Arch. Mikrobiol. 64, 289--314 (1969)

Inactivation of Candida albicans by Ultraviolet Radiation DAVID L. BUSBEE a n d A n v l x SA~ACm~I( D e p a r t m e n t of Biology, Wichita State University, Wichita, Kansas, U.S.A. Received October 1, 1968 Summary. Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells b o t h before and after irradiation. Ceils grown at 37~ after irradiation show lower survivals t h a n those grown at 25 ~ C. A t either recovery temperature, cells which h a d been cultured before irradiation at 37 ~ C are able to sustain less uv damage prior to inactivation t h a n those cultured at 25 ~ C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25 ~ C; a t low u v dosages, cells show slightly poorer recoveries on enriched medium t h a n on minimal medium whereas at higher dosages, their recoveries on b o t h kinds of media are equivalent. I n contrast, at 37 ~ C, u v treated non-budding cells are much more susceptible to inactivation t h a n budding cells; non-budding cells also express much poorer recovery on enriched medium t h a n on minimM medium a t 37~ whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivities, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37 ~ C t h a n those grown on minimal medium. The particularly poor recovery by irradiated non-budding cells at 37~ is correlated with their unique tendency to undergo a transitory filamentation when initiating growth a t t h a t temperature. Evidence is presented t h a t neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37 ~ C exhibit singular susceptibility to inhibition of recovery b y metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected b y such agents. I t is concluded t h a t recovery from irradiation a n d the instigation of cytokinesis b y non-budding cells of C. albicans result from different metabolic processes which m a y be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery b y cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to t h a t occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.

C u r r e n t u n d e r s t a n d i n g o f t h e d a m a g e s p r o d u c e d i n cells b y u l t r a v i o l e t (uv) r a d i a t i o n a n d o f t h e m e c h a n i s m s cells p o s s e s s f o r r e p a i r i n g damage stem largely from discoveries initially made in microorganisms.

290

D.L. BUSB~S and A. SA~ACgEK:

Studies on bacteria were the first to demonstrate positively that, at biologically effective uv dosages, the dimerization of adjacent pyrimidine bases on the same strand of the desoxyribonucleie acid (DNA) duplex is a cardinal event responsible for uv induced cellular inactivation (CAsTELLA~I et al., 1964), mutation (WITKI~, 1966) and inhibitions of eytokinesis (KANTORand DEEI~ING, 1967) or macromolecular syntheses ( S w ~ s o ~ and S~TLOW, 1966). Similarly, early investigations on bacteria, fungi and protozoa indicated that cells may repair uv damage if exposed to visible light after irradiation (photoreactivation; JAGG~, 1958) or if subjected to treatments which delay onset of their postirradiation division (dark repair ; STAI~LETO?N,1960). Subsequent analyses established that photoreaetivation occurs through a light-activated, enzymatic monomerization of pyrimidine dimers (Ss.TLOW et al., 1965) and that at least one major mechanism for dark repair involves enzymatic excision of damaged regions of DNA (S~TLOW and CAgRI~g, 1964) followed by localized DNA resynthesis to restore the normal base sequences (PETTIJOH~ and HANAWALT,1964). Although microorganisms continue to serve as primary tools in radiobiologieal research, they are used principally to elucidate basic radiobiologieal processes and, understandably, studies tend to focus upon a few experimentally convenient microbial forms. Thus, despite an abundant literature dealing with such selected forms, much remains to be learned of the full range of possible radiation repair processes in different microorganisms, their systematic distribution among procaryotie and eucaryotic species and the specific conditions which affect the abilities of particular microorganisms of medical or industrial significance to cope with germicidal light. The asporogenous yeast, Candida albicans, is an opportunistic pathogen normally resident on mucous membrane surfaces and widely distributed in the human population. In the course of attempts to uncover possible genetic interactions between strains of C. albicans (SA~ACH~K, 1964), we have used uv as a mutagen and have noted that the organism's susceptibility to uv inactivation is profoundly affected by its nutrition and incubation temperature both before and after irradiation. Though a great deal is known of the radiation responses of the innocuous sporogenous yeasts, Saccharomyces and Schizosaccharomyces ( J A ~ s and W~R~E~, 1965), corresponding information on the important representatives of the genus Candida is scant. In a note published in 1961 describing uv induction of pseudohypha formation in C. tropicalis, SzlLVlXu and ROSENKI~ANTZclaimed that no prior radiation studies on asporogenous yeasts had been reported. Though technically an overstatement, in substance the claim is correct. Several observations on uv killing of asporogenous yeasts, including C. albicans, under single arbitrary sets of conditions have been reported (TANNER and RYnER, 1923; LUND,

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1958), but, a p a r t from establishing the obvious l e t h a l i t y of uv, t h e y collectively c o n t r i b u t e little to the u n d e r s t a n d i n g of radiobiological processes i n these organisms. We have u n d e r t a k e n , therefore, a comprehensive s u r v e y of circumstances affecting n v i n a c t i v a t i o n of C. albicans as a general c o n t r i b u t i o n to the limited store of radiobiological i n f o r m a t i o n on asporogenous yeasts a n d for the specific purpose of i d e n t i f y i n g p r i m a r y conditions influencing the s e n s i t i v i t y of this import a u t fungal p a t h o g e n to germicidal light.

Materials and Methods Test Organism The primary test organisms included three prototrophic, wild type strains of C. albisans, strains 207, 526, and 792 (SA~ACg~K,1964), and an uv-induced methionine auxotroph of strain 526, WD-2. Certain confirmatory studies were performed on the leucine auxotroph, WD-4, and the lysine auxotroph, WD-18, also derived from strain 526. The wild type strains were obtained originally from H. F. I-IASENCLOVEn, National Institutes of Health, U.S.A., for the purpose of creating auxotrophic stocks. Strain 207 belongs to the antigen group A and strains 526 and 792 belong to the antigen group B defined by I-IAs~cLsv~n and MITCHELL (196l). Though we have procured a number of auxotrophs from each strain through uv irradiation, the strains differ markedly in mutability and the spectrum of auxotrophs yielded. Stock cultures were maintained at 5~ C upon 1.8~ agar slants containing 1~ neopeptone, 0.1 ~ yeast extract and 20/0 glucose. Media All media were constituted from the following basal solution: glucose, 20 g; KH2P Q, 1.5g; K2tIPO4, 0.1g; MgS04 • 7H~O, 0.5g; CaC1s, 0.15 g; F e S Q • 7tt20, 3rag; ZnS04• 3rag; CuS0~• 0,5rag; ~nS04x4H20, 0.4mg; (NH4)6MoT024x 4H,O, 0.15 rag; Na2B407• 10H20, 0.9 rag; inositol, 10 rag; calcium pantothenate, t.0 rag; pyridoxine hydrochloride, 1.0 rag; para-amino benzoic acid, 1.0 rag; thiamine hydrochloride, 1.0 rag; nicotinic acid, 1.0 mg; biotin, 0.02 mg; distilled water, 1000 ml. For solid media 20 g a g a r (Difeo) was added. Minimal medium was prepared by the addition of 4 g ammonium tartrate and complete media were prepared by adding either (a) 3 g neopeptone (Difco) and l g yeast extract (Difco) or (b) 50 mg each of adenine and uracil and 4 g casamino acids (Difco). Irradiated cells exhibit identical survivals upon both kinds of complete media. However, the neopeptone medium was used when necessary to grow uniform cell populations for irradiation on complete medium since strains 207 and 792 tend to form some pseudohyphae in the presence of casein hydrolysate. The casamino acid containing medium was used for postirradiation survival determination because it is better defined and would be more suited to interpretable modifications in possible future studies. Media were fully constituted, together with special supplements where indicated, prior to autoclaving at 116~ for 10 rain. Glucose and inorganic eonstitutents of the media were reagent grade. All biochemicals used were products of the highest purity available from the Calbiochem Corporation.

292

D . L . BUSBEE and A. SAxACHEK: Preparation

o f Cells f o r I r r a d i a t i o n

P~adiosensitivity of b o t h budding and fission yeasts are known to vary with different stages of the cell division cycle (JAMES and WEANER, 1965). To insure maximal homogeneity in radiosensitivity of C, albican8 populations, a variety of techniques were tested for obtaining uniform suspensions of nonbudding cells. For the wild; type test organisms, the following procedure was found best suited for t h a t purpose. Ceils growing on minimal b r o t h in shake culture at 25 ~ C were harvested b y centrifugation during log phase. A packed cell volume of 0.5 ml was washed twice in eold M/15 KH2PO 4 and resuspended in 10 ml ~ / 1 5 K H 2 P 0 ~. A 0.7 ml volume of suspended cells was evenly distributed on a standard petri dish containing either minimal or complete medium and grown to stationary phase at 25~ or 37 ~ C. Cells were washed from minimal plates with M/15 KH2PO a after 28 hrs incubation at 25 o C or 22 hrs incubation at 37 ~ C. Complete plates were harvested after 18 hrs at 25 ~ C. lV[ieroscopie examinations at 450 • magnification established t h a t suspensions so prepared consisted routinely of from 930/0 to 960/0 single cells with the remainder comprised largely of cells bearing single buds; units composed of three or four cells constituted less t h a n 1~ of each suspension. No satisfactory way was found to obtain equivalent populations at 37 ~ C on complete medium. Plate cultures grown under such conditions were superior to stationary or shake b r o t h cultures b u t still contained only ca 600/0 single cells with appreciable numbers of multi-budded cell aggregates when harvested in stationary phase. Experiments with amino acid auxotrophs required t h a t they be nitrogen depleted as well as unicellular before irradiation. Each auxotroph was grown in mininlM b r o t h containing 1 • 10 -a 5I of its required amino acid. Washed log phase cell suspensions were prepared as indicated above and distributed on plates of nitrogen-free basal medium. After 24 hrs incubation at 25 ~ C the plate populations regularly contained more t h a n 950/0 single cells. The nitrogen depleted cells do not bud when replated on minimal medium b u t express essentially 100~ viability on amino acid supplemented minimal plates.

Irradiation and Photoreaetivation

Procedures

For u v survival determinations, cells were removed from preparatory plates, washed twice in ~/15 KH2PO 4, and resuspended in 51/15 KII.,PO, to a density of ca 1.5 • 106 cells/ml. Fifty ml of suspension was irradiated in an open petri dish at a distance of 28 cm from an 8 w a t t General Electric germieidM lamp having a peak ultraviolet emission at 2537 A. The dose rate at the surface of the dish was 18 ergs/ mm2/see as a measured b y an International Light Inc. ultraviolet dosimeter, model UV 254. The suspension was stirred continuously during exposure b y means of a magnetic stirrer and one ml aliquots were removed after each interval of exposure, appropriately diluted and plated immediately. All procedures involving u v exposed cells were conducted under yellow light. To test for photoreaetivation, duplicate 0.5 ml Miquots from a n irradiated suspension were each added to 4.5 ml of W15 KI-I2P Q in 16 • 125 m m screw capped test tubes. One suspension was placed in a temperature controlled water b a t h constructed of plane, soft glass 3 m m thick and illuminated before plating for 1 h a t 25 ~ C or 37 ~ C with a 400 watt Sylvania Sun Flood lamp (type 400T4Q/CL/F) placed outside the bath, 35 em from the sample. The duplicate suspension was held under equivalent conditions in the dark before plating. Preliminary experiments h a d established t h a t severM arbitrarily selected strains of Saceharomyces a t t a i n maximal photoreaetivation in less t h a n 30 rain under these conditions of illumination.

Ultraviolet Inactivation of Candida albicans

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Survival Determinations For survival determinations on wild type strains, 10 plates were prepared routinely for each experimental treatment, 5 to be incubated at 25~ C and 5 at 37~ C. For cells plated on complete medium, colony counts were made after 3 days at 37~ C or 4 days at 25 ~ C. MinimM plates were scored one day later at each temperature. For certain experiments in which growth inhibitors were incorporated into minimal medium, counts were made after 5 days at 370 C and 7 days at 25~ C. Nitrogen depleted amino acid auxotrophs were plated following irradiation on minima] medium to evaluate recovery of cells held for periods in the absence of cell division. Immediately upon plating, 10 control plates were each overlayed with 10 ml of minimal medium supplemented with 1 • l0 -s ~I of the requisite amino acid; 5 plates were incubated at 25~ C and 5 plates at 37~ C. Other plates were held for 8 hrs or 24 hrs at 25~ C or 37~ C before overlay. For each holding time and temperature 10 plates were prepared and, following overlay, 5 were placed at 25~ C and 5 at 37~ C. Colony counts were made 4 days after overlay at 37~ C and 5 days after overlay at 250 C. M e a s u r e m e n t s of Colony Sizes For some studies, the relative growth rates of cells on different kinds of plating media were compared in terms of the diameters of 24 hrs old colonies. The technique for measurement and the rationale for the procedure have been described previously

(IRELANDand S~kRACtIEK,1968). Results Effects of Pre- a n d P o s t i r r a d i a t i o n N u t r i t i o n a n d Growth T e m p e r a t u r e s u p o n Survival Though t y p i c a l l y located on the b o d y surfaces of m a m m a l s , C. a l b i c a n s is a non-fastidious organism which m a y also occur free-living

i n soft or water. The two kinds of ecological situations provide growing cells with d i s t i n c t l y different conditions of t e m p e r a t u r e a n d n u t r i t i o n a l e n r i c h m e n t . Therefore, to make a proper general assessment of the u v susceptibility of C. albicans, it was considered desirable to compare the responses of cells grown before a n d after i r r a d i a t i o n on m i n i m a l or enriched media at 2 5 ~ or 37 ~ C. S u r v i v a l curves depicted in Fig. 1 illustrate the effects of pre- a n d p o s t i r r a d i a t i o n n u t r i t i o n a n d growth t e m p e r a t u r e s on the susceptibilities of the three wild type test strains to u v i n a c t i v a t i o n ; curves were n o t o b t a i n e d for cells prepared for i r r a d i a t i o n on enriched m e d i u m a t 3 7 ~ since the large n u m b e r of multieellular aggregates i n such p o p u l a t i o n s would preclude their use for comparisons. I t is seen t h a t , u n d e r e q u i v a l e n t conditions of temperat a r e a n d n u t r i t i o n , the three test organisms e x h i b i t v e r y similar responses to uv. Regardless of their p r e i r r a d i a t i o n growth conditions, all strains show m u c h lower survivals when p l a t e d a t 37 ~ C t h a n a t 25 ~ C on either m i n i m a l or enriched media. F o r a n y given population, i r r a d i a t e d ceils plated a t either t e m p e r a t u r e yield sigmoid s u r v i v a l curves. However,

294

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I I i I I 1 T i 20 40 60 80 1000 20 40 60 80 100 20 40 60 80 100 UV dose in seconds of exposure

Fig. I. Curves depicting the effects of pre- and postirradiation nutrition and incubation temperatures on the survival of stationary phase populations of three wild type strains of C. albicans. Preirradiation growth conditions. I: minimal medium, 25~ C; II: minimal medium, 37 ~ C; I I I : enriched medium, 25~ C. Postirradiation growth conditions. ~ minimal medium, 25 ~ C; 9 enriched medium, 25~ C; o minimal medium, 37~ C; 9 enriched medium, 37 ~ C

t h e cu r v e o b t a i n e d a t 3 7 ~ has a r e l a t i v e l y s h o r t e r shoulder an d a steeper slope during t h e e x p o n e n t i a I phase of i n a c t i v a t i o n t h a n t h a t o b t a i n e d a t 25 ~ C. Moreover, all 3 7 ~ curves show a characteristic tailing below t h e I0~ s u r v i v a l level which does n o t a p p e a r in 2 5 ~ curves. This tailing indicates t h a t each of t h e C. a l b i c a n s p o p u l a t i o n s

Ultraviolet Inaotiv~tion of Canclidaalbicans

295

tested contains a minor component which is particularly resistant to the stress t h a t the high recovery temperature imposes on uv damaged cells. The survival curves for cells grown for irradiation at 25 ~ C on minimal medium or on enriched medium exhibit very similar shoulders and exponential rates of inactivation. For either kind of population, cellular recovery is distinctly lower on enriched medium t h a n on minimal medium through the shoulder portions of the curves obtained at 25 ~ C or 37~ and during the exponential inactivation phase of the curve obtained at 37 ~ C; the differential effect of the plating medium disappears

with the transitions into the exponential inactivation phase of the 25 ~ C survival curve and the tailing portion of the 37 ~ C survival curve. The single critical consequence of the difference in preirradiation nutrition is the fact t h a t 37~ survival curves of cells grown for irradiation on enriched medium tail less severely t h a n those of minimal grown cells. Evidently, t h a t fraction of cells which is uniquely resistant to u v inacgvation at 37 ~C is relatively less resistant when produced on enriched medium than when produced on minimal medium. Cells grown for irradiation at 37~ on minimal medium resemble their 25 ~ C grown counterparts in exponential rates of inactivation when plated at 25 ~ C or 37 ~ C and in the tailing characteristics of their 37 ~ C survival curves. They differ in t h a t the cells prepared at 37 ~ C produce survival curves with shorter shoulders and show less disparity h~ postirradiation recoveries on minimal and enriched media than cells prepared at 25 ~ C. I t m a y be noted t h a t cells of strain 207 grown for irradiation at 37 ~ C yield an unique biphasic survival curve when plated at 25 ~ C. Though the curve signifies a heterogeneity in radiosensitivity peculiar to strain 207 populations under these conditions, it also shows the abbreviated shoulder and reduced cellular responsiveness to postirradiation nutritional conditions typical of corresponding curves for the other test organisms. Thus, for all strains, the generalization holds t h a t the temperature at which cells grow prior to irradiation determines the amount of u v damage they can sustain before inactivation as well as their responsiveness to postirradiation nutritional conditions. Relation of Cell Division Stage to Radioresistance Budding cells of Saccharomyces are only slightly more resistant than non-budding cells to u v but are much more resistant than nonbudding cells to ionizing radiations (ELKINDand SUTTOn, 1959). Consequently, uv survival curves for mixed suspensions of budding and nonbudding cells do not usually reflect the heterogeneity of the population whereas X - r a y curves always show a pronounced tailing due to selective 21

Arch. iVIikrobiol., Bd. 64

296

D.L. BVSB~ and A. S~a~Ac~x:

survival of radioresistant budding individuals. Since each of the basically unicellular populations of C. albicans employed for survival curve determinations contained a small percentage of budding cells, experiments were performed to determine whether these would account for the tailing peculiar to 37 ~ C survival curves. Strain 526 was cultured at 25 ~ C on minimal medium and on enriched medium to provide populations containing 5~ budding cells. A washed cell suspension from each kind of culture was divided into two portions. One portion was irradiated immediately and survivals assessed at 25~ and 3 7 ~ on minimal and enriched media. The second portion was used to inoculate a 250 ml flask containing 50 ml of broth in a 250 ml flask to a density of ca 6 • 10 ~ cells/ml. Cells initially cultivated on enriched medium were introduced into enriched broth and those grown on minimal medium into minimal broth. The flasks were incubated at 25~ with shaking until ca 500/0 of the cells possessed single buds, (i.e., l i 0 rain on minimal medium and 90 rain on enriched medium). Cells were then harvested and prepared in the usual way for irradiation. Survival curves presented in l~ig.2 reveal t h a t the preirradiation incubations increase the overall resistance of cell populations to uv. The effect is expressed in extension of the shoulders of the survival curves and decreases in rates of exponential inactivation at both 25 ~ C and 37 ~ C. However, while the tenfold increases in proportion of budding cells have no effect upon the form of the 25 ~ C survival curves, they do elevate the level at which the 37 ~ C curves begin to taft b y ca one decade. Moreover, as was noted for corresponding unbudded suspensions, the characteristic differences in cell survivals on minimal and enriched media tend to disappear as tailing progresses and the budded population taken from enriched medium shows less pronounced tailing t h a n t h a t from minimal medium. These findings collectively establish t h a t the proportion of budding cells present in irradiated populations of C. albicans will account for the radioresistant cell fraction detected when survival is scored at 37 ~ C. They also indicate t h a t a radiosensitive system must exist in non-budding cells for the elaboration of a metabolic or structurM component which is present in budding cells and is critical to survival at 37 ~ C. Damage to the system is repaired poorly at 37 ~ C. Since budding and non-budding cells do not show differential survival when grown at 25~ following irradiation, the damaged step must be either reparable or irrelevant to snrvival at the lower temperature. ELxI~]) and SVTTO~ (195.9) reported very slight differences in the uv susceptibilities of budding and non-budding cells of Saccharomyces when survivals are scored at 30 ~ C. Unpublished work in our laboratory has shown t h a t postirradiation temperatures as high as 37 ~ C decrease survival of Saccharomyces but do not accentuate the differential radio-

Ultraviolet Inactivation of Candida albicans

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l~ig. 2. Survival curves obtained for stationary phase populations of C. albicans, strain 526, containing 5~ budding cells and the same populations after a brief preirradiation growth period which increased the proportions of budding cells to 50o/o. Stationary 2)basepopulations and their corresponding pre-incubated populations grown /or irradiation on: I. minimal medium at 25 ~ C; I I enriched medium at 25 ~ C. Cells plated after irradiation at: A 37 ~ C; B 25 ~ C. Stationary phase populations plated on minimal medium (--o--) or enriched medium (--.--) and pre-ineubated populations plated on minimal medium (--o--) or enriched medium (--.--) sensitivity of cells at different stages of division. Thus, the m a r k e d influence of recovery temperature on the relative susceptibilities to u v of budding and non-budding cells of C. albicans is a distinctive p r o p e r t y of the organism and is not characteristic of all budding yeasts. Relation of Postirradiation I n c u b a t i o n Temperature to Cellular Growth Rate, Growth F o r m and Radiosensitivity I t has been frequently reported for bacteria (STAPLETO~, 1960) and Saccharomyces (JAM~s and Wnl~EI~, 1965) t h a t slow growth after irradiation encourages the recovery of u v treated cells. Restriction of growth p r e s u m a b l y allows time for repair processes to restitute damages which would otherwise contribute to aberrant and lethal cell replication. 21"

298

D.L. BUSBEE and A. SARAC~EK:

Since C. albicans grows m u c h b e t t e r a t 37 ~ C t h a n 25 ~ C, t h e g r e a t e r inactivation obtained at 37~ could be due either t o m o r e r a p i d p o s t i r r a d i a t i o n g r o w t h a t t h e higher t e m p e r a t u r e or to t h e t e m p e r a t u r e s e n s i t i v i t y of a n essential r e p a i r s y s t e m . To d i s t i n g u i s h b e t w e e n these a l t e r n a t i v e s , c o m p a r i s o n was m a d e of t h e s u r v i v a l s o f u v t r e a t e d cells a t t e m p e r a t u r e s a b o v e as well as below t h e i r o p t i m u m for growth.

Table 1. Influence o/ postirradiation incubation temperatures upon survival o/ (C. albicans) strain 526, plated on minimal or enriched media. The treated cell population was grown ]or irradiation at 25 ~ C on minimal medium and contained 5~ budding cells

uv Dose (in sec)

15 30 40 50 60

Per cent survival 25 ~ C 37~ C

40 ~ C

42 ~ C

minireal

enriched

minireal

enriched

minireal

enriched

minireal

enriched

100 96 80 58 26

100 93 73 54 25

100 82 33 3.7 1.4

93 65 16 2.9 1.1

79 46 8.3 1.4 0.87

60 17 3.9 1.2 0.84

45 3.0 1.7 0.94 0.74

12 2.2 1.4 0.96 0.73

T a b l e 1 s u m m a r i z e s s u r v i v a l values for s t r a i n 526 g r o w n for i r r a d i a t i o n on m i n i m a l p l a t e s a t 25 ~ C a n d p l a t e d a f t e r u v e x p o s u r e on m i n i m a l or enriched m e d i a a t 25 ~ C, 37 ~ C, 40 ~ C, or 42 ~ C; t h e sizes of 24 hrs old colonies p r o d u c e d b y u n i r r a d i a t e d cells are p r e s e n t e d in T a b l e 2 as indices o f r e l a t i v e g r o w t h r a t e s u n d e r these conditions. I t is seen t h a t cells grow b e t t e r a t 37 ~ C t h a n a t 25 ~ C or 40 ~ C a n d t h a t t h e p o o r e s t r a t e o f g r o w t h occurs a t 42 ~ C. Y e t for each u v dose, increasing posti r r a d i a t i o n i n c u b a t i o n t e m p e r a t u r e s cause progressive declines in overall cell s u r v i v a l s a n d e x a g g e r a t i o n o f t h e t e n d e n c y for higher s u r v i v a l s to occur on m i n i m a l m e d i u m t h a n on enriched m e d i u m . Clearly t h e depression o f s u r v i v a l b y high t e m p e r a t u r e c a n n o t be e x p l a i n e d as a d e r i v a t i v e effect of a n e n h a n c e d p o s t i r r a d i a t i o n r a t e of growth. I n s t e a d , i t a p p e a r s t h a t high t e m p e r a t u r e s d i r e c t l y n e g a t e m e t a b o l i c e v e n t s i n v o l v e d in a m e l i o r a t i n g u v d a m a g e a n d t h a t t h e effect is a c c e n t u a t e d d u r i n g cellular g r o w t h in a n enriched n u t r i t i o n a l e n v i r o n m e n t . I t is n o t e w o r t h y t h a t t h e abilities of high i n c u b a t i o n t e m p e r a t u r e s t o enhance a b s o l u t e u v l e t h a l i t y a n d to p r o m o t e differential cellular r e c o v e r y on m i n i m a l a n d enriched m e d i a diminish as s u r v i v a l s fall below ca 5~ Since t h e p r e p o n d e r a n t p o r t i o n of t h e v i a b l e p o p u l a t i o n below t h a t level consists of b u d d i n g cells, t h i s o b s e r v a t i o n is f u r t h e r evidence t h a t high g r o w t h t e m p e r a t u r e p a r t i c u l a r l y b u r d e n s t h e r e c o v e r y of n o n - b u d d i n g cells.

Ultraviolet Inactivation of Candida albicans

299

An important clue to the nature of the temperature induced stress in non-budding cells was uncovered through consideration of the relationship of cell divison stage to recovery on enriched and minimal media at 25 ~ C and 37 ~ C. I t has been reported for a variety of microorganisms t h a t enriched medium is less effective than minimal medium in supporting recovery from uv irradiation (ADLEr, 1966; JAMES and WE~h'E~, 1965). AL~I~ (1961) has interpreted this to mean t h a t highly nutritive conditions encourage radiation damaged cells to undergo a detrimental, unbalanced growth. H e r concept was reinforced and extended b y ADLE~ and I-IARDIGI~EE'S (1965) comparative studies on bacterial strains which do or which do not show such plating medium responses. They observed t h a t following low dose hTadiation, bacteria of either sort grow in their usual cellular form on minimal medium. However, only those strains which are adversely affected b y good nutrition grow as filaments on enriched medium. The filamentous growth is associated with normal nuclear replication but an absence of septation, and invariably culminates in death unless septation eventually occurs spontaneously or through special induction b y pantoyl lactone. Thus, the lethal unbalanced growth of bacteria is expressed as a specific inhibition of eytokinesis. C. albicans is naturally dimorphic and can grow alternatively in yeast or filamentous forms depending upon its eulturM circumstances. Yeast phase cells exhibit rates of cell division regularly attuned to protoplasmic growth while filamentous cells show normal protoplasmic increases accompanied by only occasional cell division. Prompted b y the findings with bacteria, we observed microseopicMly (200X) the growth form of C. albicans cells after plating on minimal or enriched media at 25 ~ C or 37 ~ C; cells of all three prototrophic strains, whether prepared at 25 ~ C or 37 ~ C, on minimal or enriched media, were found to exhibit the same pattern of responses. Irradiated or unirradiated non-budding cells grow only as typical yeast cells when plated on minimal or enriched media at 25 ~ C. Similarly, cells which are in the process of budding when plated replicate only in the yeast form on either medium at 37 ~ C as well as 25 ~ C. On the other hand, non-budding cells plated at 37~ on either minimal or enriched media invariably generate filaments ca 30 ~ to 66 ~ in length (i.e., 5 to l0 times the length of an average blastosporic cell) before reverting permanently to the typical yeast-like pattern of blastosporic growth and reproduction. Except for an absence of branching, these filaments appear identical in form to the transitory "pseudo germ tubes" described b y MAC~E~-ZIE (1964) which are produced by blastosporie cells of C. albican8 upon subcutaneous inoculation into mice. For unirradiated cells or cells exposed to less t h a n a 20 sec dose of uv, budding resumes 3 hrs after plating on enriched medium or 4 hrs after plating on minimal medium.

300

D.L. BUSB~E and A. SXaACHEX:

At higher dosages filaments grow at highly variable rates but budding ensues when they attain the same lengths as those of unirradiated cells. Buds generally arise terminally and laterally on the filaments at about the same time indicating that nuclear divisions occur in the absence of cytokinesis during filamentation. In some instances, uv exposed cells are inactivated before filamentation or in the filament state. However, inactivation is also observed in clones which have accomplished several buddings after filamentation. Thus the resumption of budding per se is not the recovery event. Evidently, non-budding cells are able to commence a normal budding process immediately when grown at 25 ~ C whereas incubation at 37 ~ C selectively inhibits one or more metabolic activities concerned in cytokinesis. Since the filamentation is temporary and cells revert permanently to a budding yeast form during growth at 37 ~ C, it is probably de novo formation of a system necessary to division that is inhibited by high temperature rather than the rate at which the system operates. This interpretation would predict that cells in the process of budding must already be equipped with this system, and is consistent with the fact that budding cells do not undergo filamentation when placed at 37 ~ C. The filamentation observed in C. albicans is clearly unlike that reported by ADLv~Rand HAnDIr (1965) in bacteria since (a) it is neither induced nor qualitatively modified by irradiation, (b) is not conditioned by the composition of the plating medium and (e) the eventual onset of cell division does not assure clonal viability. However, the fact that it occurs exclusively among non-budding cells made to initiate growth at 37~ strongly suggests that it may bear a fundamental relationship to the particularly poor radiation recovery characteristic of such cells as well as the pronounced suppression of their recovery by growth on enriched medium. Effects of Metabolic Antagonists on Survival of Irradiated Cells As an approach to determining whether protein or nucleic acid syntheses play important roles in the temperature dependent recovery of irradiated cells, survivals of uv treated cells were assessed after growth at 25 ~ C and at 37 ~ C on minimal medium containing inhibitory levels of various amino acid, purine or pyrimidine analogues. Caffeine and the acridine dye mixture, aeriflavin, were included among the agents tested because of their known abilities to inhibit the dark repair of DNA in bacteria (ALrn•, 1963). In order to relate all agents to a common standard of cytotoxicity, each was incorporated into the medium at a concentration which does not affect the viability of unirradiated cells but which reduces the average size of 24 hrs old colonies produced by such cells at 37 ~ C to ca 30~ of the size attained on minimal medium alone

301

Ultraviolet Inactivation of Candida albicans (Table2). At these concentrations, colonic similarly produced at 25 ~ C in the presence o antagonists ranged from ca 25~ to 400/0 o the size attained on minimal medium alone. Table 3 records the activities of antagonists screened against typical unicellular populations

"~

(:)

-H 5xl

<5

of strain 526 grown for irradiation on minimal

medium at 25 ~ C or at 37 ~ C and exposed to 40 or 80 see doses of uv. Despite the fact that these two kinds of cell populations differ in the extent to which they respond to nutritional enrichment after irradiation (Fig. 1), their postirradiation responses to the analogues are very similar. The purine analogues 8-azaadenine and 8-azaguanine and the pyrimidine analogues 6-azauracil and 2-thioeyCosine do not affect survival of cells plated at 25 ~ C or 37 ~ C. On the other hand, the amino acid analogues p-fluorophenylalanine and ethioninc profoundly potentiate uv inactivation of cells at 37 ~ C but show only slight effects at 25 ~ C; acriflavin and caffeine exhibit corresponding though less pronounced activities. Thus the active cornpounds interfere with metabolic functions which are particularly essential for cellular recovery at 37 ~ C. Significantly, at 37 ~ C, each compound elicits a proportionately much greater response in cells irradiated for 40 sec than in cells given 80 sec irradiation. In contrast, their slighter effects at 25~ are generally proportional at both dosage levels. Nonbudding cells have been shown to be much more sensitive than budding cells to uv inactivation at 37 ~ C but not at 25 ~ C. The

S O <5

"~

<5 ~ "~ ~ ~ ~ "~ ~ '~ ~ ~ ~ ~ ~ ~ .~ ~ c5 ~ ~ cd o ~ ~ .+ ~ ~ ~>

o. O

<5 o.

<5 r O O

-H

o. -H O

two

~q

representative unicellular cell suspensions used to test the effects of the antagonists contained 40/0 and 60/0 budding cells (Table 3). Reference to the 37~ survival curves for analogous suspensions given in Fig. 1 shows that the major fraction of each population surviving the 40 see dose would consist of non-budding cells

whereas the survivors of the 80 see dose would consist almost exclusively of budding cells.

v

<5

~q

<5

~q

~+

z O

<5

302

D.L. BvS~EE and A. S~mACa~K:

Thus, the m u c h greater effectiveness of the a n t a g o n i s t s i n reducing s u r v i v a l a t the lower dosage a t 37 ~ C, b u t n o t a t 25 ~ C, indicates t h a t t h e y d i s t u r b t h a t recovery process which is singularly critical for nonb u d d i n g cells at 37 ~ C. Table 3. Per cent survivals obtained ]or populations o] C. albicans, strain 526, grown prior to irradiation on minimal medium at 25 ~ C or 37 ~ C and plated a]ter uv ex19osure at 25 ~ C or 37~ on minimal medium contained metabolic antagonists. The population prepared /or irradiation at 25~ contained 6~ budding cells and the population prepared at 37 ~ C contained 4~ budding cells uv dose Antagonist (seconds of exposure)

40

80

Ceils grown for Cells grown for irradiation at 25~ C irradiation at 37~ C Postirradiation Postirradiation incubation 25~ C

temperature 37~ C

incubation 25~ C

temperature 37~ C

none aerittavin (50 tzg/ml) 8-azaadenine (8 X 10-~ ~)

84 73 86

34 9.5 32

45 32 48

7.1 3.8 6.9

8-azaguanine (8 X 10 -6 z~)

81

30

44

7.3

6-azauracil (5 • 104 ~) caffeine (2.5 • 10-a M) ethionine (1 • 10-~ ~) p-fluorophenylalanine (1 x 10-2 M) 2-thioeytosine (2 X 10-~ M)

85 77 79 70

33 13 3.4 1.7

43 39 37 31

7.1 4.1 1.9 1.2

81

35

43

6.2

none acriflavin (50 ~g/ml) 8-azaadenine (8 X 10-~ ~) 8-azaguanine (8 X 10-5 ~) 6-azauraeil (5 X 10-~ ~) caffeine (2.5 • 10-3 ~) ethionine (1 X 10-2 M) p-fluorophenylalanine (1 x 10-2 M) 2-thioeytosine (2 x 10-4 ~)

6.1 4.9 5.8 6.0 5.8 5.3 5.2 4.7

0.76 0.34 0.77 0.71 0.80 0.42 0.32 0.23

3.3 2.2 3.1 3.2 3.1 2.7 2.9 2.4

0.47 0.31 0.50 0.46 0.46 0.42 0.34 0.27

6.3

0.77

3.0

0.45

Yeast cells readily incorporate ethionine (MAw, 1966) or p-fluorophenylalanine (COHEN et al., 1958) to produce abnormal proteins. In bacteria, caffeine can block the dark repair of D N A b y i n h i b i t i n g the repair enzymes (SID~OPOULOS a n d SKA~;~L, 1968) whereas acriflavin blocks repair b y r e n d e r i n g D N A lesions inaccessible to the enzymes (Lv,~MA~, 1961). However, b o t h of these agents exert a b r o a d cytotoxicity which m a y also be expressed i n general d i s t u r b a n c e s i n p r o t e i n or nucleic acid syntheses ( D o v n ~ u et al., 1964; LOVELess et al., 1954). To

Ultraviolet Inactivation of Candida albicans

303

d e t e r m i n e w h e t h e r caffeine or aeriflavin affect a different k i n d of cellular r e p a i r s y s t e m t h a n ethionine or p - f l u o r o p h e n y l a l a n i n e , t h e four a g e n t s were t e s t e d singly a n d in all possible p a h ' e d c o m b i n a t i o n s for effects u p o n i r r a d i a t e d cells a t 25 ~ C a n d 37 ~ C. R e s u l t s p r e s e n t e d in T a b l e 4 Table 4. Survival o] irradiated cells o/C. albicans, strain 526, exposed to a 40 sec dose o/ uv and plated at 25 ~ C or 37 ~ C on minimal medium containing metabolic antagonists, singly or in paired combinations. The treated population was grown/or irradiation at 25 ~ C on minimal medium and contained 8~ budding cells Antagonist (concentration)

:Per cent survival 25 ~ C

37 ~ C

none acriflavin (50 ~xg/ml) caffeine (2.5 • 10-~ ~) ethionine (1 • 10 2~) lo-fluorophenylalanine (1 • 10-2 ~)

80 69 74 80 70

28 9.2 15 3.5 1.2

acriflavin (100 ~xg/ral) caffeine (5 • 10-~ ~) ethionine (2 • 10-2 ~) p-fluorophenylalanine (2 • 10-2 ~)

36 56 79 68

4.4 11 3.0 0.82

acriflavin (50 fzg/ml) ~- caffeine (2.5 • 10-3 ~) acriflavin (50 fzg/ml) ~- ethionine (1 • 10-2 ~) acriflavin (50 ~g/ml) -p p-fluorophenylalanine (1 • 10-2 ~) caffeine (2.5 • l0 -3 ~I) + ethionine (1 • 10-2 M) caffeine (2.5 • 10-3 ~) ~- p-fluorophenylManine (1 • 10-2 ~) ethionine (1 • l0 -2 ~) ~- p-fluorophenylalanine (1 • 10-2 ~)

67 70 65 77 70 69

8.6 3.9 0.98 3.8 1.0 1.4

show t h a t , a t b o t h t e m p e r a t u r e s , e n h a n c e m e n t of u v i n d u c e d l e t h a l i t y b y each c o m b i n a t i o n is r o u g h l y e q u i v a l e n t to t h a t caused b y t h e m o r e a c t i v e i n d i v i d u a l a g e n t present. I f a n y t w o of t h e a g e n t s d i s t u r b e d s e p a r a t e r e c o v e r y processes, t h e i r effects when c o m b i n e d w o u l d be e x p e c t e d to be either a d d i t i v e or s y n e r g i s t i c ; t h e absence of i n t e r a c t i o n i n d i c a t e s t h a t all four a g e n t s interfere w i t h t h e s a m e r e c o v e r y process, t h o u g h p r o b a b l y b y different means. F r o m t h e p r e - e m i n e n t a c t i v i t i e s of t h e a m i n o a c i d a n t a g o n i s t s , i t can be concluded t h a t f o r m a t i o n o f f u n c t i o n a l p r o t e i n s is essential to cellular r e c o v e r y a n d t h a t t h e a c t i v i t i e s of caffeine a n d acriflavin reflect general d i s r u p t i o n o f such s y n t h e s i s r a t h e r t h a n specific i n h i b i t i o n s of D N A repair. The f a c t t h a t these a g e n t s are as i n h i b i t o r y to t h e g r o w t h of u n i r r a d i a t e d cells a t 25 ~ C as a t 37 ~ C i n d i c a t e s t h a t t h e y i n d u c e similar cellular d i s t u r b a n c e s a t b o t h t e m p e r a tures. Theb: abilities to enhance i n a c t i v a t i o n of i r r a d i a t e d n o n - b u d d i n g cells a t 37 ~ C, specificially, implies t h a t a n o m a l o u s p r o t e i n s y n t h e s i s a d d s

304

D.L. BUSB]~E and A. S~Ac~Ex:

additional stress to a recovery system which is already debilitated in such cells by the initiation of growth at 37 ~ C. I t was noted above that growth at 37 ~ C specifically retards the de novo formation in non-budding cells of a metabolic system required for normal cytokinesis. Direct microscopic examinations established, however, that the metabolic antagonists which augment uv lethality for non-budding cells at 37 ~ C also retard their growth but do not qualitatively modify the usual course of their filamentation and eventual resumption of budding. This differential effect of the metabolic antagonists upon systems involved in achieving cytokinesis and recovery from irradiation establishes that the two systems are not identical but does not exclude that possibility that they are related through a crucial temperature sensitive step. Recovery by Non-dividing Cells Bacteria and sporogenous yeasts will undergo extensive recovery from lethal uv damage if held irradiation under conditions which suspend cell division (CAsT~LLACCIet al., 1964; PATriCK et al., 1964). Customarily, cells are held simply in buffer solution and repair is accomplished through endogenous resources. Bacterial recovery is known to result from the direct restitution of damaged segments of DNA (S~TLow and C ~ I E ~ , 1964; PETTIJO~I~ and ttA~AWAL% 1964). Though the exact repair mechanism in the yeasts is still uncertain, there are substantial indications that it may differ from that in bacteria (see Discussion). We have shown that recovery from uv irradiation in actively growing cells of C. albicans is temperature sensitive and requires synthesis of functional protein. I t was of interest to determine whether repair can also occur under conditions precluding postirradiation growth and replication and, if so, whether the requirements for recovery differ from those pertaining to actively dividing cells. The prototrophic test strains were not suited to such studies since their cells contained sufficient endogenous reserves to support at least one postirradiation division in phosphate buffer. The cells could not be used for irradiation after depletion of their reserves in buffer or nitrogen-free basal medium because of the presence of buds and their development of a stickiness which causes large-scale cellular aggregation. However, an adequate procedure for suspending postirradiation division was designed employing amino acid auxotrophs. When depleted of nitrogen reserves by 24 hrs incubation at 25 ~ C on plates of nitrogen-free basal medium, the auxotrophs yield unicellular populations with typically fewer than 5~ budding cells. The cells are essentially 1000/0 viable; they undergo some inactivation if maintained on the nitrogen-free basal medium for an additional 24 hrs but retain complete viability without any overt sign of growth for

Ultraviolet Inactivation of Candida albicans

305

a t least 48 hrs a t 25 ~ C or 37 ~ C on m i n i m a l m e d i u m . F o r q u a n t i t a t i v e studies following i r r a d i a t i o n , t h e y c a n n o t be held for e x t e n d e d periods in buffer or m i n i m a l b r o t h since t h e y b e c o m e s t i c k y w i t h i n a few hours a n d t e n d to a g g r e g a t e a n d adhere to t h e surface of t h e glass container. To c i r c u m v e n t this problem, dilutions of i r r a d i a t e d cells were s t r e a k e d u p o n m i n i m a l plates a n d held in a n o n - d i v i d i n g s t a t e for specified periods before being o v e r l a y e d w i t h a m i n o acid s u p p l e m e n t e d m i n i m a l m e d i u m to p e r m i t t h e i r o u t g r o w t h as colonies.

Table 5. Recovery by uv treated cells o/ the methionine auxotroph o/C. albicans, WD-2, when held in a non-dividing state at 25 ~ C or 37 ~ C be/ore initiating division at 25 ~ C or 37 ~ C. The treated WD-2 population was depleted o] nitrogen, reserves at 25 ~ C prior to irradiation and contained 97~ non-budding cells. ~ollowing exposure, cells were plated on m i n i m a l medium and overlayed immediately or after 8 or 24 hrs holding at 25 ~ C or 37 ~ C with m i n i m a l medium containing 1 • 10 -3 M methionine to permit outgrowth o/ colonies. JFor each holding condition 10 plates were prepared; after overlay, 5 plates were incubated at 25 ~ C and 5 plates at 37 ~ C

uv Dose (in sec)

Post-irradiation treatment

Per cent survival

Holding Growth temperature temperature ~ ~

0 hrs ho]ding

40

-

-

25 37 -

-

25 37 60

-

2

-

5

37 -

-

25 37 80

- -

2

5

37 -

-

2

5

37

37 37 37 25 25 25

29 --83 ---

37 37 37 25 25 25

1.2 --20 ---

37 37 37 25 25 25

0.13 --2.0 ---

8 hrs holding

-

-

-

43 31 -

-

- -

-

-

14 1.9 - -

21 14 -

-

84 52 -

3.3 1.2

-

69 34 -

80 62

-

24 hrs holding

-

21 3.6 - -

0.46 0.15 -

-

0.99 0.13 -

2.3 1.5

-

2.3 0.34

Tab l e 5 c o m p a r e s t h e r e c o v e r y of d e p l e t e d cells of t h e m e t h i o n i n e a u x o t r o p h W D - 2 , i r r a d i a t e d a t t h r e e u v dosages a n d held in a nond i v i d i n g s t a t e a t 25 ~ C or 37~ prior to g r o w t h a t either t e m p e r a t u r e .

306

D.L. BuSJ~Ev~and A. SARACgEK:

Effects of holding are evident after 8 hrs but are most pronounced after 24 hrs; continued holding up to 48 hrs is without additional effect. I t is seen t h a t cells held at 25 ~ C before being allowed to grow at 37 ~ C show much greater recovery than cells grown at 37 ~ C immediately following irradiation; holding at 37~ only slightly increases survival during subsequent growth at 37 ~ C. Thus, the repair system which functions well during postirradiation growth at 25~ but poorly at 37~ also functions in non-dividing cells. Interestingly, this repair in non-dividing cells is associated with a change in the cellular potential for cytokinesis during subsequent growth at 37 ~C. I f the non-budding WD-2 cells which had been held for 24 hrs at 37 ~ C are washed from holding plates and streaked upon methionine containing medium at 37 ~ C, they exhibit the typical pattern of transitory filamentous growth. I n contrast, cells held at 25~ for 8 or 24 hrs do not filament when allowed to begin growth at 37 ~C. Thus, the holding at 25 ~ C not only encourages non-budding cells to recover from irradiation but allows them to undergo a metabolic adjustment which restores their capacities for normal cell division when initiating growth at 37 ~ C. As might be expected, cells held at 25~ before growth at 25~ show about the same survival as those grown immediately after irradiation at t h a t temperature. However, cells held at 37 ~ C before growth a t 25 ~ C exhibit slightly greater recovery than those grown at 37 ~ C without delay but significantly lower recovery than cells grown at 25 ~ C immediately after exposure. This would indicate t h a t during holding at 37 ~ C, most of the uv damage initially sustained b y a cell becomes fixed into a form which is no longer reparable b y the recovery system which operates in cells undergoing immediate postirradiation growth at 25 ~ C. The plate-holding recovery observed in WD-2 is not a peculiar feature of methionine auxotrophy since equivalent effects have been obtained with the leueine auxotroph, WD-4, and lysine auxotroph, WD-18. I n spite of the fact t h a t methionine depleted WD-2 cells lack the protein synthesizing capacity to initiate budding on minimal medium, their recovery from uv damage at 25 ~ C and their fixation of damage into an irreparable form at 37 ~ C indicates t h a t they m a y accomplish sufficient protein formation to modify their radiation responses. To test this possibility, p-fluorophenylalanine, ethionine, acriflavin and caffeine were assayed for effects on recovery of WD-2 during plate holding. Irradiated cells were plated on minimal medium with or without one of the agents; plates were overlayed immediately or after 24 hrs holding with corresponding medium supplemented with methionine. Cells were held at 25 ~ C or 37 ~ C and, for each holding temperature, survivals were assessed after outgrowth at 25 ~ C or 37 ~ C.

Ultraviolet Inactivation of Candida albicans

307

Table 6. E]]ects o~ metabolic antagonists on recovery o] uv treated cells o/the methionine auxotroph o/C. albicans, WD-2, during 24 hrs holding in a non-dividing state at 25 ~ C or 37 ~ C prior to initiating division at 25 ~ C or 37 ~ C. The treated WD-2 population was depleted o/ nitrogen reserves at 25 ~ C prior to irradiation and contained 95o/0 non-budding cells. Cells were irradiated /or 40 sec and plated on minimal medium containing one o/several antagonists. Plates were overlayed immediately or after 24 hrs holding at 25 ~ C or 37 ~ C with corresponding medium suplemented with 1 • -~ M methionine. P o t each holding condition 10 plates were prepared; after overlay, 5 plates were incubated at 25 ~ C and 5 plates at 37 ~ C Antagonist (concentration)

Postirradiation t r e a t m e n t

Per cen~ survival

Holding time in hrs

Holding temperature o C

Growth temperature o C

none

0 24 24 0 24 24

-25 37 -25 37

25 25 25 37 37 37

86 84 49 34 71 36

aeriflavin (50 ~g/ml)

0 24 24 0 24 24

-25 37 -25 37

25 25 25 37 37 37

70 73 14 5.2 42 4.9

caffeine (2.5 • 10-3 N)

0 24 24 0 24 24

-25 37 -25 37

25 25 25 37 37 37

79 77 26 8.2 52 8.5

ethionine (1 • 10 -2 ~)

0 24 24 0 24 24

-25 37 -25 37

25 25 25 37 37 37

51 44 1.1 0.32 22 0.19

p-fluorophenylalanine (1 • 10 -2 ~)

0 24 24 0 24 24

-25 37 -25 37

25 25 25 37 37 37

64 64 5.7 0.83 28 0.71

Data presented in Table 6 shows that each agent slightly reduces t h e e x t e n t o f p l a t e - h o l d i n g r e c o v e r y a t 25 ~ C b u t s h a r p l y p o t e n t i a t e s c e l l u l a r i n a c t i v a t i o n d u r i n g h o l d i n g a t 37 ~ C. F u r t h e r m o r e , w h i l e

308

D.L. BUSBEE and A. SAR~C~EK:

p-fluorophenylalanine was superior to ethionine in promoting inactivation of u v exposed prototrophie cells (Table 4), ethionine is the more active for WD-2 cells. Since both antagonists probably act through incorporation into protein, the enhanced effectiveness of the methionine analogue, ethionine, in cells specifically deficient for methionine is not surprising. These observations indicate t h a t protein formation capable of affecting recovery from u v t r e a t m e n t does occur in C. albicans cells under conditions of amino acid starvation which preclude budding. The source of new protein in such cells is not obvious. The auxotrophs we have tested for plate-holding recovery all have absolute genetic blocks as adjudged by their inabilities to grow on minimal medium. However, it is conceivable t h a t each m a y have a minute leakiness inapparent by the criterion of growth and division or t h a t their depletion in preparation for irradiation leaves sufficient labile protein to provide amino acids for traces of de novo protein syntheses. I n any event, the fact that non-dividing cells attain the same high level of recovery at 25 ~ C as actively growing cells establishes t h a t cytokinesis is not an essential element in the recovery process. Moreover, the observation t h a t cells can fix lethal damage at 37 ~ C without undergoing filamentation shows t h a t filamentous growth is not the direct cause of poor radiation repair at 37 ~ C and favors the view t h a t the two events m a y be derivative effects of a common metabolic fault occurring at t h a t temperature. Absence of Photoreactivation Non-budding cells of the three wild type test organisms grown prior to uv exposure at 25 ~ C on minimal medium do not photoreactivate when exposed to visible light for a period twice as long as is necessary to achieve maximal photoreactivation of Saccharomyces. I n view of findings by ]~OLINGand SETLOW (1967) t h a t the level of photoreactivating enzyme in Saccharomyces cells varies with their growth stage, additional observations were made on logarithmic phase and stationary phase cells of strain 526 grown at 25 ~ C or 37 ~ C on minimal or enriched media and held at 25 ~ C or 37 ~ C during postirradiation illumination. No indication of photoreactivation or light induced inactivation was obtained under any set of conditions.

Discussion Properties of the cellular structures which sustain uv damage and the metabolic systems responsible for repairing damage are genetically determined (ADLEr, 1966). Considering the great genetic variability naturally encountered among microorganisms, valid generalizations concerning the radiobiological characteristics of a particular species

Ultraviolet inactivation of Candida albicans

309

cannot be drawn from the behavior of any arbitrarily chosen strain. Accordingly, our investigations of C. albicans were conducted upon three prototrophic strains of independent clinical origins and differing in antigenic composition and mutability. The responses of these diverse strains to conditions affecting uv inactivation are strikingly similar and, therefore, may be considered representative for C. albicans as a species. C. albicans exhibits higher survivals ff grown after irradiation at 25~ than at 37 ~C; though the radiosensitivities of non-budding and budding cells are alike at 25 ~ C, non-budding cells are much more radiosensitive than budding cells at 37 ~ C. The postirradiation responses are not due to the relative growth rates of cells at 25~ and 37~ but involve the direct depression of a recovery process by the higher temperature. Comparisons of the effects of metabolic antagonists on uv treated cells plated at 25~ and 37~ indicate that the high temperature interferes with the ability of non-budding cells to form one or more proteins essential for recovery and that the presence of the preformed protein in budding cells accounts for their relative resistance to inactivation at 37 ~ C. The characteristically poor recovery exhibited by irradiated nonbudding cells at 37~ is correlated with an anomaly in their division mechanism also expressed at that temperature. Whether irradiated or not, non-budding cells plated at 25 ~ C and budding cells plated at 25 ~ C or 37 ~ C enter immediately into typical blastosporie division. In contrast, non-budding cells plated at 37~ initially undergo a unique, transitory filamentous growth before reverting permanently to a blastosporic growth form. The eventual resumption and maintenance of blastosporic reproduction at 37 ~C indicates that the high growth temperature impedes de novo formation in the non-budding cell of a metabolic system required for cytokinesis but does not disturb operation of the system once it is formed. The fact that growth at 37 ~ C also stresses the formation by nonbudding cells of protein required for repair of radiation damage but does not affect the repair system already present in budding cells suggests that the potentials for achieving cytokinesis and recovery from irradiation m a y be different manifestations of the same metabolic process. This possibility is supported by our observation that uv treated non-budding cells held for 24 hrs at 25 ~C in a non-dividing state accomplish physiological readjustments which not only increase their viability but also eliminate their proclivities for filamentation when growth is allowed to proceed at 37 ~ C. However, we have also noted that antagonists of protein syntheses which sharply diminish recovery of irradiated nonbudding cells at 37 ~ C do not influence the course of filamentation and resumption of budding by such cells. Though this signifies that the

310

D.L. BVSBVEand A. S~mAcm~x:

overall processes involved in repair of radiation damage and the achievement of cytokinesis must have separate components, the correlation between poor recovery from irradiation and defective cytokinesis at 37 ~ C indicates that the two processes may share a common temperature sensitive step. Filamentation can be induced easily in C. albicans through a variety of nutritional or physical manipulations (ScH~Ra and W~Avv,~, 1953) and further assessment of the putative association of cellular growth form with radiosensitivity is planned. C. albicans does not photoreactivate and we have obtained no substantial indication of dark repair of DNA in the organism. Removal of uv induced photochemical products from DNA and the subsequent special DNA repair-replication can be demonstrated in bacteria by direct physico-ehemical analyses (Sv,TLow and CAI~RI~a, 1964; PETTIJOH~ and HA~CAW~T, 1964). Such analyses are not feasible for yeast because of the very small quantity of DNA per cell and the fact that the DNA cannot be labeled specifically. However, it is known that nuclear repair in bacteria is reflected in a marked recovery of uv treated cells when held under non-nutritive conditions which do not permit cell division, (CAsTELLA~r et al., 1964). Similar recovery by non-dividing yeast cell s therefore, could be taken as presumptive evidence of DNA repair. Amino acid auxotrophs of U. albicans which are prevented from dividing by starvation for their required nutrilites undergo much better recovery from uv damage if held at 25 ~ C than at 37 ~ C. However, the extent of the recovery during holding at 25 ~ C approaches but does not exceed that attained when cells are allowed to divide at 25 ~ C immediately following irradiation. I f the high survival associated with incubation at 25~ were due to DNA repair, these findings would be unusual in indicating that maximal DNA repair can occur while cells are actively engaged in their postirradiation division. We have presented evidence that recovery during holding at 25 ~ C is due to a residual capacity for protein synthesis in the amino acid depleted cells and it would seem more probable that recovery represents correction of physiological disturbances rather than DNA damage. The most comprehensive studies of dark repair in yeasts have been conducted by PATI~ICK and associates on Saccharomyces. PATricK et al. (1964) reported that diploid or polyploid cells harvested after 2 to 3 days growth on malt agar show much recovery from uv or X-irradiations if suspended with aeration for 1 or more days in distilled water or phosphate buffer (liquid holding); haploid cells exhibit little or no recovery. They assume that the recovery process in yeast is analogous to the DNA dark repair which irradiated bacteria accomplish during liqnid holding. Since C. albicans is asporogenous, it would be tempting to attribute its seeming lack of a DNA repair system to the fact that it is haploid. However,

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only single haploid, diploid and polyploid strains were examined by PATRICK et al. (1964) and the generality of the absence of repair in haploids is moot. Furthermore, though they attributed the recovery observed in Saccharomyces to moderation of DNA damage, their data are readily open to other interpretation. For bacteria, photoreaetivation eliminates all of the uv damage which can be restituted by liquid holding (CAsTELLA~-I et al., 1964). In the yeast system, however, liquid holding was found to promote recovery to about the same extent in photoreaetivated and non-photoreactivated cells. Since photoreaetivation reflects the monomerization of pyrimidine dimers in DNA, the magnitude of dark recovery in yeast is dearly not related to the amount of DNA damage initially present. We have found consistently that logarithmic or stationary phase cells of Saccharomyces from minimal or enriched media have enough endogenous carbon and nitrogen reserves to undergo at least one full cell division when aerated in distilled water or buffer. Despite the fact that PATnlCK et al. (1964) did not detect budding in recovery suspensions microscopically, their notation that unirradiated control suspensions acquire within 6 hrs a cell fraction which can cause tailing of X-ray survival curves signifies that some budding does occur in such suspensions. This, together with the observation by PATt~ICI~and HAYNES (1964) that repair in yeasts during liquid holding is generally favored by physical and metabolic conditions which are optimal for growth in a nutrient environment, suggests that the recovery may involve simply a normalization of crucial physiological disturbances achieved through a modified form of growth during liquid holding. Consonant with such a possibility, they also reported that ehloramphenicol inhibits recovery and that adenosine triphosphate, but not adenosine monophosphate, stimulates recovery from either uv or X-ray damages during liquid holding. Yeast cells are normally impermeable to nueleotides (DEMAIX, 1964) and refractory to inhibition of protein synthesis by ehloramphenieol because of the inability of their ribosomes to bind the antibiotic (VAzQVEZ, 1964). The findings of PATI~ICK and HAYNES must mean, therefore, that even low doses of ionizing or non-ionizing radiations can render cells freely permeable to nueleotides and modify ribosomal structure so as to create a novel affinity for ehloramphenicol. Either of these drastic non-genetic alterations could have profound consequences for cell survival and be subject to metabolic correction. Most recently, PATnlCK and HAYNES (1968) have shown that liquid holding of either X-ray or uv treated cells slightly increases their sensitivities to subsequent X-ray exposure and markedly reduces their sensitivities to subsequent uv irradiation. They speculate that these changes probably relate to the occurrence during liquid holding of a partial correction of DNA damage in a way yet to be determined. However, it is known that aeration of 22

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Saccharomyces cells in a non-nutrient suspension leads to breakdown of much of their functional protein and ribonucleic acid, partial depletion of their nueleotide pools and exhaustion of their free amino acid pools (SPIEGELMAN et al., 1955). Such radical shifts in the macromolecular composition and overall physiological potential of cells subjected to liquid holding might offer plausible explanations for changes in cellular radioresistance without need to invoke the possibility of special corrective events occuring at the DNA level. Cellular inactivation by uv undoubtedly involves a complex of nongenetic as well as genetic damages. I n contrast to yeast, bacteria typically contain little or no labile protein and a very meager amino acid pool (HARris, 1958). Consequently, their relative metabolic quiescence during liquid holding might allow the occurrence of DNA repair to be expressed directly in increased survival whereas the extensive degradatire and biosynthetic changes which occur in yeasts under such conditions might modify the entire panoply of damages so as to obscure the specific contribution of DNA repair in the overall recovery process. For yeasts, mutation would seem to be a more sensitive biological indicator than cellular inactivation for DNA repair since induction of mutational lesions and their correction must involve events occuring within DNA (WITKIN, 1966). SA~AC~EK and BIS~ (1963) have demonstrated that haploid cells of Saccharomyces, depleted of endogenous nitrogen reserves before irradiation, do show a slight repair of nv induced pre-mutational lesions during liquid holding. We are presently investigating the possibility of a comparable repair process in C. albicans. Acknowledgement. These studies were aided in part by a contract AT (11-1)-1772 with the U.S. Atomic Energy Commission.

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Protein Synthesis (Ed. R.B. ROBERTS), pp. 100--108. London: Pergamon Press 1958. DElWAIN,A. L.: Nutrition of "adenineless" auxotrophs of yeasts. J. Baet. SS, 339--345 (1964). DOt~)N~Y, C. 0., B. F. WmT~, and B. J. BRUCV.:Acriflavin modification of nucleic acid formation. Mutation induction and survival in ultraviolet light-exposed bacteria. Biochem. biophys. Res. Commun. 15, 70--75 (1964). ELKIND, M. M., and H. SUTTON: The relationship between division and X-ray sensitivity, ultraviolet sensitivity and photoreactivation in yeast. Radiat. Res. 10, 283--295 (1959). HARRIS, G.: Nitrogen metabolism. In: The Chemistry and Biology of Yeasts (Ed. A. H. COOK),pp. 437--533. New York: Academic Press 1958. HASENCLEVER,I-I.F., and W.O. MITCttELL: Antigenic studies of Candida I. Observation of two antigenic groups in Candida albicans. J. Bact. 82, 570--573 (1961). IRELAND, 1~., and A. SAI~C~EK: A unique minute-rough colouial variant of Candida albicans. Mycopath. Myeo]. Appl. (in press). JAoG~, J.: Photoreactivation. Bact. Rev. 22, 99--142 (1958). JAzzEs, A. P., and M. M. WEI~N~R: The radiobio]ogy of yeast. Radiat. Rot. 5, 359--382 (1965). KANTOR,G. J., and R.A. DEERING: Recovery of division ability in ultraviolet irradiated Escherichia coli induced by photoreactivation, photoproteetion and liquid holding treatment. J. Bact. 94, 1946--1950 (1967). LERMAN, L. S. : Structural considerations in the interaction of DNA and acridines. J. molec. Biol. 3, 18--30 (1961). LOVELESS, L. E., E. SPOERL, and T. H. WEIS~aN: A survey of effects of chemicals on division and growth of yeasts and Escherichia coll. J. Bact. 68, 637--644 (1954). LVND, A.: Ecology of yeasts. In: The Chemistry and Biology of Yeasts. (Ed. A. H. CooK), pp. 63--91. New York: Academic Press 1958. I~ACKEIqZIE,D. W. R. : Morphogenesis of Candida albicans in vivo. Sabouraudia 3, 225--232 (1964). MAw, G. A. : Incorporation and distribution of ethionine-sulfur in the protein of ethionine-sensitiveand ethionine-resistantyeasts. Arch. Biochem. llS, 2 9 1 - 301 (1966). PATRICK,M.H., and 1%.H. HAYNES: Dark recovery phenomena in yeast II. Conditions that modify the recovery process. Radiat. Res. 28, 564--579 (1964). -- -- Repair-induced changes in yeast radiosensitivity. J. Baet. 95, 1350--1354 (1968). PATRICK,~V[.i . , R. ]{. HAYNES, and R. B. URETZ: Dark recovery phenomen~ in yeast. 1. Comparative effects with various inactivating agents. Radiat. Res. ,Ol, 144--163 (1964). PETTIJOHN, D., and P. HANAWALT:Evidence for repair-replication of ultraviolet damaged DNA in bacteria. J. molec. Biol. 9, 395--410 (1964). SARAeH]~K,A. : Pseudoprototrophs and the absence of genetic interactions between auxotrophie strains of Candida albicans. Microbio]. Genet. Bull. 20, 15--17 (1964). --, and J. T. BISH: Postirradiation protein synthesis and the inductions of cytoplasmic and genic mutations in Saccharomyces by ultraviolet radiation. 28, 450--462 (1963). SCltERI~, G.H., and R.H. WEAV~I~: The dimorphism phenomenon in yeasts. Baet. Rev. 17, 51--91 (1953). 22*

~1~

~.~.]t~

and A. ~ C ~

: Ultr'z~z,l b l ~ ~-~'4~vation of Candida ~

SETLOW,.~. ~., ~ad W. L. C A ~ : The disa~]oeav~.ce of thymine dimers ~r DNA:: am emr~r-eorrecting mec];m~sm. Proc. ~ t ~ e a d . ~4. (Wash.) 51,226--~L (1964).. SETLOW, 3. ~.,, :~L E. BOLING, and F . J . BOLLU]M=The' chemical nature of photo~ reactivab~]e3egons in DNA. Proc. n~at. Acad. ScL (,q~.ash.}~ , 1430--1436 (1965). SIDEROPOULOS,d~. ,S., and D. M. SHANI~EL:Mechanism. of caffeine enhancement of mutations ~indueed by sublethal uIJtraviolet dosag.~s. J. Baet. 96, 198--204 (1968). [SPIEGEL~fAN,S.., ]~. O. HALVORSON,and R. BE~-IsI~r: Free a~fino acids and the enzyme forming mechanism. In: Amino Acid MetaboliSm (Eds. W. D. McELRoY and H. B. GLAss), pp. 124--170. Baltimore: John Hopkins Press 1955. 'STAPLETO~',G.. E.= Protection and recovery in bacteria and fungi. Ir~: Radiation Protecg~on and Recovery (Ed. A. H(~LAENDEt~), pF. 87--116. New York: Pergamon Press 1960. :Sw]~NSON,P. A , and R. B. S~Tnow: Effects of ultraviolet radiation on macromoleeu]ar synthes~s in Escherichia coli. J. molee. Biol. 15, 201--~19 (1966). SZ~LVlNYI,A., and U. ROSENKRANTZ: Radiation effects on yeast of tho genus Candida Berkhout. Nature (Lond.) 190, 1~12--1213 (1961}. T . ~ E ~ , F. W., and E. I~u Action of ultraviolet light oll yeast like fungi. Bot. Gaz. 75, 309--317 (1923). V~zQv]~z, D. : Uptake and binding of chloramp,henicol by sensitive and resistant organisms. NaCre (Lond.) 203, 257--258 (1964). ~V~TKIN,E.M.: t~adiation-induced mutations and their repair. Science 1~,~ 1345--1362 (1966). Prof. Dr. A. SARAC~F~K Wichita State University Wichita, Kansas 67208, U.S.A.