Mycopathologia vol. 68, 2: I05-120, 1979

POPULATION CHANGES INDUCED IN CANDIDA ALBICANS BY NALIDIXIC ACID Alvin SARACHEK Department of Biological Sciences, Wichita State University, Wichita, Kansas 67208, USA

Abstract

Introduction

Cells of Candida albicans plated on media containing nalidixic acid (Nal) either die, adapt physiologically to Nal-tolerance or mutate to Nal-resistance. The fraction of a population exhibiting each response depends on the growth phase of cells when plated and their nitrogen and carbon nutrition and growth temperatures before and after plating. Nal induces Nal-resistant mutants in very high frequency but only at 37 C on plates containing i) glucose as primary carbon source and ii) adenine, a sulfur amino acid or a representative of the glutamic acid family of amino acids. Nal does not affect either forward mutation to caffeine-resistance or reverse mutation from histidine auxotrophy to prototrophy. Nal-resistant mutants produce minute colonies on Nal-free medium, respire oxidatively and are unusually sensitive to inhibitors of oxidative phosphorylation. They revert spontaneously to wild type at very high rates but can be propagated indefinitely in the absence of Nal by serial selection and replating of minute colonies. Cellular inactivation and induction of Nal-resistant mutants are greatly affected by specific inhibitors of mitochondrial macromolecular syntheses. The presence of chloramphenicol or erythromycin during exposure to Nal prevents cell death and mutation but has no effect on adaptation to Nal-tolerance. Growth on acriflavin or ethidium bromide enhances resistance of cells to inactivation when subsequently plated on Nal containing media. It is concluded that Nal-induced cellular inactivation and mutation to Nal-resistance, but not adaptation to Nal-tolerance, result from damages to the mitochondrion which are fixed or promoted by macromolecular syntheses within the mitochondrion. Implications of these findings for the therapeutic use of Nal are discussed.

The antibacterial agent, nalidixic acid (Nal), causes a selective and lethal inhibition of deoxyribonucleic acid (DNA) synthesis in procaryotic cells (6). In eucaryotic microorganisms, generally, it preferentially inhibits replication of the procaryote-like DNA of chloroplasts (22, 28, 30) or mitochondria (20, 39), and at somewhat higher levels coordinately depresses the syntheses of nuclear DNA, total cell protein and ribonucleic acid (7, 25). A number of investigators have noted immediate but temporary inhibitions of cell division or overall macromolecular synthesis in cultures of Saccharomyces cerevisiae exposed to Nal (12, 25, 38). Depending on the yeast strain, Nal concentration and cultural conditions employed, the inhibitory period varies in duration from ca. one to ten hours and may or may not be accompanied by the death of a fraction of the treated population. In all cases, however, inhibited cells eventually develop tolerance to Nal and resume a normal rate of growth in its presence. In contrast, Sobieski and Brewer (35) have reported that Nal induces in the pathogenic yeast, Candida albicans, a persistent inhibition of macromolecular synthesis which is not associated with either cellular inactivation or physiological adaptation to the drug. Although they regard their findings as signifying a fundamental difference in the mechanisms of Nal toxicity for C. albicans and S. cerevisiae, that interpretation is equivocal since their studies were conducted with a single strain of C. albicans exposed to Nal for no more than five hours under growth conditions unlike those used in stfldies of S. cerevisiae. While attempting to determine whether acquisition of resistance to Nal could be used as an indicator of forward mutation in C. albicans, I observed that cells plated on Nal-containing medium either die or else undergo mutation or physiological adaptation to ~lal resistance. The relative frequencies of these responses depend on the growth stage and nutritional history of the inoculum as well as 105

the Nal concentration and carbon and nitrogen sources in the plating medium, and the temperature at which plates are incubated. The present report describes these relationships and presents evidence that i) Nal directly induces an extrachromosomal sort of mutation to Nal resistance and that ii) the lethal and mutagenic, but not the adaptive, responses to Nal depend upon mitochondrial protein synthesis.

Materials and methods

Test Organisms: The wild type strains of C. albicans used, 207, 526, 792 and B-311, were obtained originally from H. F. Hasenclever, National Institute of Health, USA and have been maintained in our laboratory for a number of years. The mutant test organisms are derivatives of strain 526; the histidine auxtotroph, WD-24, was obtained through ultraviolet irradiation and the nalidixic acid resistant variants, M2, M3 and M5, were induced by growth on nalidixic acid at 37 C on enriched-dextrose (ED) medium.

Media: All media were constituted from the following basal ingredients: CaC12 92H20, 0.04 g; KH2PO 4, 2.0 g; MgSO 4 9 7H20, 0.5 g; FeSO 4 9 7H20, 3 mg; ZnSO 4 97H20, 3 mg; CuSO 4 9 5HzO, 0.5 mg; MnSO 4 9H20, 0.4 mg; (NH4)6MoTO24, 0.15 mg; NazB207, 0.9 mg; biotin, 0.02 mg; inositol, 10 mg; nicotinic acid, 1 mg; pantothenic acid, 1 mg; para-aminobenzoic acid, 1 mg; pyridoxine, 1 mg; thiamine, 1 mg. For solid media, 16 g agar was added. Minimal medium (M) was prepared by addition of 3 mg/ml ammonium tartrate; enriched medium (E) was prepared by additions of 3 mg/ml casamino acids (Difco) and 50 pg/ml each of adenine and uracil. The primary carbon sources were either 2 ~ (W/V) dextrose (MD or ED) or 2 ~o (V/V) glycerol (MG or EG). Where indicated, chloramphenicol or erythromycin were added directly to preautoclaved media cooled to ca. 50 C: filter sterilized preparations of sodium azide or dinitrophenol were added similarly to preautoclaved media. Nal, dissolved in 0.1 N KOH, and all other indicated supplements were added to media prior to autoclaving. Media were adjusted to pH 6.8 with KOH prior to autoclaving at 118 C for l0 min. Nalidixic acid was provided by the Sterling-Winthrop 106

Research Institute; erythromycin was obtained from the Chemical Division, Abbott Laboratories and chloramphenicol from Parke-Davis and Company. Other materials used were obtained from Mallinkrodt, Baker or Calbiochem Corporation and were of the highest grades available from these sources.

Preparation of cells for experimental use: Stationary phase populations of strains with wild type sensitivity to Nal were grown on ED or MD plates as described by Busbee and Sarachek (2). To obtain exponential phase cells, individual plates were inoculated initially with ca 2,000 ceils: ED plates were harvested after 20 hr. at 25 C or 16 hr. at 37 C; MD plates were harvested at 30 hr. at 25 C or 22 hr. at 37 C. Where indicated, broth grown cells were cultivated with shaking in 250 ml or 500 rnl Ehrlenmyer flasks containing 50 ml or 100 ml of medium respectively. Though agar grown inocula were used routinely, agar or broth grown cells give comparable responses. Preparations of Nalresistant mutant cells were made by picking and pooling individual five-day-old mutant colonies grown at 25 C on ED. Typically such suspensions consist of 80 ~ or more single unbudded cells and are comparable in appearance to agar-grown, stationary phase wild type populations. In all instances cells were harvested and washed in 2 ~ ..033M KHzPO 4 before use.

Population analyses: For routine determinations of responses to Nal, washed cell suspensions were plated in quintuplicate on Nalcontaining media; plates were scored for colony counts and colony characteristics after five days incubation. When necessary, colony sizes were measured at magnifications of from 0.7 x to 30X by means of an ocular micrometer fitted on a stereoscopic microscope. Procedures for determining i) forward and back mutation frequencies in WD-24 and ii) reversion rates of Nal-resistant variants to Nal-sensitivity are described in the appropriate sections under RESULTS.

Cellular respiratory character&tics: Qualitative spectral analyses for cytochromes were made on frozen cell suspensions using a Beck (London) model 26-3657 prism spectroscope according to the method of Sherman and Slonimski (33). Spectra for strains 526, M2,

conditions. Fig. 1 illustrates the plating efficiencies of exponential and stationary phase populations of C. albicans 526 grown at 25 C or 37 C on MD or ED when plated on graded concentrations of Nal in MD or ED and incubated at 25 C or 37 C. In general, colony production on Nal is greatest on plates incubated at 25 C. At that temperature, all inocula plate with ca. 100 % efficiency on as much as 500 #g/ml Nal; only small incremental decreases in proportions of colony-forming cells are noted as Nal concentrations increase to 800 #g/ml, the highest level of drug fully soluble in plating media. The decreases are unaffected by pro-or post-plating nutrition, but are greater for (i) stationary phase than exponential phase cells or (ii) inocula initially grown at 37 C rather than 25 C. However, the absolute sensitivities of all inocula, as well as the differential sensitivities between inocula, are slight on 25 C plates; even at 800 #g/ml Nal, the

M3 and M5 were determined on cells harvested from colonies grown on ED for 4 days at 25 C or 3 days at 37 C. Oxidative respiratory competencies of mutant and wild type colonies were compared by means of the tetrazolium overlay technique of Ogur et al (27).

Results Platin9 efficiencies on N a l

9

Preliminary attempts to develop a plating medium for selective isolation of Nal-resistant mutants of C. albicans established that the plating efficiencies of wild type populations (i.e., proportions of plated cells which produce macroscopic colonies) on a given level of Nal varies enormously with pre-and post-plating growth

IO0

8I.A'

#\

-! i

E

i

1

12A

"

Z

1,4. l,U

',,

10

"-

0 Z

#,

I r~ '

!B 2OO

4OO

~)0

8OO

200

400

600

800

NALIDIXIC ACID CONC. IMg/ml] Fig. 1. Plating efficiencies of C. albicans 526 under various pro- and post-planning growth conchtions. lnoculum ."

i) grown at 25 C (open symbols) or 37 C (closed symbols) ii) stationary phase population: MD grown (diamond); ED grown (triangle) iii) exponential phase population: MD grown (square); EDgrown (circle) Plate 9rowth conditions:

i) medium: MD, 1A and 1B; ED, 2A and 2B ii) temperature: 25 C, 1A and 2A; 37 C, IB and 2B

107

on Nal noted for strain 526 are representative for the species (Table 1). Populations of strain 526 deposited on millipore filter discs and placed on media containing 500/~g/ml Nal were found to show no differences in colony production after 5 days at 37 C if held on Nal throughout that period or if transferred to Nal-free medium after two days contact with Nal; the effects are the same on ED or MD plates. Evidently failure to produce colonies on Nal is due to inactivation rather than inhibition of cells. Estimates of the kinetics of inactivation at 37 C were made by holding cells on millipore discs on Nal-containing media for intervals up to 24 hours before transfer to Nal-free medium to complete five days incubation at 25 C. Fig. 2 presents survival curves for 25 C, ED grown, exponential and stationary phase populations on ED or MD plates containing Nal in concentrations ranging from 400 to 700 /~g/ml. Each curve exhibits a shoulder preceding an exponential phase of inactivation. Both the rapidity

plating efficiency for the most sensitive inoculum is as high as ca. 60 ~ and ranges up to ca. 90 ~o for the most resistant population. In contrast, on 37 C plates, cells are much more susceptible to Nal generally and plating efficiencies depend markedly on the backgrounds of the inocula and the nutritional quality of the plates. As on 25 C plates, inocula are more sensitive if initially prepared at 37 C than 25 C. However, all inocula plate with 100 ~ efficiencies only at Nal concentrations up to 200 ~tg/ml. Declines in colony production beyond that level are generally much more significant on ED than on MD plates. On ED plates, stationary phase or MD grown populations are more Nal-sensitive than corresponding exponential or ED grown ones; these relations are reversed on the MD plate. A survey of the abilities of four additional arbitrarily selected strains of C. albicans to plate on 500 #g/ml Nal indicates that while strains differ in absolute in susceptibilities to the drug, the interactions between plating efficiencies

Table 1. Plating efficiencieson 500 #g/ml Nal for three wild type stains of C. albicans under various pre- and post-plating growth conditions. Values given represent means and 95 ~ confidence limits based on 3 to 5 determinations. Inocolum

Post-platin 9 Growth Conditions 25 C

_Strain 201

Growth Conditions Temp, 25 C

07 C

Growth P h a s e exponential

25 C

25 C

95 _+ 2.9

98 _+2.8

19 • 4.3

94 • 3.3

0.80 • 0.41

stationary

MD

92 • 4.4

93 +_ 3,7

71 • 9,4

0.21 • 0.09

ED

91 _+4.8

89 • 4.3

27 • 6,0

0.37 • 0.12

exponential

MD

92 _+ 3.7

94 • 4.9

9 _+ 3.2

0,41 • 0.08

ED

93 • 4.1

87 ~ 8.0

0,23 • O.lO

0.64 • 0,18

MD

90 • 3.9

92 _+ 5.4

27 +- 5.4

0.08 ! 0.03

ED

95 • 4.0

94 • 3.9

14 • 4.3

0.12 • 0.06

MD

97 +- 3.9

98 • 2.7

65 -+ 7.3

4,4 • l.OO

ED

99 • 2.0

96 _+2.5

n

9.5 • 3.i

MD

iO0 t 1.8

96 • 2.6

98 ~ 4,0

1.5 ~: 0,45

ED

100 • 1.5

I00 ~ 1.0

88 • 4.7

2.2 :F 0.73

MD

96 • 2.8

95 -+ 1.8

35 ~ 6,5

4,0 _+0.91

ED

97 -+ 2.8

99 • O.gl

29 -+ 6.5

7.7 ~ 2.8

MD

I00 _+ 3.3

97 • 3.8

73 • 9.1

l.l

ED

96 ~ 3.4

96 • 3.7

39 • 7.8

1,9 ! 0.53

exponential

exponential

exponential

stationary

37 C

exponential

stationary

108

LD

Medium

92 _+3.8

stationary

B311

MD

ED

stationary

37 C

37 C LD

MD

stationary

792

MO

~- 2.9

1.3 ! 0.55 l.l

~ 0.66

• 0.48

MD

95 • 4.2

98 +_ 3.8

44 _+ 8.4

1.3 _+ 0.62

El)

99 • 1.0

100 _+ l.O

1.8 _+ 0.61

2.5 • 0.60

M~)

97 -+ 3,7

94 • 3.6

94 • 3.6

0.40 _4 0.09

ED

98 • 2.9

93 -+ 0.9

65 -+ 6.8

0.61 "- 0 , I I

MD

95 _+ 3.9

9g • l.O

19 • 7.2

0.08 • 0.08

ED

94 _+ 5.1

100 • 0.28

0,66 +_ 0,21

1.7 -+ 0,30

MD

93 • 4.6

91 -+ 4.1

56 -+ 5.3

0.19 -+ 0.04

ED

96 • 3.7

97 -+ 2.6

31 • 4.l

0.51 • 0.08

..,,,I

16

2'4

-

16

2'4

TIME OF INCUBATION ON NAL [hr] Fig. 2. Survival curves for cells of C. albicans 526 deposited on millipore filters discs and held at 37 ~ on Nal-supplemented MD or ED plates for intervals before transfer to corresponding Nal-free media to complete five days incubation at 25 ~ Each disc was flushed initially with 10 ml of Nal-containing broth equivalent in composition to the Nal agar on which the disc was to be plated. Stationary phase (dark symbols) or exponential phase (open symbols) populations plated on MD, (A) or ED, (B) : Nal concentrations (#g/ml); 400 (circle), 500 (square), 600 (triangle) ; 700 (diamond).

of the onset of inactivation and the final rate of inactivation increase with increasing Nal concentrations. At a given Nal concentration, exponential cells are inactivated more rapidly on MD medium but less rapidly on ED medium than stationary cells. Significantly, the relative inactivation rates correlate with relative plating efficiencies (Fig. 1) under the various conditions tested; the two different classes of response, therefore, are parallel expressions of cellular sensitivity to Nal, Adaptation and mutation to Nal resistance:

Generally, colonies arising on a Nal-supplemented plate are uniform in size and have smooth surfaces and entire margins. However, those produced at 37 C on ED plates containing inactivating levels of Nal (i.e., 300 /~g/ml or more) vary considerably in size; they appear to be regular in form and smooth in texture through three days incubation but are somewhat rugose with highly irregular margins after 5 days of incubation (Fig. 3). Cells in colonies produced on any level of Nal, under any set of growth conditions, are blastosphoric and re-plate with ca. 100 % efficiencies at 37 C.on ED test plates containing 500 ~g/ml Nal. However, smooth, regular colonies contain only cells

with transient tolerance to Nal whereas irregular ones consist largely of Nal-resistant (Nal R) mutants. When replated on Nal-free media, smooth, regular colonies produce colonies of uniform size, wild type in form and growth rate and composed entirely of cells which have reverted to full Nal-sensitivity, as indicated by subsequent plating efficiencies on Nal test plates. To determine how rapidly tolerance to Nal is lost in the absence of the drug, drug adapted cells from five-day-old Nal grown, regular colonies were inoculated into Nal-free broth and tested at intervals of incubation for loss of ability to plate at 37 C on ED + 500 #g/ml Nal. Comparisons were made with colonies initially produced at 25 C on MD or ED containing 500 /~g/ml Nal; deadaptations were followed in MD or ED broths at 25 C and 37 C. Table 2 shows that adapted populations retain full Nal-resistance for ca. two generations in the absence of Nal; drug sensitivity then rapidly reappears, attaining a level usual for unadapted populations by about the fourth generation. The general course of deadaptation is not notably affected by the differences in cultural conditions during adaptation to Nal or during deadaptation. The rapid and uniform transitions of populations to Nal-sensitivity indicate that Nal-tolerance is physiological rather than a genetic 109

Fig. 3. (A-C) Five day old colonies of C. albicans 526 produced on 500/~g/ml Nal under various growth conditions: (A) on ED at 25 ~ (B) on ED at 37 ~ (C) on EG at 37 ~ (D) Colonies produced after 5 days at 25 ~ by a clonal population of the Nal R mutant, M3, plated on Nal-free ED. Small colonies are NalR clones; large colonies arise from wild type revertants typically present in NalRpopulations.

adaptation. Yet persistence of Nal resistance through two generations on Nal-free media implies either that the molecular changes underlying adaptation are very stable or that Nal-grown cells bind sufficient quantities of Nal to sustain induction of resistance through two or more divisions in the drug's absence. The irregular colonies produced at 37 C on high Nal ED plates are heterogeneous in composition. Replatings on Nal-free medium show that each contains some cells which form large, regular colonies but a preponderance ( > 60 ~ ) of stable, small colony producing variants. Cells of the large colonies exhibit wild type sensitivity to Nal while those in the small colonies retain drug resistance. The small colonies of Nal R mutants eventually generate papillae or sectors of rapidly growing Nal-sensitive revertants upon protracted incubation on Nal-free media (i.e., > 8 days at 25 C or > 5 days at 37 C on ED medium). The revertants show full wild type growth rates and susceptibilities to Nal; evidently they arise through precise correction to the Nal R genetic lesion or through secondary suppressor mutations which compensate fully for the physiological defect caused by that lesion. Despite the reversion, Nal R clones can be propagated indefinitely in the absence of Nal by successive replatings of young Nal R colonies and selection of small colony forms. Reversion rates differ for Nal R of independent origin. Revertant ll0

frequencies among thirty 5-day-old small colonies obtained as independent primary isolates at 25 C on ED were found to range from ca. 0.4 ~ to 15 ~. For a given NalR isolate, however, the revertant frequency in five-dayold colonies remains quite constant through successive platings in the absence of Nal. To determine whether cultural conditions required for induction of Nal R variants also affect their reversion, fluctuation analyses were performed to estimate reversion rates of two Nal R strains, M3 and M5, at 25 C and at 37 C on MD or ED with or without 500/~g/ml Nal. In outline, ca. 500 Nal R cells were spread on an agar plate and incubated under specified test conditions until microeolonies containing ca. 150 to 400 cells were produced. Eighty colonies were then excised and individually respread on a single ED plate. After five days' incubation at 25 C, each plate was scanned for occurrence of large colony revertants and counted to establish the actual number of cells in the original micro-colony. Reversion rates were comPuted according to the formula (21): a = - ( I n 2) (ln p)/N where a = reversion rate per cell division cycle, p = fraction of colonies containing no revertants, N = the mean number of cells in the set of colonies sampled. Findings summarized in Table 3 show that reversion rates of Nal R variants are not notably influenced by growth

Table 2. Declines in plating efficiencies at 37 ~ on ED+500 /~g/ml for Naladapted populations during growth on Nal-free ED or MD at 25 ~ or 37 ~ Cells adapted to Nal at 25 C on*

Conditions for Deadaptationt Medium

MD

Temperature

Plating Efficiencies Number Of Generations

MD

25 C

0,9 1.8 3.6 4.5

98 89 40 Z,2

ED

25 C

1.2 2,3 3.4 4.2

I00 lO0 36 3.7

MD

37 C

0.7 1.6 3.1 4.0

I00 97 62 5.l

ED

37 C

0.8 I. 6 3.3 4.3

100 89 31 2,9

MD

25 C

l.l 2.6 3,7 4.5

gl lO0 27 2.5

ED

25 C

1.2 2.4 3.5 4.2

98 96 41 3.2

MD

37 C

l.g 2.5 3.8 4.6

lO0 87 19 2.1

ED

37 C

1.4 2, 7 3.7 4.6

96 98 28 Z.5

*Nal adapted ce~Is are from five day old colonies produced on either ED or MD with 500 ug/ml Nal. fCells from three equivalent Nal adapted colonies were pooled, inoculated into broth and plated at Intervals of incubation onto ED with or without Nal to estimate increases in cell numbers and changes in plating efficiencies on Nat.

temperature or nitrogen nutrition. They also reveal that, despite the high rate and apparent precision of spontaneous reversed of Nal-induced Nal R variants, Nal itself does not alter the incidence of reversion. Table 3. Rates of reversion to wild type for the Nal R mutants M3 and M5 during growth at 25 ~ or 37 ~ on MD or ED, with or without Nal. Growth Conditions Temp. 25 C

37 C

Media

M3

M5

MD

2.9xi0 -4

8.1xlO -4

ED

3.8x10 -4

8.9xIO -4

MD+500 ug/ml Nal

2.0xlO -4

7.0xlO -4

ED+500 ug/ml Nal

2,7xi0 -4

7.2xi0 -4

MD

1,8x]O -4

6.9xi0 -4

ED

3.2x10 -4

9.OxlO-4

MD§

~g/ml Nal

3.0xlO -4

8.2.10 -4

ED+500 ~,g/ml Nal

2 . 2 x i 0 -4

5.3x10 -4

Nutrilites requiredfor induction of Nal R mutants." Induction ofNal Rmutants occurs on enriched, but not on minimal, medium. To identify the nitrogenous components of ED necessary for mutagenesis, a cell population grown to stationary phase at 25 C on MD was screened for survival and colonial characteristics when plated at 37 C on a series of MD + 500 #g/ml Nal plates supplemented with either adenine, uracil or one of 19 amino acids. Ordinarily, under these conditions, cells plate with very high efficiency on MD, producing regular, mutant-free colonies; on ED, their plating effectiveness is very low and only'very irregular, mutant-rich colonies arise. Control data given in Table 4 show that survival on unsupplemented MD is c a . 100 • higher than on ED; colonie's on MD are uniform while those on ED exhibit the irregularity indicative of mutagenesis. In the presence of either adenine, a sulfur amino acid (cysteine or rhethionine) or a member of the glutamic acid family of biosynthetically related amino acids, (arginine, glutamic acid, ornithine, or proline),

111

Table 4. Plating efficiencies and form of colonies produced after 5 days by stationary phase cells of strain 526 plated at 37 ~ on ED or MD with or without supplements of adenine, uracil or one of 19 amino acids. The inoculum was grown at 25 ~ on MD. Cysteine was used at 4x 10-* M concentration: all other supplements were 1 • 10 -3 M. Colony form: smooth and regular ( - ) , increasing degree of irregulatiry (+ through +++). Media MD

ED

Supplement

Plating Efficiency

none

81

l-alanine

SO

l-arginine

22

l-aspartic acid

69

Colony Form

Nal and nuclear #ene mutations:

++

l-cysteine

29

+

l-glutamic acid

38

+

glycine

86

]-histidine

80

l-isoleucine

82

l-leucine

79

l-lysine

74

l-methionine

22

++

l-ornithine

29

++

l-phenylalanine

82

l-pro)ine

31

l-serine

7~

l-threonine

82

i-try~tophane

87

]-tyrosine

84

l-wline

7S

adenine

19

uracil

84

none

0.77

+

++

+++

survival on M D declines and colonies produced are irregular. These survival reductions and colonial irregularities, though notable, are less extreme than those occurring on ED. Replatings of regular and irregular colonies onto Nal-free ED confirmed the presence of Nal R mutants in irregular colonies only. Though invarianly present in such colonies, their frequencies were much lower in the slightly irregular colonies from invariably supplemented plates (ca. 0.5 ~o to 20 ~o per colony) than in the highly irregular colonies produced on ED ( > 60 per colony). Except for cysteine, which is severely growthinhibiting at concentrations above 5 x 10-4M, each nitrogen supplement was found to have the same effect over a concentration range up to 5 x 10- 3M. Furthermore, a combination of all amino acids within a biosynthetically 112

related group exerts no greater effect than the most active amino acid within the group. However, adenine and amino acids from different families additively decrease survival and enhance colonial irregularity when combined; plating efficiency and colony form on MD containing arginine, methionine and adenine are essentially identical to those on full ED.

The high frequency with which Nal R mutants are induced at 37 C on ED suggests that epigenetic or extrachromosomal events rather than nuclear gene mutations may be involved. To determine the possible mutagenicity of Nal for nuclear genes, a histidine auxtotroph, WD-24, derived from strain 526 was tested for reversion to prototrophy and for forward mutation to caffeine resistance during growth on Nal. ED grown stationary phase cells were plated so as to produce ca. 500 colonies per plate on ED or histidine-supplemented MD, with or without 500/~g/ml Nal. Plates were incubated at 25 C or 37 C until macroscopic colonies containing from 1 x 106 to 5 x 106 cells per colony were produced. For each treatment, cells were washed from 10 replicate plates after colony counts were made and the pooled population was assayed, at 25 C, according to previously described procedures (31), for frequencies of prototrophs and of cells capable of growing in the presence of 500 #g/ml caffeine. The average number of cell generations achieved during each treatment was computed on the assumptions that i) individual colonies arose from single cells and ii) all cells in colonies were viable and recovered during harvesting. Data given in Table 5 show that Nal does not detectably affect back or forward mutations in WD-24 during 18 to 22 generations in the presence of the drug under any of the growth conditions tested. WD-24, like 526, yields Nal R containing colonies during growth at 37 C on ED with 500 /~g/ml Nal. The absence of effects on reversion to prototrophy or mutation to caffeine-resistance under these conditions indicates that Nal R variants are not due to chromosomal gene mutations. Mitochondrial functions and responses to Nal."

Nal selectively depresses mitochondrial D N A synthesis in Klyveromyces lactis (20) and S. cerevisiae (39). It induces respiration-deficient mutants through structural changes in mitochondrial D N A in at least some strains of S. cerevisiae (3, 12) but inhibits the activity of other agents

Table 5. Frequencies of prototrophs and caffeine-resistant mutants in populations of the histidine auxtrotroph, WD-24, after growth on MD or ED, with or without Nal at 25 ~ or 37 ~ Generations Competed

Test Growth Conditions

Mutants/lO 8 Cells

~Test~Medium

~nePratrotrQhp~*

Medium MD

none

25 C

18.5

37 C

19,2

II

25 C

22.0

12

77

37 C

20.2

lO

64

25 C

20.7

12

67

37 C

23.0

7

48

25 C

19.6

13

80

37 C

21.6

lO

52

500 ~g/ml

ED

Resis_tant~

Mal

none

500 ug/ml

8

56 74

* screened on MD + 0.5 ~g/ml histidine $ screened on MD + 25 ug/ml histidine and 500 ug/ml caffeine

Table 6. Plating efficiencies of glucose or glycerol grown populations of strain 526 when plated at 37 ~ on 500 #g/ml Nal in glucose or glycerol media with or without 4 mg/ml chloramphenicol or erythromycin. Plating Media MD

Inoculum:

MD +

HD +

Chl

Ery

96

ED

ED +

ED +

Chl

Ery

MG

MG + Chl

1.0

I00

MG EG + Ery__

99

31

I00

99

EG + Chl

EG + E~

1.6

I00

I00

Exponential phase c e l l s grown at 25 C on MD

33

97

ED

1.2

94

99

2.2

96

97

1.4

97

99

2.4

I00

97

MG

37

98

100

0.84

97

I00

33

97

97

1.0

98

lOO

EG

1.0

97

I00

2.2

I00

96

1.2

96

I00

2.0

98

98

MD

92

100

98

0.33

89

97

90

98

iO0

0.40

I00

96

ED

47

I00

97

0.55

94

100

51

97

97

0.50

97

100

MG

89

I00

I00

0.41

95

96

94

I00

98

0.47

I00

95

EG

50

g6

99

0.63

98

97

53

98

I00

0.54

97

96

Stationary phase cells grown at 25 C on

which can also induce such mutants (39). Moreover, growth inhibition ofS. cerevisiae by Nal depends markedly on the cellular state of respiratory adaptation. It is greatest when the oxidative respiratory system is fully derepressed by growth on oxidative substrates, such as glycerol, and least when respiration is maximally repressed, either by mutation to respiratory deficiency or by growth on high ievels of glucose (3). Although the mechanisms underlying these observations are unknown, collectively they signify that mitochondrial activity is crucial in determining the toxicity of Nal for yeasts. To detect a possible bearing of mitochondrial functions on sensitivity of C. albicans to Nal, glucose and glycerol grown log phase cells were compared for plating efficiencies at 37 C on 500 #g/ml Nal. Responses

were tested on minimal and complete media, each containing either glucose or glycerol as primary carbon source, with or without a 4 mg/ml supplement of erythromycin or chloramphenicol, specific inhibitors of mitochondrial protein synthesis in yeasts (19). Results given in Table 6 show that sensitivity to Nal is unaffected by carbon nutrition before or after plating and, therefore, is independent of the state of respiratory repression. In contrast, chloramphenicol or erythromycin render cells completely resistant to inactivation by Nal on all media. In S. r these antibiotics prevent utilization of oxidative carbon sources by blocking formation of the terminal respiration chain (19). The effects noted here cannot be attributed to complete suppression of oxidative

113

respiration since cell growth is essentially unaffected on glucose and only partially depressed on glycerol by either antibiotic (Table 7). They do establish, however, that some aspect of mitochondrial protein synthesis is essential to cellular inactivation by Nal. Induction of Nal Rmutants is profoundly affected by the presence of chloramphenicol or erythromycin and the nature of the carbon source in the inducing growth medium. Colonies produced by wild type cells at 37 C on ED + 500/~g/ml Nal are normally irregular in size and form indicating induction of such mutants. On EG + 500 /~g/ml Nal, at 37 C, colonies are variable in size but very regular in form (Fig. 3). When replated on Nal-free medium they give no evidence of minute Nal R variants. Similarly, colonies arising after five days at 37 C on ED + 500 #g/ml Nal supplemented with either antibiotic are uniform in size, regular in form and give no indication of Nal R variants when screened on Nal-free medium. Failure to detect Nal R mutants under these growth conditions could indicate either that i) they are not induced or that ii) their outgrowth or survival in developing colonies is suppressed. To test these alternatives, colony growths were compared for wild type strain 526 and two Nal R derivatives of 526, M3 and M5, at 37 C on ED and EG with or without 500 #g/ml Nal, in the presence or absence of each antibiotic. Table 7 shows that Nal R mutants grow well on EG. In the absence of Nal, NalR mutants are more resistant than the wild type to growth inhibition by chloramphenicol or erythrornycin on either dextrose or glycerol as carbon source. In the presence of Nal, the

antibiotics significantly promote growth of the wild type but not that of the mutants. However, the antibiotics are not notably more inhibitory to the mutants in the presence of Nal than in its absence. Thus failure to find Nal Rmutants within wild type colonies developing at 37 C on EG or on ED containing Nal and antibiotic cannot be attributed to selective inhibition or destruction of the mutants and induction of Nal R mutants requires growth on glucose as well as particular nitrogenous nutrients. Significantly, the mutant-free wild type colonies produced on EG or on ED plus Nal and antibiotic replate with ca. 100 ~o efficiences at 37 C on ED + 500 #g/ml Nal test plates. The tolerance of cells taken from Nal plus antibiotic plates is not an artifact due to an intracellular carry-over of antibiotic since cells grown on either antibiotic without exposure to Nal exhibit the characteristic low plating efficiencies on Nal at 37 C. Thus adaptation to Nal-tolerance occurs on oxidative as well as fermentative carbon sources and is not blocked by disturbances in mitochondrial protein synthesis which prevent mutation and cellular inactivation by Nal. Effects o f azide and dinitrophenol on susceptibility to Nal :

The characteristic coordinate inhibition by Nal of RNA, DNA and protein synthesis in euearyotes (7, 25, 35) suggests that the drug directly affects metabolic activity essential to all three processes. Genetic deficiency for such a central function could explain the coincidence between the low growth rate and Nal insensitivity of Nal R

Table 7. Effects of chloramphenicol or erythromycin (4 mg/ml) on size of colonies of strain 526 and its NalRderivatives, M2, M3 and M5, after 2 and 4 days growth at 37 ~ on ED or EG with or without 500/~g/ml Nal. Colony Sizes (mm)* Without Nal Strains.

ED 4 da~

526

~

~

~ 3.0

4.2

EG

4 day

2.8

22day_~

EG + Chloram

EG + E ~ t h r o

~

2 daZ

4 da~

4.0

1.3

3.7

0.69

1.8

0.72

.70

3.1

.72

2.9

.72

2.8

0.17

l.O

0,15

0.94

0.16

0.96

M3

,47

2.5

.45

2.2

.45

2.2

0.11

0.87

0,II

O.gl

0.13

0.96

2.8

0.28

1.2

0,22

1.0

0.25

l,.O

1.2

5.1

Ed + Erythro

M2

M5

3.4

ED + Chloram

3.2

].l

3.0

1.0

2.1

with 500 ug/ml Nat+ 526

.32

2.0

0.97

3.4

1.2

4.0

0.08

0.78

0,38

I.I

0.33

1.3

M2

.30

1.5

0.38

1.3

0.31

1.6

0.11

0.81

0.12

0.82

0.12

0.78

M3

.25

l.l

0.27

1.4

0.28

1.4

o.og

0.77

0. I I

0.83

O. lO

0.11

M5

.26

1.2

0.23

l.l

0.26

1.3

O.14

0.95

0,11

O.gl

0.13

0.16

*Each v a l ~ is the average for 30 randomly selected colonies, Standard errors for readings on ED or EG plates containing Nal alone were 15% and l l ~ of the corresponding mean value. Standard errors for a l l other treatments ranged from ca. I% to 4% of the mean values. +Plating efficiencies on media without antibiotics:

114

strain 526, ca. 2.5%; M2, M3, M5, ca. I00%

mutants of C. albicans. An impaired cellular respiration would account reasonably for these phenomena. In S. cerevisiae, Nal does block formation of the oxidative respiratory apparatus (23) and is proportionately much more inhibitory to growth of wild type cells than of respiratory deficient mutants (3). The possibility that the oxidative respiratory system is a critical Nal sensitive target is further supported by Yamabe's observations (40, 41), that Nal will complex in vitro with cytochrome c and modify the rate ofcytochrome reduction by inorganic electron donors. However, the responses of C. albicans to Nal reported in the present study cannot be ascribed readily to induced respiratory deficiency since the drug is equally toxic to cells on oxidative or fermentative carbon sources (Table 6). Moreover, unlike the respirationdeficient mutants of S. cerevisiae, the minute Nal Rmutants on C. albicans grow well on glycerol carbon and I have noted that five-day-old Nal g colonies grown on either ED or EG reduce tetrazolium as rapidly as corresponding wild type colonies when tested by the overlay technique of Ogur et al (27). Spectroscopic examinations of strain 526 and its two Nal Rderivatives, M3 and M5, also reveal no differences between Nal n and wild type cells; both kinds of cells ghow distinct bands for cytochrome a (601 nm) and cytoclirome b (550 nm) as well as cytochrome c (547-553

rim). Though Nal may not block oxidative respiration in C. albicans, it might intrude in the terminal respiratory chain in such a way as to diminish production of high energy compounds. Kot et al (17) have described a slowgrowing mutant of C. albicans, possibly induced by acriflavin, which respires normally and has an intact cytochrome chain but is defective in oxidative phosphorylation. The observation that Nal R mutants are more resistant than wild type to growth inhibition by chloramphenicol or erythromycin indicates that genetic resistance to Nal is associated with change in the functional properties of mitochondria. To probe the possibility that Nal may interfere with energy production, wild type and Nal-resistant cells were compared for sensitivity to two inhibitors of oxidative phosphorylation, 2, 4, dinitrophenol and sodium azide, in the presence and ih the absence of Nal. Data presented in Table 8 show that Nal Rmutants are much more sensitive than wild type cells to each of the inhibitors. Furthermore, Nal significantly potentiates the inhibitor's toxicities for wild type cells but not for Nal-resistant mutants. These findings suggest that a Nal-sensitive step in oxidative phosphorylation exists in wild type cells but is severely debilitated or lacking in Nal-resistant mutants.

Table 8. Plating efficiencies at 25C and 37C of strain 526 and its Nal Rderivatives, M3 and M5~ on ED containing various concentrations of sodium azide or 2,4 dinitrophenol with or without N al. ~ement '~al

Ii,9/ml) Na Azide

Strains* 526 25 C

M3 37 C

M5

25C

37C

none

lO0

I00

97

98

52

I00

50

none

125

93

75

67

35

43

14

none

150

90

63

36

21

none

175

84

45

]l

Tlone

200

73

25

2,1

25 C

370

18

7,3

3.2

4

0.99

1.I

0.71

0.08

250

100

90

81

85

49

97

48

250

125

72

50

59

33

46

I0

250

150

22

35

27

11

250

175

2.4

0.08

8.8

2.7

3.3

0,74

200

0.I

>0.0!

1.2

0,13

0.58

0,05

250 Nal

9.3

5.5

~,4 dinitrophenol_

none

600

98

I00

91

93

84

87

nene

700

I00

97

80

74

58

55

none

800

95

80

58

47

none

900

78

53

24

250

600

70

55

84

250

700

II

250

800

3,1

250

900

0.12 >0.01

1,5

21 8.9

1.3

80

80

72 41

7

77

69

44

l.O

47

42

I7

8.0

8.6

l.]

6,4

7.5 0.85

*Inocula consisted of stationary phase ce115 grown a t 25 C on ED.

Effects of mitochondrial mutagens on susceptibility to Nal." Respiration deficient, petite mutants of S. cerevisiae arise through defects in mitochondrial DNA (19). Like Nal Rmutants of C. albicans, they grow slowly, are induced by growth of wild type cells on Nal and are resistant to growth inhibition by Nal. However, the two kinds of mutants differ in a number of significant properties; unlike petites, Nal R variants revert to wild type in high frequency, grow well on glycerol, reduce tetrazolium and exhibit a wild type cytochrome spectrum. Still, the observations that i) Nal induces Nal R variants in high frequency while not effecting forward or back mutation of nuclear genes and that ii) inhibitors of mitochondrial protein syntheses block induction of Nal* cells suggest that Nal R mutants of C. albicans, like petite Saccharomyces, are extrachromosomal, presumably mitochondrial, in origin. To test this possibility, three potent inducers of petites in S. cerevisiae, ultraviolet radiation (2537 ~), ethidium bromide and acriflavin (26), and a number of 115

amino acid, purine and pyrimidine analogues which are ineffective as inducers of petites (unreported observations in our laboratory) were compared for abilities to produce Nal "mutants of C. albicans in the absence of Nal. Stationary phase cells were exposed to uv doses of 270 and 540 ergs/mm z according to procedures described previously (31); the lower dose has negligible effect on survival while the higher dose causes ca. 25 % inactivation for populations plated at 37 C on ED and ca. 35 % inactivation for cells plated on ED + 500/~g/ml Nal. For treatments with chemical agents, cells were grown for ca. 4.5 to 6.0 generations at 37 C in MD broth containing a test agent concentration predetermined to extend the cell generation time ca. by 1.7X to 2.0X. Treated and control populations were plated and incubated i) for three days at 25 C on ED with no Nal for evidence of induced minute colony variants and ii) for five days at 37 C on ED and on MD containing 500 #g/ml Nal to detect improved plating effectiveness on Nal resulting from treatment. Minute NalR-like colonies were not detected on Nal-free plates following any of the treatments; occasional smallish colonies formed by irradiated cells proved, on testing, to be due to transitory, uv induced growth inhibitions. In contrast, exposing cells to acriflavin, ethidium bromide or uv, but not to any of the other test agents, greatly increased their plating effectiveness on ED or MD containing Nal (Table 9). The enhanced survival cannot be ascribed to the presence of induced Nal R mutants in treated populations since colonies produced on MD

with Nal are, as usual, uniform in size and form, and when replated on Nal-free medium give rise only to large regular colonies comprised entirely of cells with wild type sensitivity to Nal. Evidently the treatments render cells physiologically resistant t o inactivation when exposed to Nal. Low-dose UV (16), acriflavin (8, 32) and ethidium bromide (1, 8, 11) have each been reported to preferentially inhibit DNA, RNA and protein syntheses in yeast mitochondria. Like chloramphenicol or erythromycin, these petite-inducing agents probably favor cells' adapting to Nal rather than dying on initial contact with the drug because of their abilities to disturb mitochondrial macromolecular syntheses.

Discussion

During growth on Nal, cells of C. albicans either 1) die, ii) adapt physiologically to Nal-tolerance or ii) mutate to Nalresistance. The relative frequency of each effect for a given population depends on i) the concentration of Nal, ii) the division stage of cells when first exposed to the drug, and iii) temperature and nutritional growth conditions of cells before and during exposure. These heterogeneous responses within N al treated populations indicate that interpretations of the mechanism of Nal's action based on analyses of the composite behaviors of whole cultures may be misleading. It is possible, for example, that the enigmatic multiple, successive growth lags and

Table 9. Plating efficiencies on either MD or ED containing 500 ~g/ml Nal for strain 526 after ultraviolet irradiation or after growth in the presence of various antimetabolites. Treatment Growth in MDbroth supplementedwith (pg/ml)

No. of Generations

Plating Efficiencies on ED +_500 pg/Inl Nal

MD + 5.00 ug/ml Na.l 9.0

none

5.2

acriflavin, 40

4.4

80

8-azaadenine, I00

5.8

11

8-azaguanine, 200

6.2

6-azauraci|, 250

4.8

12 13

l-azetidine-2 carboxy|ic acid, 250

5.2

caffeine, 200

6.0

ethidium bromide, 20

5.7

ultraviolet radiation* ___CDos~___

Cell Survival

0

100%

9.4

8.8 29

7.6

] .2 64 1.3

I .S 1.0 1.3 0.9 63

1.9

270 ergs/mm2

97%

Ig

14

540 ergs/mm2

74%

31

33

*Irradiated cell suspension was prepared from an exponential phase population grown at 25 C on MD.

116

recovery phases previously noted in Nal treated cultures ofS. cerevisiae (12, 25, 38) actually represent the dynamics of induced cellular heterogeneity rather than uniform transitions in the physiological states of all cells. Though the present study was not designed to identify precisely the molecular events responsible for the alternative cellular responses of C. albicans to Nal, the findings do support the following inferences. Relationships between cellular inactivation, adaptation to Nal and mutation to Nal resistance.

Low concentrations of Nal selectively inhibit DNA synthesis in bacteria by interfering with the nicking-closing activity of the gyrase compon )t of the DNA replication complex (9, 36) ; at higher concentrations the drug impedes protein and RNA syntheses as well (6). Since low concentrations are bacteriocidal while higher ones are only bacteriostatic (6), the lo&ality ofN al seems to result from unbalanced growth accompanying inhibition of DNA synthesis. Superficially, Nal affects eucaryotes differently than bacteria. Previous workers have demonstrated that Nal is inhibitory for several species of yeasts (12, 20, 35) and the protozoan, Tetrahymena Pyr~[ormis (7), only at levels 10X to 100X higher than those effective against procaryotes and that its toxicity for these organisms is associated with coordinate inhibition of overall macromolecular syntheses. The failure of Nal to selectively inhibit nuclear DNA synthesis in eucaryotes probably reflects the fact that the conformational changes in supercoiled DNA necessary for chromosomal replication are achieved by the histones of chromatin rather than a gyrase-like enzyme (10). On the other hand, replication of the procaryote-like organelle DNA's of eucaryotes seem uniquely susceptible to Nal. Nal inhibits synthesis and promotes degradation of chloroplast DNA in Chlamydamonas reinhardtii (30) and Euylena 9racilis (22, 28). The effect leads to irreversible loss of photosynthetic capability and occurs at drug concentrations inconsequential for growth processes, generally. Similarly, the quantities of mitochondrial DNA in cultures of the yeasts, Klyveromyces lactis (20), and S. cerevisiae (39), decline progressively during successive generations of growth in the presence of Nal. Though the distribution of mitochondrial DNA losses among individual cells has not been tested directly, the fact that Nal does induce in S. cerevisiae a low frequency of petite mutants deficient for mitochondrial DNA (3, 12) implies that such losses do occur discontinuously within treated populations.

Chloramphenicol or erythromycin, specific inhibitors of mitochondrial protein synthesis, block both cellular inactivation and induction of Nal R mutants in C. albicans if present when cells are exposed to Nal; the antibiotics do not prevent physiological adaptation to Nal resistance. Rescue from inactivation or mutation does not require total cessation of mitochondrial protein synthesis since it occurs at antibiotic concentrations which allow sufficient mitochondrial development to enable cells to grow well on oxidatively respired carbon sources (Table 7). Furthermore, treatments with acriflavin, ethidium bromide or low-dose UV, agents which preferentially disrupt mitochondrial protein and/or RNA syntheses, render cells tolerant to subsequent contact with Nal alone while nonspecific metabolic inhibitors do not (Table 9). Evidently Na-induced cellular inactivation or mutation require mitochondrial RNA and protein synthesis whereas development of tolerance does not. Thus if such syntheses are at least temporarily disadvantaged during initial contact with Nal, inactivation and mutation are circumvented and establishment of a state of Nal tolerance favored. It would be reasonable to propose that disturbance of mitochondrial DNA synthesis is essential for both mutation and inactivation of C. albicans by Nal and that concomitant mitochondrial protein and RNA synthesis are necessary either to foster entry of the drug into the organelle or, as in bacteria (5, 6), to fix the critical DNA lesions induced. Nature of the NalR mutations:

Tests with forward and back mutation systems indicate that Nal is not mutagenic for nuclear genes of C. albicans (Table 5). Yet it can induce Nal-resistant mutants en masse during growth at 37 C in the presence of adenine, a sulfurcontaining amino acid or one of four representatives of the glutamic acid family of biosynthetically related amino acids (Table 4). The induction is prevented by the inhibitors of mitochondrial protein synthesis, chloramphenicol or erythromycin, at concentrations having negligible effects on cell growth. Nal R mutants are capable of oxidative respiration and have normal cytochrome spectra but are slow growing, sensitive to inhibitors of oxidative phosphorylation and exhibit a high spontaneous rate of reversion to wild type. Their origin and physiological properties suggest that they constitute a special class of mitochondrial mutants. Previous workers have attributed intercalation into DNA as the most likely explanation for Nal-induced i) frame shift mutations in phage T 4 (37), 117

as well as ii) selective inhibition of transcription of specific chromosomal loci ofE. coli (4, 34) and of whole phage S13 genomes in infected host cells (29). Each of these phenomena occurs under conditions interpreted to indicate that some metabolically transformed product of Nal is the actual intercalating agent. The special growth conditions required for production of Nal R mutants similarly implies that metabolic activity, probably within the mitochondrion, is necessary to render Nal mutagenic for C. albicans. If the mutagenesis also is due to intercalation, then Nal R cells can be assumed to bear frame shift changes in mitochondrial DNA. Single base additions or deletions are most probable; gross deletions s/Jch as are found in petite mutants of S. cerevisiae can be discounted since Nal ~ strains readily revert to wild type spontaneously. But whether frameshifts or base pair changes, the Nal-induced mutations in Nal R strains might be excepted also to be specifically revertable by Nal. The finding that they are not (Table 3), may signify that Nal R cells are physiologically unable to process the drug into a mutagenic form. The Yamabe-Shimizu model:

Mutations to ~qal-resistance in E. coli have been mapped at two loci: mutations at one locus cause low level resistance by decreasing cellular permeability to Nal; mutations at the other elicit high level resistance by rendering DNA gyrase insensitive to the drug (9). Although DNA gyrase activity in eucaryotes has not been reported, it is reasonable to suspect that replication of the procaryote-like DNA of organelles may involve such an enzyme. Modifying Nal-susceptibility of C. albicans by mutation or inhibition of mitochondrial macromolecular synthesis may also reflect changes either in the permeability of the mitochondrion to Nal or the Nal-sensitivity of its DNA synthesizing apparatus. On the other hand, Yamabe and Shimizu (42) have suggested that inhibition of bacterial D N A synthesis is only one of several secondary effects of a particular sort of binding of Nal to the cell membrane. Their model imputes a special role to the terminal respiratory chain and offers the most plausible general explanation for conditions affecting Nal resistance in S. cerevisiae as well as C. albicans. Yamabe (40, 41) observed that Nal and a number of its structural analogues increase the rate of non-enzymatic electron transfer from Fe § 2 to cytochrome c in a model in vitro system: the extents of acceleration for various compounds parallel their relative anti-bacterial activities. Since absorption spectra indicated formation of 118

Fe+Z-Nal-cytochrome c complexes, Yamabe and Shimizu (42) proposed that the quinone-like Nal molecule probably associates in vivo with the terminal respiratory components of the bacterial cell membrane in such a way as to interfere with both the energy yielding electron transport system and the membrane-bound apparatus for DNA synthesis. That interpretation is consistent with the observations that certain mutants of E. coli defective for oxidative respiration are coincidentally Nal-resistant (15, 18) and that, in eucaryotic cells, i) chloroplasts and mitochondria are primary Nal-sensitive targets (3, 12, 22, 23), ii) replication of organelle DNA is selectively inhibited by the drug (20, 28, 39), and iii) various kinds of macro molecular syntheses are coordinately depressed by Nal (7, 25, 35), presumably because of an overall cellular deficiency for energy. More specifically, it reasonably relates the Nal resistance of Candida and Saccharomyces mutants to their special physiological properties. The resistance of petite mutants of S. cerevisiae (3) to growth inhibition by Nal, for example, might be ascribed to their cytochrome deficiency, lack of requirement for mitochondrial DNA synthesis and/or their principle dependence on substrate rather than oxidative phosphorylation as source of utilizable energy. Similarly, the sensitivities to inhibitors of oxidative phosphorylation characteristic of the slow growing Nal R mutants of C. albicans would imply that the mitochondrial membrane sites which usually interact with Nal are genetically altered in Nal R cells. Possible clinical significances: C. albicans is a normal commensal resident of the human alimentary tract. Instances of systemic candidiasis very often involve invasion of the kidney or bladder. Nal is commonly prescribed for treatment of bacterial infections of the urinary tract and is administered orally in dosages ranging from 2 to 5 g per diem for several days (13, 24). More than 80 ~ of the ingested drug is excreted in urine, largely in metabolically altered forms which retain the same antibacterial activity as Nal (13). The spectrum of Nal derivatives produced by man, other animals or various fungi appear to be alike (14) and probably include the particular material(s) responsible for mutagenesis in C. albicans. During a therapeutic regimin with Nal, indeterminate but undoubtedly high concentrations of the drug must occur regionally within the intestinal tract and the levels of Nal and its modified form in urine often exceel 500/~g ml (13, 24). The temperature and free amino acids of theintestinal contents would favor inactiva-

tion of C. albicans and induction of debilitated Nal R variants among the survivors; reestablishment of the yeast flora should occur quickly following treatment through reversion of Nal R cells. Unlike most antibacterial agents, therefore, Nal should impart no selective natural advantage on the resident Candida population. Moreover, the levels of Nal obtained in urine under acceptable protocols suggest that the drug could contribute to control of candiduria. Thus while Nal is not ordinarily considered an antifungal agent, it may be useful, perhaps in conjunction with such agents, in treatment of yeast infections.

11.

12.

13. 14.

15.

Acknowledgement The author is indebted to Mrs. Roberta Pettriess for efficient and reliable assistance in the performance of a number of experiments. This study was aided in part by a grant from the American Cancer Society, Kansas Division.

16.

References

18.

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