30 h

qo2 normal oxidase

(mM)

(mg/ml)

(pl 02 rain -~ mg dry wt. -~)

1.8 1.0

0.62 0.68

0,96 1,32

1.8

0.78

0.01

I'0

5~

Control + antimycin A 9 • 10 .3 + salicyl hydroxamic acid 3.5

9

0"5

0

5

10

15

20

25

30

35

40

45

50

time offer addition el KCN (minutesl

Fig. 2. The effect of chloramphenicol on the expression of the alternate oxidase, Steady state cultures of C. albicans ATCC 10 261 were obtained in a N-limiting glucose medium as described in "Materials and Methods". The alternate respiration was expressed as described in the legend to Figure 1. 9 respiration of cells obtained from a culture with /~ = 0.2; [] respiration o f cells from a culture with # = 0.16 and grown in the presence of chloramphenicol (3 mg/ml final concentration); O respiration of cells from a culture with # = 0.2 but preincubated 30 min with chloramphenicol (3 mg/ml final concentration) before adding the cyanide. After 40 min salicyl hydroxamic acid was added to the oxygen electrode to a final concentration of 3 m M SHAM

-

2,0

SNTIMYCINA c)

1.5

i

~" 1.o

0"5

0

I

1

E

I

I

I

I

b

O

5

10

15

20

25

30

35

40

45

50

time after addition ef onfimydnA(minutes)

Fig. 3. Expression of the alternate oxidase with antimycin A. The conditions of the experiment are exactly the same as those described in Figure 2 except that antimycin A (final concentration of I p M) was used to express the alternate respiration. 9 respiration of cells with # = 0.2; [] respiration of cells with g = 0.16 grown in the presence of chloramphenicol (3 mg/ml); 9 respiration of cells with # = 0.2, these cells were preincubated for 30 rain with chloramphenicol (3 mg/ml) before adding the antimycin. After 40 rain salicyl hydroxamic acid was added to the oxygen electrode to a final concentration of 3 m M

the alternate respiration as control cells. Similarly when glucose grown cells were preincubated with chloramphenicol the alternate oxidase was still expressed with the same lag phase. In these experiments

qo2 alternate oxidase

The inhibitors were added to the flasks after autoclaving the media; salicyl hydroxamic acid and antimycin A were added as methanolic solutions. Sodium azide and potassium cyanide were added as solids

it made no difference whether cyanide or antimycin A was used to unmask the alternate oxidase. C. albicans ATCC 10261 was also grown as a batch culture in the presence of chloramphenicol and cycloheximide. With final concentrations of chloramphenicol of 3 mg/ml and 6 mg/ml the alternate oxidase reached values of 200 ~o and 160 % respectively of the normal oxidase. After 30 h of growth dry weight values were 1.8 mg/ml (3 mg/ml chloramphenicol) and 1.4 mg/ml (6 mg/ml chloramphenicol) compared with a control value of 2.5 mg/ml. With cycloheximide (10 mg/ml final concentration) the dry weight after 30 h was only 0.2 mg/ml but after 60 h a dry weight of 1.8 mg/ml was obtained. The qo2 for the alternate oxidase at 60 h was 0.54 compared with 0.79 for the normal oxidase. When cells of C. albicans were preincubated for 30 min with cycloheximide (10 mg/ml) before the addition of cyanide the alternate oxidase was still expressed. It should be noted, however, that with this concentration of cycloheximide, only 7 0 ~ of the alternate oxidase was expressed compared to a control. Cycloheximide (up to a concentration of 25 mg/ml) had no effect on the respiratory activity of cells that had the alternate oxidase expressed. In view of the effect of cyanide and antimycin on the respiration of C. albicans it was of some interest to determine the effect of these reagents on growth. C. albicans will not grow in the presence of 4 mM azide or 0.5 mM cyanide. As expected salicyl hydroxamic acid did not affect the growth curve and after 30 h the growth yield (1.8 mg/ml) was the same as the control (Table 1). Alternate oxidase activity could not be expressed in the salicyl hydroxamic acid grown cells. Antimycin caused a distinct lag in the growth; after 30 h the growth yield was only 1.0 mg/ml but by 40 h the yields in the control and antimycin cultures were the same (2 mg/ml). The respiration of the anti-

64

Arch. Microbiol., Vol. 116 (1978) Table 2. The effectof sodium azideon the respirationof C. albicans z.o-

KCN

Time

_~,,~ 1'5 - 1

(rain)

@2 Inhibipl 02 tion rag- 1min- x (~)

A Controlcells

T= 0

0.99

-

B A+Naazide(4mM)

T= 0 T = 35 T = 65

0.03 0.03 0.03

97 97 97

C

T= 0 T = 40 T = 60

0.06 0.85 1.05

94 15 --

T = 60

0

100

T= 0 T= 0 T= 0

0.58 0.31 0

45 70 100

l

0

N 1'0

@ t:~ [I.5

5

~O

15

ZO

25

30

35

@

A+KCN(0.3mM)

C' C' + salicyl hydroxamic acid

time after addition of KCN{minutes)

Fig. 4. Expression of alternate respiration in yeast and mycelial cells of C. albieans. The conditions for the production of the yeast and mycelial forms of C. albicans CMI 45348 were as described previously (Shepherd and Sullivan, 1976). The qo~-values were determined in an appropriate dilution of spent medium. Potassium cyanide was added to a final concentration of 0.4 mM after the control qo~-valueswere determined. O yeast cells; 9 mixed yeast and pseudomycelialcells; 9 mycelialculture

mycin grown cells was not inhibited but stimulated by addition of either cyanide or antimycin A to the oxygen electrode; this respiration was sensitive to salicycl hydroxamic acid. A number of strains of C. albicans were examined for alternate oxidase and all of these exhibited the alternate oxidase and it was always cryptic in nature. Among the strains examined were ATCC 10259 and 10261, C M I 4 5 3 4 8 , and several strains obtained from patients of Dunedin Public Hospital. With these different strains, however, the amount of alternate respiration expressed varied between 40 % and 200 % relative to the normal respiration. The morphology of the culture of C. albicans did not affect the expression of the alternate oxidase (Fig. 4): mycelial, yeast and mixed yeast, pseudomycelial cultures of C. albicans 45 348 all possessed an alternate oxidase which was unmasked with cyanide after a 10 rain lag. The stability of the normal and alternate oxidases w e r e examined in two different ways. Cells from a nitrogen-limiting glucose culture ( # = 0 . 1 8 , Fig. l) were stored at 4 ~ C (i) as recovered from the chemostat and (ii) after washing three times by centrifuging and then suspending in 0.1 M KH2PO4-Na2HPO4 buffer pH 5.6. After 7 days the cells in the spent media retained 50 % of both the alternate and normal oxidase while the cells in the phosphate buffer had 60 7o of the normal oxidase but only 22% of the alternate oxidase could be expressed.

D C ' + Na azide (4 raM) C ' + Na azide (8 raM) C ' + Na azide (12 raM)

The oxygen electrode chamber contained 3.0 ml of 0.1 M KH2PO4Na2HPO~, pH 5.6 buffer and 0.6 mg dry weight of cells obtained from batch cultures and suspended in the above buffer, The final concentrations of sodium azide, salicyl hydroxamic acid and potassium cyanide are given in the table

Changing the temperature of incubation for the expression of the alternate oxidase did not affect the cryptic nature of the activity. The alternate respiration was at least 100 % of that of the normal oxidase after 25 rain at 25 ~ 30 ~ 37 ~ C but the qo~ values were 0.98, 1.1 and 1.6 respectively. The effect of sodium azide on the respiration of C. albicans 10261 is shown in Table 1. Cytochrome c oxidase was inhibited 97 % by 4 m M azide but the alternate oxidase was not expressedwith prolonged incubation. Itwas subsequently found that the alternate oxidase in cells incubated with cyanide was inhibited by sodium azide but a concentration of 12raM was required for 100% inhibition (Table 2). The Ki(0.5) for the normal and alternate oxidase was 0.1 m M and 6 m M respectively, where Ki ~o.5) is equal to the concentration of sodium azide necessary for 50 % inhibition. This large difference in inhibitor sensitivity by sodium azide provide further evidence that the two oxidase are quite distinct. Respiration o f Spheroplasts and Isolated Mitochondria

Spheroplasts exhibited lower endogenous respiratory rates than intact cells (qo2 of 0 . 2 - 0.4 gl 02 mg- 1 min- 1 cf. 0 . 4 - 1 gl 02 mg -1 min-1). Incubation of spheroplasts with cyanide elicited cyanide-insensitive respiration. The time course for the expression of the alternate respiration was similar to that observed with whole cells but the maximum rate obtained was only 66 %

M. G. Shepherd et al. : The Alternate Respiratory Pathway of Candida albicans Table 3.

Endogenous respiration of spheroplasts

Additions

Time (rain)

Cyanide

0 20 30 40

Plus salicylhydroxamic acid

Fresh After J2 h preparation storage (qo2 ~I 02 ~mg-1 min -~) 0.24 OA 0.12 0.13 0.16

0.21 0.05 0.07 0.10 0.14

0.01

0.05

Spheroplasts were prepared from strain ATCC 10 261 as described in "Materials and Methods" and assayed either on the day of preparation or after 12 h storage at 4~C. The endogenous respiration was measured for 5 rain; cyanide was added (0.3 mM final concentration) and the rate of respiration was recorded at the times indicated. Salicyl hydroxamic acid was added at 40 min to a final concentration of 3.5 mM

65

succinate 0.3-1.3 and NADH, 1.3-5.0. In contrast to the whole cells and spheroplasts, when the respiration of mitochondria was perturbed by the addition to substrates or inhibitors a new steady state was established immediately and this rate did not alter with incubation times of up to 30 rain. Inhibition by cyanide or antimycin varied 7 7 - 1 0 0 ~o depending on the substrate (Table 4). The residual respiration was further inhibited by the addition of salicyl hydroxamic acid. Mitochondria were also isolated from cells grown in the presence of antimycin. These mitochondria were largely insensitive to cyanide and antimycin but the respiration was significantly inhibited by salicyl hydroxamic acid. The mitochondria isolated from spheroplasts were effectively uncoupled as judged by the absence of respiratory control with added ADP. DISCUSSION

Table 4.

Respiration of isolated mitochondria

Additions

Growth conditions normal

+ antimycin

qo~ ~tl O2 .rag protein -1. min -1 Malate pyruvate + cyanide + salicylhydroxamate Succinate + antimycin A + cyanide + salicylhydroxamate NADH + cyanide + salicylhydroxamate

0.77 0.18 0.11 0.3 0.13 0 2.18 0.13 0

] .24 0.89 0.41 2.48 2.48 2.06 0.28 2.75 2.48 0.96

Mitochondria were prepared from carbon-limiting chemostat cultures of C. albicans ATCC ]0261,/~ = 0.11, and batch cultures of the same organism supplemented with 9A ~M antimycin as described in "Methods". Each assay was carried out with one of the following substrates: 4 mM malate plus 4 mM pyruvate, 80 mM succinate, or 0.6 mM NADH. The assays were initiated by the addition of mitochondria and the rate of respiration was measured for 2 - 3 min. Inhibitors were then added consecutively as indicated and respiration was followed for 2 - 3 min before the next addition. The inhibitors tested (final concentrations in parenthesis) were: cyanide, (0.3 mM) salicylhydroxamicacid, (3.5 mM) and antimycin A, (5.4 gM)

of the original respiration (Table 3). Also as shown in Table 3 the spheroplasts could be stored for 12 h at 4~ without affecting the endogenous respiration of the expression of alternate oxidase. Mitochondria isolated from spheroplasts had negligible endogenous respiration. Malate plus pyruvate, succinate, and N A D H stimulated respiration. The rates varied within the following ranges: malate plus pyruvate qo~ (gl 0 2 . mg protein -t . min -1) 0.3-0,8,

Although the existence of cyanide insensitive respiration in Candida albicans has been observed (Kot et al., t975, 1976; Ward and Nickerson, 1958) the cryptic nature of this respiration has not hitherto been described. In the early studies on respiration in C. albicans, oxygen uptake was measured by manometry and the time scale of these observations could explain why the unmasking of the alternate oxidase was missed. In the manometry experiments of Ward and Nickerson (1958) and Chiewet al. (1972) a lag is apparent in the presence of cyanide; this could have been attributed to manometry equilibration. Kot et al. (1976) used an oxygen electrode but only measured respiration in the presence of each inhibition for a 5-min period before another addition was made to the system. The data of Figures 1 - 3 have clearly established that a 2 0 - 3 0 min period is required for the alternate respiration to be fully expressed. The alternate oxidase appears to be constitutive since it was present at all stages of a batch culture and at all the growth rates tested in the chemostat (Fig. 1). In contrast, cyanide-insensitive respiration in C. lipolytica only appeared in aged cells under conditions of glucose depletion (Henry et al., 1974). Studies on C. utilis by Downie and Garland (1973), showed that this organism developed cyanide- and antimycin A-insensitive mitochondrial respiration and lost cytochrome oxidase activity when grown in Culimiting continuous culture. An interesting feature was revealed during the reculturing of these Cu-limited cells in the presence of Cu 2§ for 20 generations; the cytochrome-c oxidase reappeared but the alternate oxidase was retained and appeared to have become constitutive. All strains of C. albicans tested thus far possessed the cryptic alternate oxidase. It thus appears

66

that this activity is a normal constitutive feature of all strains of this species. Furthermore, the numerous reports of cyanide-insensitive respiration in other species of candida; C. glabrata (Herrman, 1970), C. lipolytica (Henry et al., 1974), C. tropicatis (Teranishi et al., 1974), and C. utilis (Haddock and Garland, 1971; Downie and Garland, 1973), suggest that a branched respiratory chain may be ubiquitous within this genus. The rate of expression of the alternate respiration was essentially constant although the respiration rate for both the normal and alternate respiration decreased with increasing age of the culture (Fig. 1). The alternate oxidase could be expressed with both yeast and mycelial cultures of C. albicans (Fig. 4) which indicates that the morphology of the organism does not affect the respiratory properties. Increasing the temperature of incubation from 2 5 - 3 7 ~ increased both the rate of expression and the final level of the alternate oxidase. Although the lag period was only some 20 min it was of interest to determine whether or not the inhibitor of mitochondrial protein synthesis, chloramphenicol affected the appearance of the alternate respiration. It was found (Figs. 2 and 3) that the presence of chloramphenicol in either the growth medium or in the assay mixture had little effect on the rate of expression of the alternate respiration, but the final value attained with chloramphenicol in the assay mixture was some 90 ~ higher than the control value. There was certainly no inhibition of the alternate oxidase which indicates that mitochondrial protein synthesis is not required for the expression of the alternate oxidase. It is possible that chloramphenicol prevented the synthesis of some inhibiting component of the respiratory chain or a repressor of the cyanide insensitive pathway. These results are in accordance with the observations of Edwards et al. (1974) and Lambowitz et al. (1972) that chloramphenicol treated cells produced higher rates of alternate respiration. Cells grown in the presence of cycloheximide also express the alternate oxidase and incubation of C. aIbicans with cycloheximide allowed 70 ~ expression of this activity. These results indicate that cytoplasmic protein synthesis is not essential for the expression of this activity as suggested by Henry et al. (1974). Both antimycin A (Fig. 3) and cyanide elicited the expression of the alternate oxidase and salicyl hydroxamic acid was a potent inhibitor of this respiration; but not of normal respiration. Derivatives of hydroxamic acid have been shown to inhibit the cyanide insensitive respiration reported for all species (Henry and Nyns, 1975). This observation plus the fact that the K~(o.5) values of sodium azide for the normal and alternate respiration were so different are

Arch. Microbiol., Vol. 116 (1978) (a)

S H A M , NaN3 / - X ,, ar~timycin A / b ~ cl - ~ c - ~ a a 3 ~ O z

NADH~Fp~CoQ

t

~O2

i

/ K C N , NaN3 N A D H ~ Fp ~ CoQ ~ b --* ca ~ c --+ aa3 -+02 (b)

S H A M , NaN3

~ o2 ntimycin A NADH-~Fp~CoQ~b@c~ NADH

/ /

/ t!

~c-~aa3~O2 I~CN, NaN3

N A D H ~ Fp ~ CoQ -~ b -~ cl --+ c ---, aa3 ~ 02 S H A M , NaN3

/

Malate

e D~L c~-G3P ar r / "~ N A D H -~ Fp ~ C o Q / ~ *b~'~-~ -~ c --, aa3 --* 02 / / 7" / pyruvate ~ / / / / antimycin/A K C N , NaN3 NADH / NADH~Fp--,CoQ~b~cl--,c~aa3~02 Scheme 1. The respiratory chain of C. albicans. Two possible models for the electron transport chain of C. albicans from N A D H

strongly indicative of two distinct terminal oxidases. In an earlier study (Chiew et al., 1972) we established in respiratory particles of C. albicans the presence of the classical cytochrome c oxidase and an electron transport chain containing cytochromes a, a3, b and c. A proposed respiratory chain for C. albicans is shown in Scheme 1. If the electron transport chain is branched as suggested the pathway to the alternate oxidase must originate between coenzyme Q and cytochrome b in order to account for respiration in the presence of antimycin and the succinate stimulated respiration in mitochondria which were inhibited by salicyl hydroxamate (Table 4). Downie and Garland (1973) have proposed a similar scheme for a variant of C. utilis which arose during Cu-limited growth. The above scheme accounts for the pattern of inhibition and substrate utilization by mitochondria. It could also accommodate the effect of electron transport chain inhibitors on growth. Lack of respiratory control in isolated mitochondria could have been due to a number of factors including damage during isolation, the use of thioglycollate, impurities in the isolation medium, the conditions required to disrupt the cells and glucose catabolite repression. It has been reported that these factors can affect the integrity of isolated mitochondria (Kovacetal., 1972; Lloyd, 1974). The endogenous respiration of spheroplasts was much lower

M. G. Sheoherd et al. : The Alternate Respiratory Pathway of Candida albicans

than that of intact cells but the respiratory rates of the isolated mitochondria compare favourably with preparations from C. utilis (Light and Garland, 1971). Unmasking of the alternate oxidase activity was not observed when mitochondria were treated with either antimycin A or cyanide; it is possible that the isolated mitochondria were damaged during isolation. Haddock and Garland (1971) reported that the cyanide insensitive respiration in mitochondria prepared from sulphate-recovered cells of C. utilis was lost when mitochondria were converted into submitochondrial particles. Clear evidence that the alternate pathway in C. albicans is located in the mitochondria was obtained with cells grown in the presence of antimycin A. Respiration in these mitochondria was not inhibited significantly by cyanide or antimycin but salicyl hydroxamate inhibited 6 0 - 9 0 ~o of the respiration with various substrates (Table 4). Oxidation of external N A D H by yeast mitochondria has been well documentated and has been attributed to a rotenoneinsensitive N A D H dehydrogenase accessible from the outside of the inner membrane (von Jagow and Klingenberg, 1970; Light and Garland, 1971) and an antimycin-insensitive N A D H oxidation pathway in the outer mitochondrial membrane (Ohnishi, 1972). Cyanide inhibited the growth of C. albicans and this implies that the cytochrome c-cytochromeaa region of the respiratory chain is essential for growth. Growth in the presence of antimycin, after an extended lag phase, could be accounted for by respiration involving the alternate oxidase and a by-pass around the antimycin block. Downie and Garland (1973) concluded that the mitochondria from the antimycincyanide resistant variant of C. utilis was completely dependent on the alternate pathway and that ATP synthesis occurs only at site I. The role of the cryptic alternate oxidase in C. albicans remains to be established because this pathway was not detected under normal physiological conditions. Acknowledgements. This work was supported in part by a grant from the Medical Research Council of New Zealand.

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