Temperature effect on Leishmania enriettii in vitro

EXPERIME~AL

PARASITOLOGY 16, 36-52 (1965)

Temperature

Effect

Charles

on

Leishmania

L. Greenhlatt

and

enriettii Philip

in

Vitro

Glaser

Sational Institute of Arthritis and Metabolic Diseases; Xational Institutes of Health; Public Health Service; Department of Health, Education, and Welfare; Bethesda, Maryland (Submitted

for publication,

13 September 1963)

GREEKBLATT, C. L., AND GLASER, P. 1965. Temperature effect on Leishmania enriettii in vitro. Experimental Parasitology 16, 36-52. Temperature is an important factor in regulating the morphogenesis and growth of hemoflagellates. Leishmania enriettii is limited to growth in the colder parts of the body and does not grow above 37°C 1960. Journal of Protozoology 7, Suppl. abstract [as cited in de Castro and Pinto. lo]. The influence of various temperatures on L. enriettii is examined from the point of view of morphology, activity, growth, respiration, phosphate metabolism, and retention of metabolites. At temperatures above 3O”C, there is a decline in viability in spite of an initially elevated respiratory capacity. Between 35°C and 37”C, marked morphological changes occur including flagellar loss and inclusion body formation. These inclusions are lipid in nature. Survival is not significantly impaired for about 20 hours, during which time a division occurs. Acute exposure to elevated temperaas tures does not impair respiration, nor does it uncouple oxidative phosphorylation measured by Paa uptake. However, if the organisms are suspended in a simplified medium, inorganic phosphorus accumulates and leaks to the outside. Other substances leak as well. An analysis of these materials utilizing column and thin-layer chromatography and electrophoresis reveals a general loss including guanosine, uracil, hypoxanthine, ribose, and at least thirteen amino acids. Kinetic analysis of the leakage by study of the increase in ultraviolet absorption outside of the cells shows a straight line arrhenius plot from 10” to 4O”C, suggesting that no cataclysmic event (lysis) occurs. The increase in permeability in this range is nearly a thirtyfold change, while respiration increases only twofold. The role of enhanced permeability is discussed as it relates to intracellular parasitism.

manioid forms at 37°C. Trager (1953) reported obtaining rounded aflagellate forms of Leishmania donovani at 37”C, while similar cultures at 25°C yielded flagellated leptomonads. Thus the environmental temperature seems to be a crucial factor in determining the morphology of the parasite. The environmental temperature may explain the localization of lesions in Leishmania enriettii infections. In the guinea pig, this parasite produces lesions in cooler parts of the body: the skin, ears, pharynx, nose, peritesticular areas, and the periarticular areas, where the temperature may be as much as 10” lower than rectal temperature (Medina, 1946;

Recent studies of Trager (1953), de Castro and Pinto (1960), Neva et al. (1961), and Trejos et al. (1963) have stressed the role of temperature in the development of hemoflagellates. These articles suggest a hypothesis that helps to explain the morphological changes and location of lesions produced by the parasites studied. Thus Keva et al. have noted that at 38°C Schizotrypanum cruzi is leishmanioid within cells, while at 33” it is elongate. Trejos et al. have presented studies at 26°C where metacyclic forms of the parasite are found-as one would expect to find in the insect host-in spite of the fact that the same medium and host cell harbor leish36

TEMPERATURE

EFFECT

Muniz and Medina, 1948). Peripheral lesions occur, even when the parasites are injected intraperitoneally or systemically (Medina, 1946; Paraense, 1952; Guima&es, 1952, Adler and Halff, 1954). The possible role of temperature is supported by the findings of Pereira et al. [as cited in de Castro and Pinto (1960)] that the parasitic lesions of L. enriettii are healed in animals kept between 37” and 38°C. Trejos (unpublished observations) has confirmed these studies. In agreement with these data, de Castro and Pinto (1960) have found survival and division forms of the organism in tissue cultures at 32” and 34’C, but not at 37°C. Baker and Gutierrez Ballesteros (1957) successfully treated ulcers of the guinea pig produced by L. enriettii by vapor heat, a process by which skin temperatures are raised to 40”-45”C for 4-5 hours. They also reported the treatment of two human cases of leishmaniasis. Gutierrez Ballesteros ( 1959) extended these findings to five more human cases in which the infective agent was diagnosed as Leishmania braziliensis. These findings seem reasonable since Senekjie and Zebouni in 1941 demonstrated that five species of Leishmania, including L. braziliensis, are killed in 15-30 minutes at 40°C or almost at once at 45°C. In this study we shall attempt to explore the effects of elevated temperatures on L. enriettii. We shall first note the alterations in body form, motility, growth, and adaptive capacity up to 40°C. The second portion of the study is an attempt to examine certain aspects of the physiology of the organisms: their respiration, phosphate metabolism, and retention of metabolites. MATERIALS

AND METHODS

The strain of L. enriettii, derived from the original strain (Medina, 1946)) was brought to El Salvador by Dr. Alfonso Trejos. It was maintained by weekly transfers in Locke’s solution over the blood agar medium of Senekjie (1943) during the course of these studies.

OF

L. enriettii

in vitro

37

Locke’s solution in the majority of studies was modified by the addition of Na2HP04 to increase its buffer capacity so that its final composition per liter was: NaCl, 8 gm; KCl, 0.2 gm; CaCl2, 0.2 gm; KH2P04, 0.3 gm; Naz HP04, 0.4 gm; and glucose, 2.5 gm. The final pH was 7.2. Certain other modifications of the salt solution will be described in the text. In the growth medium penicillin and streptomycin were utilized at 100 units per milliliter. Screw-cap tubes ( 150 X 10 mm) were utilized in the growth studies, while the organisms for the respiration and chemical studies were grown in l-liter Erlenmeyer flasks; approximately 100 ml of the blood agar base and 100-200 ml of Locke’s solution were used as the overlay. Organisms were harvested by low-speed centrifugation (450g) at 10°C and washed as described in the text. Temperatures were recorded throughout the incubations and are presented as the maximum and minimum deviation from the mean. Photomicrographs were taken with Leitz ortholux accessories and Adox KB-14 film. The phase optics used in the observation of inclusion granules consisted of the Leitz Heine condenser, and a Leitz apo phase objective, 90x, N.A. of 1.15. Respiration was measured in S-ml Warburg vessels with 10% KOH in the center well. Suspensions of about 1 X 10s organisms per milliliter were used. The organisms were counted in the Levy blood-counting chamber for the respiration and growth studies. We determined residual glucose by the Glucostat method (Worthington Biochemical Corporation) . Phosphate determinations were performed by a standard molybdate method (Gomori, 1942). De-proteinized solutions (5% v/v perchloric acid was used for extraction of the organisms) were properly adjusted to volumes that gave readings from 1 to 40 pg of phosphate in a final volume of about 12 ml. These were mixed with 1 ml of 2.5% sodium molybdate in 2.5 N sulfuric acid. One ml of 1% Elon in sodium bisulfite was used for reduc-

38

GREENBLATT

AND GLASER

tion, and the samples were read after 20 lar). Solvent systems for the sugars were minutes at room temperature by using a 630 water-saturated phenol and butanol: pyridine: mu interference filter in the Klett calorimeter. water (6:4:3). Amino acid samples1 were Acid-labile phosphate was determined by analyzed both by high-voltage paper electrothe method of Crane and Lipmann (1953). phoresis and a Phoenix amino acid analyzer. The extractions were carried out at 0°C for Varsol was used as the organic phase, and one-half hour. The pellets were washed a formate and borate buffers were used as the electrolyte phases. second time in .S$ perchloric acid. Twenty mg of washed Norit A was utilized per milliRESULTS liter of perchloric acid extract to adsorb the Descriptive Aspects nucleotides. Acid-labile phosphate was determined after hydrolysis of the pellet with 0.1 Cultures of L. enriettii at room temperaN HCl at 100” for 20 minutes. ture” in Locke’s solution over Senekjie’s In the phosphate tracer studies, Paz-labeled (1943) blood agar base, with an inoculum phosphoric acid was utilized. The medium, size near S-10 >( lo5 cells per milliliter, grow perchloric acid extract, and acid-labile frac- most actively for the first several days and tions were counted by the liquid scintillation display a gradually decreasing rate thereafter method using the solution described by (Fig. 3). Cultures during the early logarithBray (1960) as the scintillator and a Packard mic phase show considerable numbers of counter. They were referred to the phosphate motile, duplicating stubby organisms, a madeterminations and expressed as specific jority of which appear by phase or light activity. microscopy to contain an essentially homoThe spectra in the leakage studies were geneous cytoplasm except for the nucleus and measured with a Cary recording spectro- kinetoplast. About 30% of these organisms photometer. The spectra of the whole organ- will show a small refractile granule or two. As isms were obtained by using ground quartz the culture advances in age, longer highly plates immediately adjacent to the cuvette motile organisms increase in number. Finally, (before the photomultiplier tube) and posi- as the stationary phase is approached, organtioning the cuvette as near as possible to the isms begin to appear which are swollen in photomultiplier (Shibata et al., 1954). Pro- shape, contain many refractile inclusions, and tein was determined by the biuret reaction have shortened flagella or lack them alto(Gornall et al., 1949). gether. Figures lA-1D are Giemsa and phase Separation and desalting of the materials preparations of organisms from a young detected in the external medium were per- active culture. Both elongate organisms and formed on Dowex exchange resins. The anion biflagellate forms are present. Rosettes are exchange material was the formate form of also a common feature. Dowex 1, 8% crosslinked, 200-400 mesh. The cation material was Dowex 50, hydrogen form, Structural Changes at Elevated Temperatures 12% crosslinked, 200 mesh. If such a young culture, as described above Standard techniques of thin-layer chroma- is placed at 37°C a number of alterations tography were utilized, with various aqueous occurs. solvent systems and cellulose powder (Brink1 These determinations were performed through man MN 300 G) as the stationary phase. Solvent systems utilized for the nucleic acid the kindness of Dr. Leo Levenbook. 2 In the earlier studies conducted in El Salvador, derivatives included that of Paladini and room temperature was 26” -t Z”C, while in the Leloir (1952), Levenbook (1955), and Pabst United States where the majority of studies were System I (Pabst Laboratories OR-17 Circu- conducted it was 23’ -C 1°C.

TEMPERATURE

EFFECT

OF

L. N’iettii

in

VitYO

FIG. 1. (A and B): Giemsa-stained individuals of a young, room-temperature culture of L. e~iettii. In Fig. 1B the organism is dividing. Fixation was by methanol after air-drying. (C and D): Phase photomicrographs of individuals from a room-temperature culture. (E): Giemsastained cells after 24 hours at 37°C. (F): Phase photomicrographs of cells at 37°C for 24 hours; note the refractile inclusions. (G and H): Phase photograph and an absorption image of cells at 37°C for 24 hours stained with l:lO,OOO Janus green B. Figure 1H was taken through a 546my interference filter with open condenser diaphragm. This combination effectively eliminates the refraction image but emphasizes the absorption of the kinetoplast. Those cells which are moribund equivalents” appear stained. are somewhat overstained, so that more than the “mitochondrial (I) : A dividing cell stained with Janus green B.

39

40

GREENBLATT

Figure 1E is a Giemsa-stained preparation of parasites maintained at 37°C for 24 hours. The features to note are the swelling, loss or shortening of flagella, and changes in cytoplasmic density. Phase photographs (Fig. 1F and 1G) of a similar culture show another important feature: the organisms at 37°C are filled with refractile inclusions that are similar to the so-called volutin granules. We have had difficulty determining how the flagella are lost. Studies by phase microscopy reveal at the most about one third as many broken flagella lying about in the medium as parasites that lack the appendage. Giemsa preparations usually reveal more organisms with flagella than do phase optics, so it seems that in some cases, the flagellum may not be lost but is folded back or coiled. Cultures vary in the percent of flagella lost, so that the age of the culture as well as the substrate may be an important factor. Electron micrographs (Dr. Bruce Wetzel, unpublished observations) show many flagella in the interstices between the cells in control cultures, while the interstices are essentially empty in cultures which have been at 37°C for 20 hours. Forms intermediate between those with and without flagella are seen, many of which have shortened flagella, or stubby, often motile, projections at the anterior end. In the electron micrographs these projections often contain what appears to be a fragmenting flagellum. Although it is often difficult to be certain whether an organism has inclusions, we have attempted to count them in cultures at an elevated temperature and at room temperature. Error would be biased toward designating an organism normal when it actually has a small inclusion body. Unfortunately the distinction is more than simply a problem of recognition. As Ormerod (1958) has pointed out, these cytoplasmic granules are widely distributed in size, and at their smallest dimensions they approach (or go beyond) the limits of resolution of the light microscope. In cultures of L. enriettii with about 60-7070 of organisms negative for

AND GLASER

granules by phase microscopy, dark-field examination shows that every organism has granules. The dark field, acting as an ultramicroscope, can discriminate objects beyond the limits of resolution of the light microscope. Thus inclusion granules are clearly defined by optics as well as by physiology. A second factor also seems important. As Ormerod (1958) has noted, larger inclusions appear in a clearer, less refractile cytoplasm. Possibly a more refractile cytoplasm tends to obscure small inclusions, while in the swollen organisms with a clearer cytoplasm they are more distinct. Within these limitations, examination of a control culture (26’C) by phase optics revealed that about 3570 of the cells contained granules on the second day after inoculation. At that time a similar culture was placed at 37’C, and within 20 hours 100% of these cells had inclusions. The control culture remained between 35 and 50% granule-free until the fourth day, when granular cells began to increase. By the sixth day nearly every cell in the room temperature culture showed granules. The nature of these granules is still not completely clear. However, from their appearance by electron microscopy, their solubility in methanol, and their staining by oil red 0, it is likely they are lipid containing. Motility Functionally the organisms have been altered as well. The loss of flagella implies that motility would be lost; it is, but at a rate more rapid than the loss of the flagella. Figure 2 shows the loss in translocation of organisms at 24.5” and 365°C in terms of percentage of motile members. Some cultures show a slight threshhold in time before motility begins to decrease, while in others it seems to decrease from the onset. Translocation has been utilized here because it is somewhat easier to tabulate than flagellar action. However, there is a gradation between ability to move and weak ineffectual flagellar motion. In Fig. 2, for example, when motility of the

TEMPERATURE

EFFECT

OF L.

HZYif3ttii

in

I/‘&!?‘0

41

loss of activity, there is also an apparent decrease in average activity per organism. A culture at 37°C compared with another at room temperature simply seems less energetic, even before individual members are totally immobilized. Growth

and Reversibility

It would appear that the parasites at 37°C are dead, irreversibly “damaged,” or at least resemble the members of aged nondividing cultures. To test this aspect, we have studied the growth of cultures at a number of temperatures. Hemacytometer counts were used to estimate cell number. Though there is significant error, visual discrimination seemed preferable to other techniques (such as particle counting or turbidity measurements in an all-liquid medium) to distinguish the organisms from the red blood cells and other debris, and to estimate organisms per rosette. In Figs. 3A and 3B it can be seen that the cultures at temperatures above 30°C increase , * , I 01 less than cultures near twenty. At 34’C IO 20 30 40 there is continuous but slow growth, and TIME IN HOURS above this point to about 38°C the organisms FIG. 2. A tabulation of motile and nonmoMe orseem capable of approximately one division. ganisms.(a) Cells at 36.5”C; (0) cells at 24.5”C. About 250 organismswere counted for each point. At 39°C there is no further growth. It should Incubation was in Locke’ssolution with glucoseused be pointed out that, though the growth curves in the original inoculum, i.e., it was equilibrated with of the organisms at 22’ and 28°C are similar, the blood agar base. the organisms at the higher temperature showed considerable degenerative changes organisms at the elevated temperature had after 100 hours (decreased activity, loss of reached 4970, about 81% of the cells still flagella, and granules), while those at 22°C retained some motion of the flagellum or of began to show considerable “ageing” in the the anterior projection. When translocation next 50 hours. These growth patterns are not was nil at 22 hours, nearly 2% of the cells related to the presence of streptomycin or could still weakly move their flagella. Aflagelpenicillin in the growth medium, since cullate organisms in the presence of the Senekjie tures free of the antibiotics act in the same blood agar or Locke’s solution equilibrated fashion. with the agar may continue to vibrate their The question also arises if there might posstubby projections for a considerable period. In cultures in Locke’s solution without the sibly be a maximum population level which can be attained at elevated temperatures blood agar, flagellar loss is not as prominent, but paralysis with an immobile flagellum is rather than an intrinsic effect within the organism. Figure 4 demonstrates that the efmuch more common. Although the counting of percentage of fect is on the organism. By successive dilumotile organisms is one valid measure of the tions of the organism one does not obtain a final

42

GREENBLATT

AND

GLASER

323!02"C

FIG. 3. Growth curves of L. enriettZi at different temperatures. Each count is presented as a multiple of the original inoculum. A11 inocula in (A) were initially between 2.3 and 4.0 x 10” organisms per milliliter, while those in (B) were between 7.4 and 13.2 x 105 organisms per milliliter. The vertical bars indicate the standard deviations.

but instead about a 1.5 times increase in the number of organisms at 37°C no matter what the inoculum size. Therefore it can be said that increases in temperature produce an inhibition of division, but not necessarily in the first division, until the incubation temperature is above 37°C. It may be that this continued division after incubation at an elevated temperature does not represent new synthesis but only the completion of divisions already initiated. As noted earlier, a large number of duplicating forms is found in active cultures. How long is the temperature effect nondamaging to the essential growth mechanisms of the organisms? After varying lengths of incubation at 35.7 “C, organisms were returned to room temperature. Their growth curves (Fig. 5) demonstrate that for about 24 hours, at least, parasite death is not apparent; but thereafter a lag does occur, which suggests the presence of nonviable members. Supporting the presence of this early reversibility is the fact that parasites returned to lower temperatures after exposures to elevated temperatures constant

number

of the organisms,

for 24 hours seem to “bounce” back toward control values in terms of motile members and those without inclusion granules. Organisms with longer exposures do not seem capable of the same degree of reversibility. Physiological Temperature

Aspects

and Respiratory

Capacity

It seemed reasonable to examine the respiratory capacity of the organisms at elevated temperatures, since a decrease in respiratory capacity and in available chemical energy (adenosine triphosphate; ATP) could clearly explain the loss of motility and failure to divide. Studies were performed with the Warburg apparatus. Organisms were cultured at room temperature and then harvested and resuspended in fresh Locke’s solution prior to determination of respiration at different temperatures. In the experiment shown in Fig. 6, glucose was omitted from the Locke’s solution used for the growth and respiratory measurements, so that the values can be considered as those for “endogenous rate” of organisms acutely exposed to varying temperatures. One

TEMPERATURE

EFFECT

OF

L. f?Zf’iettii in

43

l’itY0

6ALL

4-

TUBES

AT 3ZO*Q5V

FIG. 6. The effect of temperature on the endogenous respiratory rate of organisms grown on Senekjie’s medium in Locke’s solution with glucose deleted. Glucose was also lacking in the Warburg flask. There were 3.3 x 10s organisms per milliliter in a volume of 0.5 ml.

I

!

observes a twofold increase from 10” to 37°C and then a very sudden drop. The values given were obtained in runs of 60-90 minutes, for at both lower and upper temperatures, the rates of respiration began to drop after longer periods in the Warburg vessel. Organisms in the standard glucose-containing Locke’s solution show a similar percentage increase in respiratory activity over this temperature range, as noted below. Respiration in the region of inactivation and lowered growth po-

I

‘Ii.-+ 1, 20

10s ’ 0

I 40

TIME

I 00

I 60

IN

HOURS

FIG. 4. Growth curves of L. enriettii at different inoculum sizes.

at 37.O”C

% 2

; 69 HOURS AT 357°C.

s

m-2'

21 HOURS AT 35.7'C.

IO5 0

/ 20

45 HOURS AT 35.7'c.

I 40

60

so

TIME IN HOURS

I 00

I20

0

20

40

60

SO

100

I20

,

1

140

IM)

TIME IN HOURS

FIG. 5. A study of recovery of growth after incubation at an elevated temperature. Tubes were returned to 24°C after 21, 45, and 69 hours at 35.7”C. In the left-hand figure, A’s indicate the culture exposed to 35.7"C.

44

GREENBLATT

tential, even at 37”C, is still higher than at room temperature. This, however, does not indicate that the higher rate is sustained at elevated temperatures. In other studies the organisms were maintained under culture conditions, at counts between IO6 and lOi organisms per milliliter, rather than near 10s organisms per milliliter. Here, because of the necessity of the prolonged harvesting technique, cells were kept at room temperature, and then transferred to 37” at varying times before harvest. Harvest was then conducted simultaneously, and each sample was counted and its respiration was measured in the Warburg apparatus at 22°C (Fig. 7) in fresh Locke’s solution. If a similar temperature coefficient of respiration (between 1.8 and 2.0) is considered and no decrease is assumed for the control, the organisms at the elevated temperature would have fallen to the level of the room temperature organisms by 10 or 11 hours, that is, for this I

I

I

I

I

I

AND GLASER

period the organisms at 37°C would be consuming more than the organisms at 22’-23’C, though their respiratory capacity is decreasing. In attempts to simplify the harvesting procedure, organisms were kept at higher population densities in Locke’s solution over the blood agar base or in Locke’s solution alone. In this way the organisms could be studied continuously and respiration could be measured at the appropriate temperatures. Unfortunately it is difficult to provide enough glucose under these circumstances for long periods of time without altering our standard growth conditions ( lo7 organisms utilize about 0.1 mg glucose per hour, under culture conditions). However, we shall have to make use of these conditions for two later aspects of the work, and will therefore take note of such experiments now. In Locke’s solution over blood agar with the organisms at a concentration of near 2.0 X IO8 per milliliter, the organisms at the elevated temperature fell to one half of their respiratory level at about 10 hours, crossing over the room temperature controls. In Locke’s solution alone the crossover occurred much earlier, at 2 hours. Over blood agar the room temperature organisms nearly maintained their respiratory capacity. In Locke’s solution alone (in the Warburg vessel) the respiratory rate of the organisms at room temperature also dropped. Values of oxygen consumption for endogenous respiration at room temperature were about 30 ~102 per hour for 10” organisms, and 200 ul in the presence of 0.25% glucose. The oxygen consumption rises to about 55 and 400 1~1for endogenous and glucose-supported respiration at 37”C, respectively. These values are close to those reported by Zeledon (1960). Studies

FIG. 7. Incubation in culture medium without prior harvesting. Individual flasks were preincubated at 37°C at varying times before their simultaneous harvest and measurement of oxygen uptake at 22°C. Cell density in the Warburg vessels was 4.0 X 10s organisms per milliliter.

of Mitochondrial

Persistence

It should also be possible to attempt visualization of the mitochondria of L. enriettii exposed to elevated temperatures. As typical mitochondria, the kinetoplasts take up Janus green very strongly (Shipley, 1916). The ma-

TEMPERATURE

EFFECT

OF L.

jor site of dye uptake is at the kinetoplast (Figs. 1H and 11). A few absorbing particles are seen along the cell periphery. Some confusion between the absorption of the dyecontaining kinetoplast and the highly refractile inclusions can be avoided by using full working apertures and a green filter to enhance the absorption image. Control cultures have fully staining kinetoplasts in about 80% of the organisms. After 24 hours of exposure to 37°C this percentage is not altered. However, there is a difference between the control and organisms at 37°C when they are observed on standing under the cover slip at room temperature. Those at the elevated temperature show relatively less destaining so that in 6 hours 68% of an elevated temperature culture showed stained kinetoplasts as compared to 22% of a room temperature culture. After 22 hours, 34% of the 37°C organisms were stained, and only 2% of the organisms preincubated at room temperature displayed stained kinetoplasts. This difference in destaining was presumably due to the more rapid utilization of oxygen by the room-tem-

I

tW’it?ttii

if2

perature organisms. The persistence of kinetoplast structure is also confirmed by electron microscopy (Wetzel) . Measurements of Inorganic Uptake

w t B

IA

B ACID LABILE

PHOSPHATE

36.82 02°C

/PELLET

z I 5 200- o ZOO] \ L I d’ 2 2 I 1 2 g I / 5 ’ looloo! 4 / 2 ; 5 s Y

/

,A+--

I 50

--A ACID LABILE PHOSPHATE

I 100

I 150 TIME

FIG. 8.

Phosphate

Radioactive phosphate (P”“) was added to Locke’s solution in the form of phosphoric acid (0.2 uc per umole). The uptake of the phosphate was followed as the inorganic and organic nucleotide phosphate. The amount of radioactive phosphate was 0.3 yc per milliliter. It was incubated with 6.0 X 10’ organisms per milliliter. The organisms were twice extracted with cold 5% perchloric acid (PCA) ; this extract was then mixed with Norit A, so that nucleotides were adsorbed, and the inorganic phosphate remained in solution. The Norit was washed with cold water, and the acid-labile phosphate was hydrolyzed free with 1N HCl and counted. One % of the total count was taken up by the organisms. Figure 8 shows (in organisms at 37°C suspended in Locke’s solution with glucose) the rapid equilibration of the PCA pool and the

8 El50

1

4.5

Vh’O

I 200 IN

I 50

I 100

I 150

I 200

MINUTES

(A) The uptake of P32 by cells at 36.8”C. 36.8”C. The values for the perchloric acid-extractable material and the acid-labile phosphate are given as specific activities (see Table I) ; the pellet counts are only relative. The first plotted point represents the initial aliquot and is placed at the midpoint of the first centrifugation period. (B) Comparison of the acid-labile phosphate specific activities at 36.8” and 22.8”C. ~28°C. The first plotted point is as described in Fig. 8A.

46

GREENELATT

slower rise in the acid hydrolyzable phosphate and the PCA insoluble phosphate of the residue. Figure 8B is a comparison of the specific activities of the cells at 23°C and those at 37°C. Note that the latter have nearly twice the rate of incorporation as the former at first, and then, much as the respiratory rates approached each other, so do the rates of phosphorylation. (For respiratory rates under these conditions, see earlier discussion of the effects of medium on the O:! consumption.) One thing not apparent in the specific activities is that there were differences in the absolute amounts of acid-labile phosphate in the cells at the two temperatures. The 23°C organisms had somewhat more acid-labile phosphate at the earlier times. These values then became nearly equal at the “crossover” time. Though the 37°C organisms had more nucleotide phosphate turnover, they had somewhat less in absolute amount. At this point, it seems fair to say that, in the immediate realm of phosphate turnover, there is no direct uncoupling of oxidative phosphorylation from respiration and no profound change in steady-state levels of nucleotide. We shall say something more of nucleotide levels later. However, we did note a chemical difference in phosphate metabolism between cells at 37” and 22”-23’C. This was brought to our attention in attempts to measure phosphate uptake of organisms suspended in Locke’s solution containing phosphate. After varying periods of incubation at 37”C, there was no net uptake of phosphate; instead, there was a loss to the external medium. Though there was no a priori reason to expect, in supposedly steady-state organisms, an increase in phosphate, neither was there reason to expect an outflow. We therefore put the organisms under a stress in regard to their inorganic phosphate. Harvested pellets were washed three times in phosphate-free Locke’s solution (0.1 M tris buffer at pH 7.2 replaced the phosphate). These cells were then suspended at a population density of 8.9 X lo7 organisms per milli-

AND

GLASER

liter and were placed at 36.8”C.

Under

these

conditions it is possible to discern an outflow of phosphate from the organisms. This is greater from the cells at the higher temperature than the cells near 22°C (Fig. 9). Inorganic phosphate is increased in the pellet of

t2 g

/ / / /368+

50

03’C

P’ /

5

If 9 i B B

a

PERCHLORIC ACID EXTUACT OF THE PELLET

60

IN THE MEDIUM

40-

P /

2 “w g

,

568’

/ 03’C

a’

JO/’

I ,*’

ZZJf

oz*c

mu* 20

0

. 50 TIME

100 IN

150

MINUTES

FIG. 9. The lower portion demonstrates the outflow of inorganic phosphate to the external medium

at 36.8”C when the organisms are in a “phosphatefree” medium. The outflow is attended by an increase in the inorganic phosphate in the perchloric acidextractable material, as seen in the upper portion of the chart.

the organisms at the higher temperatures, while the nucleotide (acid-labile phosphorus) fraction gradually decreased. In the section to follow this outflow will be further described. Retension

of Metabolites

The character of cell leakage. Once we found a prominent phosphorus efflux, it seemed appropriate to explore the possibility that a specific nucleotide could be found to be escaping (remembering that lower nucleotide levels occurred at first in the tracer study at 37’C). Figure 10 shows ultraviolet spectra of the external medium of two batches of organisms, one incubated at 37.7”C and the other

TEMPERATURE

EFFECT

OF L. L.

e?Wiettii f??WitTttiiit’2 in

TIME

vit?-0

47

IN HOURS

200 MINUTES

WAVELENGTH

IN

m/.~

FIG. 10. Ultraviolet spectra of the initial aliquot of the external medium (Locke’s solution with glucose) after resuspension of washed organisms, and then after 200 minutes at 22.2’ and 37.7”C. The inset shows intermediate points (as change in optical density at 258 rnbb) plotted against time.

at 22.2 ’ C. In this experiment the medium was of necessity simplified so that it consisted of the buffered Locke’s solution with glucose. The organisms were washed four times, prior to the beginning of the experiment, so that no hemoglobin and little residual ultraviolet absorption was noted in the final washings. It can be seen that, though the 22°C organisms show some increase in absorption, it is at a lower level than that observed in the medium of the 37°C organisms. The inset of Fig. 11 gives other intermediate values in this experiment. If the material in the supernatant represented gross lysate of the organisms, some protein might be expected to be present. Both trichloroacetic acid and perchloric acid fail to show a precipitate. Biurette reactions with the pellet show no decrease or only a slight one when compared to the control. When the organisms which have been incubated between 37” and 40°C are extracted with 5% perchloric acid, there is a general correlation between

the decrease in optical density of the perchloric acid-extractable materials and the increase in adsorption in the external medium. However, at other temperatures the relationship is a complex one. At 30” and 23°C there is actually an increase in optical density of the perchloric acid extract, which probably represents hydrolysis of the structural proteins and nucleic acids, in the face of limiting permeability. Small amounts of hydrolysis may show relatively large changes in optical density due to the increased absorption of monomers compared to polymers. Finally, at 12°C there is a decrease in the perchloric acid-extractable material beyond that accountable for by leakage. This probably represents utilization of endogenous metabolites. Microscopic examination revealed that the organisms at 37” and 39°C are totally inactive in a few hours, while at lower temperatures (lo”-18°C) the organisms are in good condition after 20 hours and longer. Our analysis of the material which leaks is

48

GREENBLATT

AND

GLASER

FIG. 11. (A and B) : Semilogarithmic plots of the leakage data presented as a decrease in original absorption. The ultraviolet absorption of the whole organisms is taken as the zero time concentration. For comparison to the original data, see the Fig. 10 inset. (C): Results of kinetic experiments plotted as the logarithm of the slope versus the reciprocal of absolute temperature (Arrhenius plot). The circles and triangles represent two different experiments and ten separate runs. The activation energy calculated from this data is about 18,000 calories per mole. This value is probably too high for simple diffusion data. Data for respiration from Fig. 6 are plotted in the same way in the lower curve.

not yet complete. However, passage of this external medium through cation and anion exchange columns separates the 258 rnp absorbing material into three fractions: (a) A neutral fraction: about 205% of the total ultraviolet-absorbing material; it passes directly through a Dowex-1 formate form anion exchange column and a Dowex-50 hydrogen form cation exchange column. (b) An anionic fraction: it comprises about 257” of the total ultraviolet-absorbing material, and since it is adsorbed to the Dowex-1 column, it requires molar formate for elution; even then about 5% is left behind. This material once eluted passes through the Dowex 50. (c) A cationic fraction: it comprises some 50% of the total ultraviolet-absorbing material, and it passes directly through the anion exchange resin but is retarded by Dowex 50. Four M NH,OH is required for its complete elution. Table I lists our present findings regarding the contents of these fractions. So far we have

found that a considerable number of amino acids accompanies the nucleic acid bases in the effluent from the organisms. At least thirteen amino acids can be identified by electrophoresis, as well as one unknown ninhydrinpositive compound. Furthermore, ribose and possibly a uranic acid and several other sugars are also present. Thus we are dealing with a leakage involving several classes of chemical compounds. Later we shall consider whether it represents a gross lysis of the cells or something less. The kinetics of leakage. Some insight into the nature of the leakage should come from a consideration of the kinetics. There are some difficulties: for example, if we cannot exactly account for the loss of the material in the PCA fraction, it is hard to choose an appropriate zero time concentration. Whether one uses the absorption of the intact cells in the ultraviolet or the PCA-extractable fraction seems to make little difference in the relative rates. The former is used in the experiments presented in Fig. 11. The slopes are

TEMPERATURE

EFFECT

OF

TABLE Analysis Fraction Anionic

Cationic

Neutral

Identified

L. eWit?ttii in Vitro

49

I

of Escaping Materials

compounds

Chromatographic or electrophoretic system

Guanosine

Pabst System I, and Paladini and Leloir (1952).

Ribose

Phenol, and butanol:pyridine:water

Hypoxanthine

Pabst System I, and H,O at pH10, Levenbook (1955)

Amino acids (aspartic, threonine, serine, glutamic, glycine, alanine, phenylalanine, valine, leucine, isoleucine, tyrosine, arginine, and lysine)

Electrophoresis, varsol as organic phase. Formate and borate buffers.

Uracil

Pabst System I, and Paladini and Leloir (1952)

nearly linear when the logarithm of the remaining material is plotted against time. This exponential process would be expected in a first order reaction. When the log of the rate is plotted against the reciprocal of the absolute temperature, a straight line is obtained. This Arrhenius plot seems to indicate that what we are seeing in a prominent fashion at the higher temperatures occurs over the entire temperature range, and does not represent a sharp breakdown in function, as one might interpret the failure in respiratory capacity above 37’C [Fig. 11C; see von Brand (1952) for treatment of respiratory data in this fashion]. Is the leakage metabolically linked? If glucose is omitted from the Locke’s solution, leakage is increased about twofold; if one tenth of the glucose is replaced the leakage is cut back by about 50%. If one plots respiratory rate against leakage, a general correlation exists. DISCUSSION

We have attempted to survey some of the events occurring when L. enriettii is exposed to elevated temperatures. Several suggestions can be seen in this work that may help to delineate the mechanism of temperature inactivation.

(6:4:3)

Above 38°C the dramatic cut-off in respiration would apparently be the cause or effect of sudden death. The sharp fall-off probably is due to a breakdown of protein structure. In the remainder of this discussion, we shall limit ourselves to the region between approximately 30” and 37°C where death is not sudden but viability is impaired. The experiments presented make it seem doubtful that an uncoupling of oxidative phosphorylation, such as is known to occur in isolated mitochondria at 37°C (Weinbach, 1959), would explain the degeneration that becomes prominent above 30°C. Respiration, oxidative phosphorylation, and kinetoplast structure all seem to persist to 37°C. There are several hints that hydrolytic events may be important at elevated temperatures. The increase in inorganic phosphate suggests the presence of phosphatases, while the occurrence of hypoxanthine would probably argue for an adenine deaminase. These two instances, along with the increase in the perchloric acid-extractable substances, make it likely that a general increase in hydrolytic activity occurs in the organisms at elevated temperatures. The subcellular particle called the lysosome, described by de Duve

50

GREENBLATT

(1959), has been found to contain an entire battery of hydrolytic enzymes. It is interesting that at 37°C and a pH of 5, the lysosome enzymes are activated (de Duve, 1959). In future studies we will attempt to assay enzymic activity in these cells. We have stressed the pronounced leakage that these organisms display with increasing temperature. We do not think that it simply reflects gross lysis of the cells. Electron and phase microscopy do not reveal gross cell lysis, although the cells seem somewhat fragile on air-drying. Moreover, the straight line Arrhenius plot suggests that leakage occurs at temperatures from 10” to 40°C without any drastic alteration observed only at elevated temperature. Nor does it seem that the leakage only reflects the increased hydrolytic activity at elevated temperature. The kinetics of the latter are too erratic while the former are too uniform. It could be argued that enzymic inactivation might play a role, but this would of necessity require inhibition of enzymes in several areas of metabolism since the leakage is of such a general nature. This would be the case unless permeability itself were directly related to a temperature-sensitive enzyme. Such an enzyme that “keeps things in” would of necessity have its temperature optimum at temperatures near lO”C, and this seems unlikely. This does not imply that metabolic processes and leakage are not tied together, or that dead organisms are not more permeable than live ones. Thus we have been studying cell death (or inactivation) and cell permeability; the question is which comes first? Over the temperature range from 10” to 40°C, leakage increases nearly thirtyfold, while respiration only doubles. Figure 12 demonstrates that there is a wider and wider divergence between these two functions. The respiratory increase can possibly supply sufficient metabolic energy at lower temperatures, but higher temperatures may call upon a new mechanism. Though it is dangerous to extrapolate from artificial culture conditions

AND GLASER

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/ LEAKAGE

$240 G w200 1 2 5 1601 I P 2 120; a $

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FIG. 12. .4 linear plot of the slopes of respiration and leakage, both set at 1.0 at 10°C.

which here are far too simplified, this mechanism may be the dependence on the intracellular mode of existence. In L. enriettii, the intracellular environment might provide sufficient enrichment up to near 36”C, where the upper survival limit in viva is reached (de Castro and Pinto, 1960). Indirect evidence for this may be the observation in our respiratory studies, that the more complex the medium the more resistant the organisms. Above 38°C where the gross defects in the respiratory mechanisms seem to occur, the uppermost limit for survival under any conditions would be reached. There is, after all, nothing mysterious about leakage. As we have seen, it occurs at all the temperatures and varies only in degree. While organisms exist that are thought to be impervious, other organisms are known to require special co-factors because of their inability to retain them. Moulder (1962) has discussed Bovarnick’s studies (1953, 1957a,b) of the rickettsia in this framework. The rickettsia retain their viability when diphosphopyridine nucleotide, glutamic acid, and other factors are supplied at 34°C. Metabolites will tend to flow down the concentration gradient when-

TEMPERATURE

EFFECT

ever they are not trapped by structural or metabolic means. If the host cell (or the parasitologist) provides the substances in a high enough external concentration, no disadvantage would ensue and no net efflux will be measured; in fact, exchange might be greatly increased. As Moulder (1962) has pointed out, an increased permeability would be an advantage to the intracellular organism. Thus while our experiments have put the organisms at an extreme disadvantage for retaining metabolites, a relatively impervious cell membrane would offer no advantage within the enriched host cell environment. ACKNOWLEDGMENTS I would like to acknowledge the hospitality vf Dr. Alfonso Trejos and the Faculty of Medicine of the University of El Salvador. The kindness of Dr. Leo Levenbook made possihle the examination of the electrophoretic patterns of the amino acid samples. I am grateful to Dr. Bruce Wetzel for showing interest in the electron microscopic aspects of this problem. REFERENCES ADLER, S., AND HALFF, L. 1954. Observations on Leishmania enriettii. Muniz and Medina, 1948. Annals of Tropical Medicine and Parasitology 49, 37-41. BAKER, A. C. AND GUTIERR~Z BALLESTEROS,E. 1957. Tratamiento experimental de 10s ulceras Leishmaniasicas por eI procedimiento de1 calor de1 vapor. Rev&a Del Instituto De Salubridad y Enfermadades Tropicales 17, 115-119. BOVARNICK, M. R., ALLEN, E. G., AND PAGAN, G. 1953. The influence of diphosphopyridine nucleotide on the stability of typhus rickettsiae. Journal of Bacteriology 66, 671-675. BOVARNICK, M. R., AND ALLEN, E. G. 1957a. Reversible inactivation of typhus rickettsiae at 0°C. Journal of Bacteriology 72, 56-62. BOVARNICK, M. R., AND ALLEN, E. G. 1957b. Reversible inactivation of the toxicity and hemolytic activity of typhus rickettsiae by starvation. Journal of Bacteriology 74, 637-645. BRAY, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions. Analytical Biochemistry 1, 279-285. CRANE, R. K., AND LIPMANN, F. 1953. The effect of arsenate on aerobic phosphorylation. The Journal of Biological Chemistry 201, 235-243.

OF L.

tW’if?ttii i?Z

I/‘itYO

51

DE CASTRO,M. P., AND PINTO, S. C. 1960. Influence of the temperature on the growth of Leishmania enriettii in tissue culture. Journal of Protozoology 7 Supplement, abstract 10. DE DUVE, C. 1959. Lysosomes, a new group of cytoplasmic particles. In “Subcellular Particles” (T. Hayashi, ed.), pp. 128-159. Lord Baltimore Press, Baltimore, Maryland. GOMORI, G. 1942. A modification of the colorimetric phosphorus determination for use with the photoelectric calorimeter. Journal of Laboratory and Clinical Medicine 27, 955-960. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. 1949. Determination of serum proteins by means of the biuret reaction. The Journal of Biological Chemistry 177, 751-766. GUIMAR;\ES, J. P. 1952. ObservacBes sobre a leishmaniose do cobaio, Manquinhos, Boletim do lnstituto Oswald0 Cruz I, 4-5. GUTIERRU BALLESTEROS,E. 1959. Tratamiento de la leishmaniasis tegumentaria por medio de1 calor de vapor de agua, estudio de cinco cases humanos. Revista Del Institute De Sulubridad y Enfermadodes Tropicales 19, 317-328. LEVENBOOK, L. 1955. As cited by Wyatt, G. R. in Separation of nucleic acid components by chromatography on filter paper. In “The Nucleic Acids” (J. N. Davidson, and E. Chargaff, eds.), Vol. I, pp. 243-256. Academic Press, New York. MEDINA, H. 1946. Estudios s6bre leishmaniose. I. Primeiros cases de leishmaniose espontbnea observados em cabLios. Arquivos de Biologia e Tecnologia I, 39-74. MOULDER, J. W. 1962. “The Biochemistry of Intracellular Parasitism,” pp. 43-81. Univ. of Chicago Press, Chicago, Illinois. MUNIZ, J., AND MEDINA, H. 1948. Leishmaniose tegumentar do cobaio. 0 Hospital, Rio de Janeiro xXx111, 7-25. NEVA, F. A., MALONE, M. F., AND MYERS, B. R. 1961. Factors influencing intracellular the growth of Trypanosoma cruzi in vitro. The American Journal of Tropical Medicine and Hygiene 10, 140-154. ORMEROD, W. E. 1958. A comparative study of cytoplasmic inclusions (volutin granules) in different species of trypanosomes. Journal of General Microbiology 19, 271-288. PALADIM, A. C., AND LELOIR, L. F. 1952. Studies on uridine-diphosphate-glucose. Biochemical Journal 51, 426-430. PARAENSE, W. L. 1952. Infection of the nasal mucosa in guinea pig leishmaniasis. An& da Academia Brasileira de C&z&s 24, 307-310. PEREIRA, C., DE CASTRO, M. P., AND DE MELLO, D. 1958. As cited in DE CASTRO AND PINTO, 1960.

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H. A. 1943. Biochemical reactions, cultural characteristics and growth requirements of Trypanosoma cruzi. American Journal of Tropical Medicine 23, 523-531. SENEKJIE, H. A., AND ZEBOUNI, N. 1941. Biochemical reactions of the genus Leishmania. American Journal of Hygiene 34, Sec. C., 67-70. SHIBATA, K., BENSON, A. A., AND CALVIN, M. 1954. The absorption spectra of suspensions of living micro-organisms. Biochimica et Biophysics Acta 15, 461-470. SHIPLEY, P. G. 1916. The vital staining of mitochondria in Trypanosoma lewisi with Janus Green. Anatomical Record 10, 439-445. TRACER, W. 1953. The development of Leishmania donovani in vitro at 37°C. Effects of the kind of serum. Journal of Experimental Medicine 97, 177-188. SENEKJIE,

AND GLASER GODOY, G., GREENBWTT, C., AND CER. 1963. Effects of temperature on morphological variation of Schizotrypanum cruzi in tissue culture. Experimental Parasitology 13, 211-218. VON BRAND, T. 1952. “Chemical Physiology of Endoparasitic Animals,” pp. 155-157. Academic Press, Xew York. WEINBACII, E. 1959. Stability of oxidative phosphorylation and related reactions in isolated liver mitochondria. The Journal of Biological Chemistry 234, 1580-1586. ZELEDON, R. 1960. Comparative physiological studies on four species of hemoflagellates in culture. I. Endogenous respiration and respiration in the presence of glucose. The Journal of Protozoology 7, 146-150. TREJOS,

A.,

DILLOS,