C H L A M Y D O S P O R E P R O D U C T I O N IN C A N D I D A ALBICANS
by ALICE BOURKE HAYES
Department o/Natural Science, Loyola University, Chicago
(22.IV.1965) Candida albicans (ROBIN) BERKHOUT (51) produces both yeast and mycelial cells in vivo and in vitro (3, 25, 52, 68). Chlamydospore production is associated with the mycelial phase. It is an important criterion for identification. Therefore, the factors promoting this phenomenon are of considerable interest. The production of chlamydospores has been attributed to m a n y factors: carbon source, nitrogen source, various ions, diffusion products, high pH, tow pH, high temperature, low temperature, acids, drugs, hormones, x-rays, chelating agents, inhibitors, etc. The literature is extensive. Many factors have been studied, and each has beeu described as a causal agent in chlamydospore production. The purpose of this paper is to demonstrate through a review of the literature and laboratory observations that the apparently contradictory reports of earlier investigators are actually harmonious with the enzymatic explanation of the pathway of morphogenesis. METHODS AND MATERIALS
Chlamydospore production was observed in slide cultures. Agar blocks 1 cm square were inoculated with 0.008 ml yeast suspension, covered with a -1-1 cover slip, and incubated at room temperature in a sterile Petri dish. Medium: 1 liter distilled water, 10 cc Tween-80, 1 g ammonium sulphate, 20 g agar, 10 g carbon source. Initial pH was adjusted to 6.8 with NaOH. The carbon sources were: glucose, fructose, mannose, arabinose, xylose, ribose, rhamnose, erythrose, sedoheptulose, sucrose, maltose, trehalose, lactose, melibiose, raffinose, inulin, methanol, ethanol, cystine, asparagine, yeast ribonucleic acid. No carbon source was included in control cultures.
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Inocnlum: The inoculum was prepared from 30 clinical isolates. The isolates were identified as C. albicans b y a combination procedure using chlamydospore formation, fermentation of selected sugars, and reduction of tetrazolium. (13) Procedure: Each of the 30 isolates was subcultured twice on control medium. Then each cnlture was subcultured in duplicate on every carbon source and on control medium. After three days and again after ten days of incubation, the slides were examined for chlamydospore formation. Ten fields per slide were studied and the chlamydospores were counted for comparative evaluation. A second series (using the same medium, inoculum, and slide culture technique) investigated the effect of p H and temperature of incubation on chlamydospore production. Identical cultures were incubated at p H 3,5 and 7, and at temperatures of 15, 27 and 37 °C. The carbon sources were: xylose, arabinose, glucose, sucrose, maltose, lactose, trehalose, and raffinose. RESULTS
Each slide culture was graded according to the following criterion: 0 = chlamydospores rare 1 = 1-10 average number chlamydospores 2 = 11-25 average number chlamydospores 3 = 26-50 average number chlamydospores 4----over 50 average number chlamydospores The average grade for all cultures was recorded. The use of an average grade provides a greater range of comparison than the use of ( + ) and (--). See Table I. Presence ( + ) or absence (--) of chlamydospores in different culture conditions was recorded. See Table II. DISCUSSION
The first requisite for chlamydospore production is the formation of a mycelial growth phase. NICKERSON and co-workers (7-9, 31-43, 65, 67) have demonstrated that this is accomplished when cell division is inhibited without inhibition of growth. Cell division in C. albicans is initiated b y the enzymatic transfer of metabolic hydrogen to covalent disulfide bonds irt a polysaccharideprotein complex of the yeast cell wall. The rupture of S-S linkages in reduction to - - S H weakens the cell wall so that a bud initial can be extruded. (8) This also stimulates nuclear division, possibly through an effect on nucleic-acid synthesis. (67) Any factor which promotes enzyme activity on the cell wall favors the yeast phase, whereas any factor which inhibits this enzyme activity favors the mycelial phase and thus encourages chlamydospore formation. The pathway of morphogenesis and the factors affecting it are summarized in Scheme I.
CHLAMYDOSPORE P R O D U C T I O N IN C. ALBICANS
SCHEME I.
PATh~qAY 0F MORPHOGENESIS
FILAMENT
YEAST
T
THE CELL A. TP~H-DP.~ Production
decrease ~. exhaustion of carbon source 2.poorly available carbon source 3. decreased phosphate, etc. 4. partial anaerobism B. I. room temperature 2. slightly alkaline ~ emperature, pH extremes chelating agents and i~hlbitors
~
| ~
~SS
increase i. abundant carbon source 2. easily utilized source 3. adequate oxygen
I ~
active i. 30-37 dog. C~ 2. slightly acid
Yntracellular SS=SH ! i
x-ray
D. <
I. aminopterin 2. indoleacetic acid 3- proflavin 4- x-ray or UV \ .... J
T
Protein Disulfide Reductase
inactivated
I. auxin 2. carcinogen drugs
~
? ? RNA
damaged
I. addition of -SH 2. addition of Se, Tc 3. association with organisms with -SH diffusion products
? ? unaffected I. normal dlvieion
E,., Chlar~dosporo Production
Metabolic Hydrogen Production. Protein-disulfide-reductase activity requires the production of metabolic hydrogen in the cell. C. atbicans produces T P N H and D P N H via the tricarboxylic acid cycle. (48, 49, 61). The yeast phase is thus favored by culture conditions promoting active aerobic respiration: an abundant and easily utilized carbon source, adequate oxygen, and accessory growth factors. It has long been established that abundant carbon source favors in vivo and in vitro growth of C. albicans. (16, 29, 42, 46, 69). The yeast phase is also associated with abundant oxygen supply. (6, 69) The mycelial phase is promoted by conditions which decrease aerobic respiration. These conditions are usually provided in media designed for chlamydospore formation. Most chlamydospore media are characterized by the use of a poor carbon source (60): carrot infusion (6), potato (26), corn meal (2), rice (58), zein (50), potysaccharide (41), soil extract (1), etc. Exhaustion of the carbon source of the medium achieves the same effect. The association of filamentation with aging, and the frequent appearance of filamentation at the outer edges of a culture is considered to be due to depletion of medium. (22, 26, 21, 10, 16, 32, 52, 56) Partial anaerobism also promotes filamentation and subsequent sporulation. In practice, this is usually achieved by placing a cover
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A.s. ~AYES
slip over the culture, or by slitting the medium with the inocnlum and sealing the surface of the agar with a warm needle. (12, 14, 16, 21, 46) Lack of inorganic ions required for carbohydrate metabolism (magnesium, manganese, phosphate, and ferrous ions) promote filamentation. (22, 66, 71) Formation of excess polyphosphate also leads to the mycelial phase through binding of intracellular magnesium. (71) Our laboratory observations (Table I) support these conclusions. Readily available carbon sources such as glucose and sucrose favor the yeast phase; whereas less easily utilized sources such as melibiose and raffinose promote filamentation and chlamydospore formation. Easily utilized monosaceharides, glucose, fructose, mannose (70) support lavish yeast growth. The other monosaccharides (the pentoses and the 4- and 7-carbon sugars) promote chlamydospore formation to a greater degree than the hexoses. These carbon sources are generally poorly utilized. (5, 17, 18) TABLE I.
Ef[ect of carbon source on chlamydospore production. Carbon source
(1% COliC.) Mouosaccharides: Erythrose Arabinose Xylose Rhamllose Ribose Glucose Fructose Mannose Disaccharides: Sucrose Maltose
Average grade*
(25--30 strains) 2.2 1.0 2.1 2.2 2.3 0.9 1.2 1.6 1.9 2.7
Carbon source
(1% COliC.) Disaccharides: Trehalose Lactose Melibiose Trisaccharide: Raffirmse Polysaccharide: Inulin --SH Compounds: Asparagine Cystine Ribose nucleic acid Control, No Carbon:
Average grade*
(25--30 strains) 2.8 2.9 3.2 3.3 3.6 0.6 0.9 0.0 1.0
* 3 days incubation at room temperature; criterion of grade in text It has long been suggested that the complexity of form exhibited by C. a l b i c a n s in culture is related to the complexity of the carbon source on which it is grown. (23) It is therefore not surprising to note that the disaccharides and trisaccharides promote chlamydospore formation to a much greater degree than do the monosaccharides. Among the more complex sugars, sucrose is conspicuous. It does not produce chlamydospores as lavishly as the others, and supports extensive yeast growth. This confirms expectations, as sucrose is easily utilized. (61)
CItALMYDOSPOR~
PRODUCTION
IN
C. A L B I C A N S
91
Within 10 days under the experimental conditions described, all of the carbon sources studied supported chlamydospore formation. This supports the observation that exhaustion of carbon source induces chlamydospore formation. It further clarifies the diverse results often reported for filamentation on various sugars, since duration of incubation is obviously an important factor. Protein-Disulfide-Reductase. Even though production of metabolic hydrogen is maintained, cell division will not occur in conditions unfavorable for enzyme action. This effect was clearly demonstrated with an enzymedeficient m u t a n t which retained the filamentous form, although capable of producing metabolic hydrogen. (39, 37, 34, 65) The optimal conditions for the activity of this enzyme are: a temperature range of 30-37 °C, and a slightly acid medium. (15) It has been observed that these conditions promote the yeast phase.
(29, 52, The chlamydospore-promoting effect of several factors may be attributed to their influence on enzyme activity. (1) room temperature: Incubation of cultures at room temperature (less than optimal for enzyme activity) has been successful in promoting chlamydospore formation. (lg, 21, 52, 69) (2) slightly alkaline medium: Although Candida can grow throughout an extensive pH range, most workers find chlamydospore formation is extensive at a slightly alkaline pH. (24, 29, 57) Some observers feel that pH does not affect morphology of Candida significantly. (45, 71) Certainly our observations support the possibility of chlamydospore formation at pH 5, but we found more extensive sporulation at pH 7, a range less favorable to the activity of protein-disulfide-reductase. Where pH 5 is favorable, as in the purified polysaccharide medium (41), carbon source is an important factor. (3) temperature and pH extremes: Enzyme inactivation is frequent at temperature and pH extremes, and this m a y be the basis of the filamentation reported under such conditions. (29, 30, 56, 57) (4) chelating agents and inhibitors: These agents apparently stimulate filamentation through alteration of a metallo-flavoprotein involved in hydrogen transfer. (34, 65, 66) Surface active agents should be mentioned here. A different mechanism is involved, but the effect on the cell wall is analogous to that of enzyme action, i.e., a localized increase in plasticity (27). Decreasing surface tension thus promotes filamentation and surface active agents are frequently added to media designed to promote chlamydospore formation. (13, 53, 59, 63) Table II summarizes our laboratory observations of the influence of temperatnre and pH on chlamydospore formation. The temperature and pH conditions which are uniformly favorable to chlamydospore formation are those which are not optimal for protein-
9~
A, B. H A Y E S
TABLE II.
INTER-EFFECTS OF CARBON SOURCE, pH, AND TEMPERATURE I
CARBON SOURCE
IHp_~
xS
5 27
3'7
a5
7 e7
37
0
0
0
0
0
+
+
m
L~ Xylose
0
0
Arablnose
0
0
0
0
0
0
0
+
0
Glucose
0
0
0
0
0
0
0
+
0
Sucrose
0
0
0
0
0
0
0
+
0
Maltose
0
0
0
0
+
0
0
+
0
Lactose
0
0
0
O
+
+
0
+
+
Trehalose
0
0
0
+
+
0
+
+
0
Raffinose
0
0
0
+
+
O
+
+
0
CONTROL, No C,
0
0
0
0
0
0
0
+
0
0 = no c h l m ~ d o e p o r e s
Incubated 5 days at indlcatcd tem )erature
+ = chloa~dospores
disulfide-reductase activity, i.e., room temperature and slightly alkaline medium. It should be emphasized that temperature and p H are not unitary factors; rather they act through their influence on metabolic events within the cell. Even in the presence of utilizable carbon sources, chlamydospore formation can occur at room temperature. For example, maltose supports chlamydospore formation at room temperature. At the temperature for optimum enzyme activity, 37 °C, maltose does not support chlamydospore formation. It can also be seen that carbon sources which ordinarily promote chlamydospore formation at room temperature and slightly alkaline pH do not do so at high temperature and low pH. Lactose, trehalose, and raffinose all produce chlamydo readily at pH of 7, but none do so at pH 3. Of all the carbon sources tested, only lactose and xylose will produce chlamydospores at 37 °C. Some of the confasion surrounding identification of carbon sources which favor chlamydospore formation can be removed if incubation conditions are considered, for no single carbon source, temperature, or pH alone guarantees sporulation. Intracellular
SS-SH.
Transfer of metabolic hydrogen by protein-disulfide-reductase to the disulfide links binding the polysaccharide-protein bonds in the cell wall results ill the reduction of cell wall disulfide to sulfhydryl, which weakens the cell wall. (8) Any factors womoting a shift towards sulfhydryl in the cellular balance of SS = SH will promote cell division. The addition of - - S H or the analogous, selenium or tellurium, suppresses filamentation. (43, 44) Diffusion of - - S H m a y also explain association effects noted. (15, 40, 64)
CHLAMYDOSPORE
PRODUCTION
IN
C. A L B I C A N S
93
Agents which reduce SH level in the cell promote filamentation: (1) auxin: Filamentation promotion by auxins is directly related to inactivation of - - S H groups. (30, 52) (2) carcinogens: 9, 10-bishydroxymethyl-1, 2-benzanthracene makes - - S H groups unavailable for cysteine synthesis (52) and thereby stimulates the transformation of yeast to filament. (3) drugs: The mode of action of penicillin (4, 44, 47) and oxine (45) on C. albicans is inactivation of - - S H groups. (4) x-rays: The mycelinm-promoting influence of x-rays has been attributed to - - S H inactivation. (62) (5) aging: The loss of - - S H from the cell through release of cysteine may be another factor responsible for filamentation with aging. (64) Our laboratory investigations (See Table I) support the conclusion that addition of sulfur compounds promotes the yeast phase.
? ? Nucleic Acids ? ?, The final step in the pathway is under consideration. It has been demonstrated that - - S H level affects RHA activity in the cell, which in turn affects cell division. (28, 42) Factors that interfere with nucleic acid synthesis do promote filamentation: (1) aminopterin (42, 67); (2) indoleacetic acid (52); (3) proflavin (32); (4) x-ray and UV (62). Preliminary studies (see Table I) show that yeast RNA supports lavish yeast formation, and that no chlamydospores at all were formed on medium in which RNA was the carbon source. Chlamydospore formation. The factors described promote chlamydospore formation by initiating the mycelial phase. The subsequent formation of chlamydospores is the result of accumulation of metabolic products. (11) Complex polysaccharides and lipids cannot move through the cell wall and are accumulated within the cell. The cell wall is thickened by the deposit of polysaccharides, and a resistant cell, filled with reserve materials (the chlamydospore) is formed. Summary A review of the literature correlated with laboratory" studies of the influence of carbon source, temperature, and pH on chlamydospore formation, substantiate the enzymatic hypothesis of morphogenesis in Candida albicans.
Acknowledgements I wish to thank J. ROBERT TI~OMPSOX, M.D., and YosH TAKIMURA of the Municipal Tuberculosis Sanatorium, Chicago, for their kind assistance. I also wish to thank W. J. 1NICKERSONof Rutgers University for his helpful comments.
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