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Regulation of cell membrane permeability in Trypanosoma lewisi
EXPERIMENTAL
PARASITOLOGY
18,
231-243
Regulation
(1966)
of
Cell
Membrane
Trypanosoma
Permeability
in
lewid
H. G. du Buy Laboratory
of
Biology
of
Viruses,
National Bethesda,
Institute Maryland
of Allergy
and
Infectious Diseases,
and
Metabolic
C. L. Greenblatt Laboratory
of Physical
Biology,
National Institute Bethesda, Maryland
of Arthritis
Diseases,
J. E. Hayes, Jr. Laboratory
of Technical
Development,
National
Heart
Institute,
Bethesda,
Maryland
and D. R. Lincicome Howard (Submitted
University,
Washington,
for publication,
14 June
D. C. 1965)
Du BUY, H. G., GREENBLATT, C. L., HAYES, J. E., JR., AND LINCICOME, D. R. 1966. Regulation of cell membrane permeability in Trypanosoma lewisi. Experimental Parasitology 18, 231-243. Fluorescent properties of tetracycline (TC) were used to study permeability changes of T. lewisi in various surroundings under phase and fluorescence microscopy. Mitocbondria and the mitochondrial elements of the kinetoplast fluoresced bright yellow under fluorescence microscopy when tetracycline entered the cells. Survival time of the organisms in saline was measured in hours, and in serum in days, either with or without TC. Survival time was proportionally shortened under conditions of fluorescence microscopy. This proportional shortening of the survival times was dependent on penetration of TC and the effect of the light necessary for fluorescence microscopy. The cells in saline became immediately visible and died rapidly, while those in serum became visible within an hour, followed by death. Serum serves as a protective permeability-regulating coat around the parasites. Thus, permeability changes which required hours or days under normal conditions could be measured in minutes with tetracycline. The protection afforded by different serum fractions of rabbit and rat sera was also measured. The beta globulins, and, to a lesser degree, the albumins, prevented the penetration of tetracycline. The presence of a serum barrier was also demonstrated by washing serum-coated organisms, by the use of fluorescein-labeled serum, and by leaching experiments. Anti-T. lewisi-antiserum does not prevent tetracycline penetration. The nature of the serum barrier and the probable role of the protective lipoproteincontaining serum fractions is discussed and their significance regarding chemotherapeutic studies pointed out.
culture, as well as the mitochondrial memTetracycline (TC) penetrates the cell mem- brane, and accumulates in the mitochondria brane of higher animals in vivo and in tissue (du Buy and Showacre, 1961). It is unable to penetrate the nuclear membrane of living 1 Supported in part by Research Grant No. AIcells, or the vacuolar wall surrounding intra03409 from the National Institutes of Health to D. R. cellular bacteria and certain intracellular Lincicome. INTRODUCTION
231
232
DU BUY,
GREENBLATT,
HAYES,
AND
LINCICOME
200 X g. The pellet was again suspended and centrifuged as before. The three supernatants, all rich in parasites, were combined. This suspension, which contained the Alsever-diluted serum of the first supernatant and the Alsever-Locke’s mixture of the two others, served as stock suspension. Immediately before use, a given amount of stock suspension was recentrifuged for one minute in 1.5 ml-centrifuge tubes in an International Model MB microcapillary centrifuge The pellets were resuspended in Locke’s solution with dextrose. Equal amounts of this suspension were dispensed into the small centrifuge tubes, recentrifuged for 1 minute, and the pellets resuspended in the various media under study to the volume of the original aliquot portion. For microscopic observation, samples were removed by capillary pipettes and placed on microscope slides with cover slips. Sera and serum proteins. Blood was obtained by heart puncture of anesthetized rats or rabbits, and the blood allowed to clot for a MATERIALS AND METHODS few hours at room temperature. The serum for 10 Test organism. Osborne-Mendel rats was collected after centrifugation (N.I.H. strain, randomly mated in closed minutes at 2000 X g, and either used fresh, or, if necessary, kept frozen (- 25” C) until colony) were inoculated intraperitoneally with 1 ml of a suspension of washed T. lewisi con- use. Only in the case of mice was blood obtaining approximately 3 X IO6 cells. One tained by decapitation. Rabbit serum fractions were obtained comweek later blood was collected from these rats by heart puncture in one-fifth its volume of mercially.4 The rat serum fractions consisted of (1) the albumins and globulins represented Alsever solution.” The suspension was centrifuged for 10 respectively by the supernatant and the pellet of serum treated with 50% (NH4)$04, and minutes at about 200 X g and the superna(2) the gamma globulins which were the first tant, containing the organisms, removed. The fraction obtained by passing serum through a pellet was resuspended to the original volume, DEAE-cellulose column (Sober and Peterson, with a one-to-one mixture of Alsever solution and Locke’s buffered glucose-salt solution3 1958). Tetracycline. A stock solution, made fresh and again centrifuged for 10 minutes at about daily of TC HCI (Lederle) , 1 mg per milli3 Alsever sol., per liter: dextrose, 20.5 gm; Na liter Locke’s solution, was adjusted to pH citrate 2H,O, 8 gm; NaCI, 4 gm; citric acid, O.SS 6.8-7.2 with KOH. gm. Tetracycline binding by serum. Measure8 Locke’s sol. with dextrose, per liter: NaCI, 8 gm; KCI, 0.2 gm ; Na2HP0,, 0.4 gm; KH,PO,, 0.3 gm; ments of binding of TC to serum proteins
parasites sufficiently to be observed by fluorescence microscopy (du Buy et al., 1964). The observation that Trypanosoma lewisi in serum did not fluoresce when TC (20-100 pg per milliliter) was added, whereas those in saline were almost immediately visible, suggested the use of TC and fluorescence microscopy as tools for a study on T. lewisi, where the host environment is serum, and the formation of a protective coating around a parasite could be studied in an extracellular environment (du Buy et al., 1963a,b). This paper presents an analysis of some factors in both homologous and heterologous sera, which, on the basis of observed differences in TC penetration, are responsible for the survival of the parasites outside the host. Preliminary accounts of this work were presented at the 1963 annual meetings of the American Society of Parasitologists (du Buy et al., 1963a) and the Third Annual Meeting of the American Society for Cell Biology (du Buy et al., 1963b).
CaC12, 0.2 gm; dextrose, sterilized separately.
2.5 gm;
CaCl,
and dextrose 4
The Mann
Research
Laboratories,
Inc.
TRYPANOSOME
MEMBRANE
PERMEABILITY
233
were done by dialysis equilibrium followed by 200 mercury-xenon arc. It was used with two analysis of the dialysand and dialysate for 3-mm Schott BG12 glass filters as primary TC. Two-ml portions of various dilutions of filter, and a l-mm Schott OG5 glass filter as sera in Locke-Ringer saline were placed in barrier filter. Visking casing and dialyzed for 12 hours Photography. Photographs were taken on against 40-ml portions of TC solution in Adox KB17 film or on Kodak Royal Pan (4 X 5 sheets), both developed in Acufine at Locke-Ringer saline in glass tubes with Teflon-lined caps on a rocking dialyzer. At the room temperature. end of this equilibration period, the dialysis RESULTS casings were removed, blotted dry, and opened. The TC contents of both the inner Preliminary studies for the establishmentof indices of the degree of viability of the orgaand outer solutions were determined by the nisms under varying conditions. By fluoresmethod of Hayes and du Buy (1964). The excess TC in the inner solution over the con- cence microscopy, T. lewisi suspended in centration present in the outer solution was serum with 200 ug TC per milliliter could be detected only by the motion of fluorescent regarded as due to the binding by the serum present. particles in their immediate surroundings, Dialyzed serum. Rat or mouse sera were causedby the movement of the parasites, In dialyzed for 24 hours at 4°C against 3-4 saline, 200 ug TC per milliliter caused suffichanges of either saline or Locke’s solution on cient accumulation of the antibiotic in the a magnetic stirrer, and kept frozen until used. organismsto make them almost immediately Leaching of intracellular material. Washed appear brightly yellow fluorescent against the T. lewisi cells were suspended in Locke’s solu- black or faintly yellow background. The irtion minus glucose, divided into equal ali- radiation itself increased the permeability of the drug as indicated by an increase in tluoquots, centrifuged, and the pellets resuspended in 2.5 ml of varying concentrations of rescent intensity. (For further discussion of normal serum, antiserum, or Locke’s solution. this point, seep. 239.) The fluorescent image The number of organisms was measured either showed no specific cell wall staining (Fig. 1, by counts in a Levy chamber, or volumetriA,). Yellow dots were seen, corresponding to cally by the use of the capillary centrifuge (a the granular elements observed under phase lo/o volume of organisms corresponds to about (Fig. 1, A1), and representing, by analogy 5 X 10’ organisms per milliliter). After variwith vertebrate studies, TC-stained mitoous time intervals, the organisms were centrichondria (du Buy and Showacre, 1961). A fuged in 70 X 7-mm test tubes in a capillary dark area indicated the location of the nucentrifuge and the UV absorption of the cleus. The region of the kinetoplast showedas supernatant at 260 mu measured with a Cary a dark vacuole with a yellow crescent, prespectrophotometer, Model 14. The material sumably representing the equivalent of its released to the medium was read as the optimitochondrial and its nucleic acid material cal density difference, 260-360 mu. (Clark and Wallace, 1960). Apparently the Microscopy. Microscopy was done with a TC penetrated through the cell membrane Zeiss W microscope, which could be used and accumulated in the mitochondria and in either with a tungsten light source for phase the mitochondrial elements of the kinetomicroscopy, or with a filtered mercury arc plasts. Even after death (Fig. 1, B1, B,) and light source for fluorescence microscopy as vacuolation (Fig. 1, B3) of the organism, described elsewhere (du Buy and Showacre, these elementscould still be recognized. 1961). Trypanosomes, suspendedin salt solutions The arc source used was an Osram HBO deteriorated in a few hours, but in serum they
234
DU
BUY,
GREENBLATT,
HAYES,
FIG. 1. Al, T. Zewisi in 200 pg of TC in saline observed fluorescence microscopy. Bl, dead organism in 200 pg of TC B3, vacuolation after death.
for days (Table I, column 2). The addition of TC in both cases did not alter these findings. However, when observed under the conditions of fluorescence microscopy, in the presence of TC their survival in both saline and serum was shortened, but proportionately so. In saline the organisms became
survived
AND
LINCICOME
by phase by phase;
microscopy; AZ, same organism by B2, same organism, by fluorescence;
visible in a few seconds, in serum within an hour. Apparently the considerable damage done by maintaining the organism in saline and the gradual deterioration in serum were proportionally accelerated by the photochemical damage. The photochemical damage, in its turn, appeared directly correlated with
TRYPANOSOME
and Tetracycline
Survival
MEMBRANE
TABLE I of Trypanosoma lewisi in Rat and Mouse
Fluorescence
Absence of TC
Seruma
Presence of TC
Phase microscopy 50% survival time Medium
235
PERMEABILITY
Fluorescence microscopy Intensity
37” (hr)
25”
I To
Tbnx
Movement at T, Trans.
1%ax
50% Survival time
Undul.
25”
(set)
100%
5 days
-
-
4+
30
2+
2+
Ifr
25
4+
2+
0
30
3+
0
1+ to4+
2.5
min
0
4+
35 min
to 4+
4+
12.5
to 4+
3+
RS 5 days
10%
RS 1% RS 0.1% RS 100% MS 10% MS 1% MS 0.1% MS Locke &D Locke Saline
24
hr
to 4+
min
6 min
4
hr
ca 8
3
days
-
-
4+
4+
>
12
min
ca 8
3
days
3.5
2+
2+ to4+
4+
>
10
min
hr
20
3+
0
3+
10
3+
0
2+
4
24
1
8 hr
0
ca 1
ca 6 hr
1.5
3+
0
1+
ca4hr ca 2 hr
10 10
2+ 3+
0 0
lf 1+
to 4+
5 min 90 set
to 2+
10 set 10 set 10 set
a RS: rat serum; MS: mouse serum; Locke & D: Locke’s solution with dextrose; ITo: intensity of fluorescence at zero time, graded from 0 to 4+; TImax: time of maximal fluorescence; Lr max: intensity of fluorescence at TImax; Transl.: translocation, and Undul.: undulation at zero time, graded from 0 to 4+. Last column: 50% survival time under continuous fluorescence irradiation. Each determination is based on at least two observations.
the degree of damage to the parasites under various conditions: (1) degree of translocation, scored from 4 + (maximum translocation, with wave motion of flagella) to 0 (no intensity constituted a measureof membrane translocation) ; (2) degree of mobility of the permeability to this drug. After onset of flagellum: undulation 4 + (whipping of death, the fluorescence decreased again. By flagellum) to 0 (immobile) ; (3) adherenceto this procedure, permeability changes due to a glasssurface and clumping; and (4) vacuchanges in the environment, which might olation (often sudden) and death (no moverequire hours or days to become apparent ment, increasein refractive index) (Fig. 3,B). Decreasing values of these indices paralunder ordinary conditions, were measuredin leled the degreeof increaseof permeability to minutes by fluorescencemicroscopy. The above observations led to the establish- TC, as observed by the intensity of fluoresment of arbitrary standards, by the scoring cence of the organisms,and thus served as an of four criteria which were used as indices of index for the permeability of other substances, the rate of accumulation of the antibiotic in the organism. As judged by fluorescent intensity, this rate of accumulation was rapid in saline, slow in serum. Thus, the fluorescent
236
DU
BUY,
GREENBLATT,
HAYES,
which possess permeability characteristics comparable to those of TC. By the above criteria, the effect of various sera and their fractions on TC penetration was studied. Effect of rat serum concentration on survival and fluorescence. In this and the subsequent experiments the organisms were prepared as described under Methods. The protective effect of 4 concentrations of rat serum was compared with that of Locke’s solution with and without dextrose. The results are summarized in Table I. The first two columns show that the 50% survival time decreases, both at 37’ and 25’C, with decreasing serum concentration. A comparison of the last column with the first two shows that protection against light damage in fluorescence microscopy decreases in the same order as protection against death on standing. From columns 3-7 in Table I, it can be seen that in decreasing serum concentrations in the salt solution the parasites show more and more fluorescence, concomitant with decreasing mobility, and death. The effect of 0.1% serum is about the same as that obtained in Locke’s solution with dextrose. Thus, the protective effect of rat serum decreases as the serum concentrations decrease.
.4ND
LINCICOME
Eflect of mouse serum. The effect of mouse serum was studied similarly to that of rat serum (Table I). Both sera supplied a similar permeability barrier against TC to the parasite. Thus, the factors for survival are present in heterologous as well as homologous sera. It should be remembered, however, that the factors for reproduction are presumably not present in mouse serum (Lincicome, 1958 ; Lincicome and Francis, 1961) . The effect of dialyzed rat and mouse serum. The protective effect of dialyzed rat and mouse serum was studied as described above. Normal as well as dialyzed serum, from either rat or mouse, increased the survival time under irradiation with filtered mercury arc light used for fluorescence microscopy. In these sera the organisms survived for more than 20 minutes, concomitant with the presence of a permeability barrier against TC, as compared with a survival time of 3 minutes in Locke’s solution. Thus, the length of the survival time in the dialyzed serum as well as the failure of TC to penetrate show that in this respect the difference between dialyzed and undialyzed serum is inapparent. From this it can be concluded that a considerable amount of the dialyzable small-molecule com-
60 t
PERCENT
FIG.
horse
2. Tetracycline serum. mouse,
n
(100 @g per A horse, 0
milliliter) rat.
RAT,
binding
MOUSE
AND
by
HORSE
different
SERUM
concentrations
of
rat,
mouse,
and
TRYPANOSOME
MEMBRANE
PERMEABILITY
237
FI G. 3. A , T. lewisi in fluorescein-labeled rat serum by phase microscopy. B, The organism appears as a dark shad ow against the fluorescent background of the tagged serum.
Panents of rat serum is nonessential to the 9x7rival the parasites. ‘he t?zect of serum dialysate. Organisms (31 < 10B I?er milliliter) washed in Locke’s solu We :re added to a dialysis bag containing 5 ml of Locke’s solution, and the bag sus-
pended in 5 ml of rat serum. One ml of the suspensionof organismswas added to a test tube containing the 24-hour dialysate in Locke’s solution from an equal, nonrenewed volume of rat serum. In either case, the survival time of the or-
238
DU
BUY,
GREENBLATT,
ganisms was increased considerably as compared with that in Locke’s solution. After 4 hours the controls were dead; those in the dialysis bag showed about 5% dead organisms, and those in the dialysate, 10%. After 24 hours those in the dialysis bag still showed 5% deaths, those in dialysate, 50%. It is apparent that the serum continues to supply necessary factors, whereas the dialysate becomes depleted in time. When studied for fluorescence, the organisms from the dialysis bag became visible after 90 seconds, those in dialysate after 30 seconds. The dead organisms were immediately visible in either case. Apparently, the dialyzable factors did not supply a permeability barrier to TC as did the nondialyzable portion. The increased survival time could be explained by assuming that factors from the dialysate temporarily counterbalanced the loss of these factors from the test organisms, brought about by the absence of the protein barrier. Serum protection. Two possibilities were considered in order to explain the absence of visible fluorescence of the trypanosomes in various sera: ( 1) The TC binding by serum was sufficient to lower the concentration of free TC available to the parasites to a point that fluorescence could not be observed. In that case, fluorescence should be observable, if the TC concentration were increased to a level at which free TC would be available. (2) The nature of the serum protection is such that TC cannot penetrate the organisms. A series of experiments was performed in order to choose between these possibilities: ( 1) Binding of tetracycline by serum. The first series was designed to determine whether the TC binding by serum was dependent on the concentration of TC. The binding of TC to rat, horse, and mouse serum was studied by the dialysis equilibrium technique. At concentrations between 100 and 300 ug TC per milliliter, it was found that the fraction of TC bound to horse serum was a function of the serum concentration but not of the TC concentration (Fig. 2). Accordingly, all sub-
HAYES,
AND
LINCICOME
sequent binding measurements were made at a total TC concentration of 100 ug per milliliter. Rat and horse serum were used at serum concentrations of 0, 10, 20, 50, and loo%, and mouse serum at 0, 20, and 100%. It can be seen from Fig. 2 that the TC binding increased with increasing serum concentration, with a maximum binding of 50-60s by the undiluted sera. Similar results were obtained by Wozniak (1960) for human and dog plasma. Thus, at a concentration of 100 ug per milliliter of TC, in undiluted sera, the concentration of unbound TC is about 40 yg per milliliter, or on the basis of the results presented in Fig. 2, 80 ug per milliliter when the TC concentration is 200 ug per milliliter. This, then, is the concentration of TC immediately available to the organisms. These concentrations are sufficiently high to observe fluorescence of the organisms, if it were taken up by them. Another series of experiments was performed to determine whether the TC concentration in serum could be increased to a point that the serum protection was no longer effective. To this end, organisms were washed two times in Locke’s solution without dextrose and resuspended in Locke’s solution with 40, 80, and 120 u.g of TC per milliliter, and in 100% rat serum, with 100, 200, and 300 and 600 pg of TC per milliliter containing about 40, 80, 120, and 240 ltg of unbound TC per milliliter (Fig. 2). The organisms were visible in Locke’s solution at all TC concentrations (even in 40 pg of TC, the visibility was f within 10 seconds). The organisms remained invisible in serum, except as black shadows against the fluorescence of the serum. Apparently, although sera bind a certain amount of TC, the remaining unbound TC, even at high concentration, is unable to penetrate the organisms protected by the serum. These results, thus, support the second alternative mentioned above (this page) : the nature of the serum protection is such that TC cannot penetrate the organisms.
TRYPANOSOME
MEMBRANE
(2) Serum coating. The second series of experiments was performed to determine whether serum could be shown to coat the cells, thus producing a TC barrier. A trypanosome suspension was twice washed with Locke’s solution with glucose to remove the serum, as indicated by stainability with TC. The organisms were then resuspended in normal rat serum conjugated with fluoresceinisothiocyanate (tagged serum) and exposed to it for 20 hours in order to allow a thorough coating with the tagged serum. In the first experiment, organisms suspended in the fluorescein-labeled serum could, under fluorescence microscopy, be recognized as dark shadows against the fluorescent background of the tagged serum (Fig. 3). In the second experiment, the organisms pretreated with tagged serum as above were washed once with Locke’s solution and immediately resuspended in nontagged serum, in order to prevent furt,her removal of tagged serum. Observation by fluorescence microscopy revealed the organisms as mobile fluorescent outlines against the nonfluorescent control serum (nontagged organisms remained invisible). In the third experiment, the pretreated and washed organisms were immediately resuspended in saline. These organisms were only slightly visible for a few seconds: the fluorescein serum was not retained sufficiently to show a clear fluorescent outline. Apparently the “coat” washed off, and the organisms behaved as those washed in saline: they adhered to the glass surfaces. Another experiment, not involving tagged serum, was performed to show that the serum coat was readily removable. Saline-washed trypanosomes were exposed for 60 minutes to whole rat serum with 200 ug of TC per milliliter added, and then washed twice in Locke’s solution with glucose and TC. They were observed immediately after the washes. Fluorescence microscopy showed that these organisms behaved as those in 1% serum, i.e., they were invisible for l-2 minutes, and slowly, while
PERMEABILITY
239
losing their mobility, they became more and more visible. Apparently, the serum pretreatment only temporarily prevented the penetration of the TC into the organisms, and this serum coat was readily removable by washing, which allowed the TC to enter, deteriorating the organisms. The nature of increasing fluorescence of T. lewisi during irradiation. The increase of fluerescence observed during irradiation, either in low concentrations of serum or in Locke’s solution, could be explained if the Locke’s solution in conjunction with the radiation damage increased the permeability of the organisms, gradually allowing the TC to enter. The following experiment was performed to test this explanation. The organisms were exposed for a few hours to TC in 1% rat serum in order to allow TC to be taken up. ‘They were then washed and resuspended in salineglucose devoid of TC. Placed under the fluorescence microscope, the initial fluorescence was seen to decrease, rather than to increase, following irradiation. Apparently, without an external supply of TC no increase of fluorescence can occur. The effect of serum fractions. In order to determine which fraction of the serum afforded most protection, some serum fractions were investigated. Experiments were performed with rabbit serum and its fractions, which were commercially available, namely, serum dialysate, serum albumin (fraction 5)) and alpha, beta, and gamma globulin fractions. Experiments were also performed with rat albumin, total globulins, and gamma globulin. As controls, rat serum, Locke’s solution with dextrose, and a bovine globin fraction were used. The organisms were prepared as described under Methods, and known aliquots resuspended in various fractions. As Table II shows, rabbit serum and rabbit beta globulin were indistinguishable from rat serum: the organisms remained motile. Under fluorescence microscopy the organisms remained in. . vrsrble for at least 10 minutes. In albumin they became only faintly fluorescent, and also
240
DU
BUY,
GREENBLATT,
HAYES,
AND
LINCICOME
lewisi
in Fractions
Fluorescence
microscopy
TABLE Survival
and Tetracycline
II
of Trypanosoma
Fluorescence
Fluorescence Medium
I -PO
Rat serum (100%) Rabbit (Rb) serum (100%) Rat albumin (30 mg/ml) Rat globulinsa (50 mg/ml) Rat y globulin (42 mg/ml) Immune rat serumb Dialyzed Rb serum (lOO’j%) Rb serum albumin (30 mg/ml) Rb u-globulin (30 mg/ml) Rb P-globulin (30 mg/ml) Rb y-globulin (30 mg/ml) Rb serum dialysate Locke’s Bovine globin Q 70% clumped at T,,. b Tighter clumping than Table I.
in normal
0 0 0 0 0 l-3+ 0 0 0 0 0 0 -c k rat
globulins;
intens.
T LICLX 8 min 45 set 75 min 0 3 set 20 set lo-15 set 35 set 10 set 10 set 35 set few
survived for more than 10 minutes. In the remaining fractions they adhered to the glass, became brightly fluorescent, and died in 15 240 seconds.Bovine globin proved to be toxic. Apparently, the lipoprotein-containing fractions (beta globulins) and the lipid-containing albumin protect better than the other fractions. The e8ect of rat anti-T. lewisi-antiserum. Organismsprepared as described under Methods were resuspendedin normal rat serum and in serum from rats infected 4-6 weeks previously with T. lewisi, both sera containing 200 ug of TC per milliliter. As reported above, the organismsin normal serum translocated freely when observed by phasemicroscopy, but were invisible by UV microscopy. The organisms in antiserum, under phase, clumped immediately and adhered to the glasssurfaces, while the undulation decreasedin time. A few freeswimming organisms,coming near the clump, were suddenly attracted by the clump, and remained stuck. By UV microscopy the organisms were visible immediately and then increased in visibility after 1-5 minutes and decreasedin undulation. In contrast to normal
free
I T,,X
3+ 4+ 1+ 2+
at T,
Transl.
Undul.
4-t 4+
4+ 4+
lf <1+ 0 0 0 4+ 4+ 4-t
4+ 4+
4f 3+ 3+ 3+ swimming
and Rat Serum
Movement
2+ lf 3+
of Rabbit
4+ 3+
2+
organisms.
< 75 set
3+ 3+ 4+ 4f 4+
u
2-t For
> 15 min > 10 min > 8 min > 9 min
2+
4f 4+ 4+
0
50% Survival time
further
5min 4 min > 10 min 4 min > 10 min 70 min 60 min 15-30 min 35 min legend,
see
serum, antiserum did not provide protection against TC penetration. Loss of intracellular medium in normal
material to the external and anti-T. lewisi-rat
serum. It has been reported previously that Leishmunia enriettii leaches intracellular material to the external medium, as indicated by an increaseof UV absorption at 260 mu when organisms are kept under unfavorable conditions (Greenblatt and Glaser, 1965). Similar experiments were performed with T. Zewisi (see Methods). Figure 4 presents a representative experiment in which the leaching in normal rat serum is compared with that in antiserum. Proteins, whether of the serum or organisms,are precipitated by perchloric acid. No distinction was made between small molecules that had leached out of intact organisms or those that were releasedby lysis. It can be seenthat in antiserum the leaching is more pronounced than in normal serum. This parallels the results obtained in experiments with TC penetration into organismsin antiserum, as measured by fluorescence microscopy. These results suggesta gradual breakdown
TRYPANOSOME
MEMBRANE
MINUTES
FIG.
4.
ultraviolet cipitation vs. time.
Leaching of organisms as measured by absorption at 260mp, after protein prewith 2% perchloric acid. Units: absorbance
of the protecting membrane in antiserum, which not only allowed the extracellular TC to enter, but also the intracellular material to leave the cells. The leaching of the organisms in Locke’s solution was variable. This was probably due to the variable amount of leachable material still present in the organisms after manipulation and to their disintegration to various degrees. DISCUSSION
Mouse and rabbit serum as well as rat serum permitted survival of T. Zedsi for at least 7 days in vitro, thus making it seem that survival factors involving both small nutritive molecules and permeability-determining proteins are present in homologous and heterologous sera. However, the factors related to reproduction and infectivity are either absent in the mouse or are counteracted there (Lincicome, 1958; Lincicome and Francis, 1961). We have stressed the protein factors which lessen tetracycline penetration, and on several occasions have spoken of it as a “coat.” This does not imply that exogenous proteins are the only permeability barriers, Also, in the
PERMEABILITY
241
absence of external protein, some temporary barrier to TC exists, as indicated by the temporary protection afforded by serum dialysate or Locke’s solution. This protection seems of a metabolic nature (see below). The formation of a protective layer by the cytoplasm, either around a foreign element (thus protecting the host against the parasite and, ipso facto, also protecting the foreign element under certain conditions against the host) or around host elements which must be kept separate from their surroundings, seems to be a general phenomenon which is readily demonstrable by the presence or absence of tetracycline fluorescence. Du Buy et QJ. (1964) have demonstrated such effects with Escherichia co&, Salmonella typhosa, Bacillus cereus, Blenorrhea virus, and Toxoplasma. In each case, the extracellular parasite was permeable to tetracycline, but once within host cell cytoplasm, the antibiotic was excluded by the formation of a “capsule.” Such a capsule has also been reported by Desowitz (1954) and Desowitz and Watson (1953), and has recently been visualized by electron microscopy, as reported by Rudzinska et al. (1964), who showed encapsulated intracellular forms of Leishmania donovani. In the case of T. letisi, where the host environment is the serum, the nature of the “coating” phenomenon, which may resemble the encapsulation process, can be studied more effectively than in intracytoplasmic parasites. Perhaps the clearest expression of this hostserum protection is seen when, with the appearance of immunity, immune rat serum no longer provides permeability protection to the parasite and tetracycline enters the parasite. This occurs in spite of the presence of the full complement of rat serum proteins, and makes unlikely the explanation that serum protein per se provides protection. Instead, it seems that there is competition between the normal and immune globulins for the parasite surface. The stickiness to the glass, which occurs after removal of normal serum by washing in Locke’s solution or by addition of antiserum,
242
DU
BUY,
GREENBLATT,
HAYES,
suggestsalso that the surface charge of the parasites has changed, probably due to the exposure of new chemical groups to the external medium. The demonstration of a fluorescein-labeled serum coat does indicate that a protein layer adheres to the organisms. The association is obviously a weak one which can be disrupted simply by washing. Somedifference must exist between this coat on the living, nonpenetrable and on the dead, penetrable organism, so that maintenance of this coat can be thought of as depending on a dynamic state. Korn et al. (1965) have noted that T. lewisi does not make stearic acid, but is capable of removing it from exogenousproteins, esterifying it, and desaturating it further. This kind of reaction might well serve as a model for the transitory nature of a “protein coat.” At the moment of the stripping of the fat from the protein, the fat would serve to bind the protein to the protozoa1 surface. In this regard it is noteworthy that the fraction containing the richest source of fats, the beta globulins, is most protective. Other casesof small molecule requirements provided by macromolecules are also discussedin the above article. The results reported have implications in regard to chemotherapeutic studies. It seems obvious, as Yorke et al. (1929) have pointed out, that prospective chemotherapeutic substances must be studied for their effect in media which contain the serum of the host. Penetration of such substanceswhich might have a subsequent tidal effect when studied in, for example, Locke’s solution with glucose might be unable to penetrate in the presence of host serum, and thus be ineffective in vivo. ACKNOWLEDGMENT
The authorswish to expressappreciationto Mr. Fred Riley for technicalassistance. T.
B.,
AND
WALLACE,
F.
G.
1960.
A com-
parative study of kinetoplastultrastructurein the Trypanosomatidae. Journal of Protozoology 7, 115-124. DESOWITZ, R.
vivax. X. The activity of someblood fractions in facilitatinginfectionin the white rat. Annals of Tropical Medicine and Parasitology 46, 142151. DESOWITZ,
R.
1954.
S.,
Studies
in
Trypanosoma
AND
WATSON,
H.
J.
C.
1953.
Studieson Trypanosoma vivax. VI. The occurrence of antibodies in the seraof infectedsheep andwhite rats, and their influenceon the course of infection in white rats. Annals of Tropical Medicine DU
BUY,
and
H.
G.,
Parasitology
47, 247-257.
GREENBLATT,
C.,
LINCICOME,
D.
R.,
Tetracyclineas a tool for experimentalstudy of cell permeabilityof AND
HAYES,
J.
Trypanosoma
1963a.
lewisi.
Journal
of Parasitology
49
(Suppl.), 21-22. DU
BUY,
H.
G.,
GREENBLATT,
C.
L.,
LINCICOME,
Tetracyclineas a tool for the study of cell permeabilityof D.
R.,
AND
HAYES,
J.
1963b.
lewisi.
Trypanosoma
Journal
Cell
of
Biology
19, 21A-22A. DU
BUY,
H.
1964. studies DU
sites. BUY,
G.,
RILEY,
Science H.
E.
mitochondria
in
Parasitology D.
1964.
A.,
AND
WOZNIAK,
sp.:
A.
M.
acids and
in the
Trypanocomparative
6, 43-50. of
Trypanosoma
mouse
host.
FRANCIS,
Experi-
E.
H.
on
of
A.
ion-exchange 17, 1116-1126. on
1958. cellulose. binding
TRACER,
Leishmania
of the kinetoplast transformation.
Studies
inin
lewisi
11, 68-76.
structure
role
1961.
sera
P. A., AND
11, 166-191. PETERSON, E.
1960.
A
Research Growth
7,
LEES, fatty
Parasitology
the
Proceedings L. A.
AND
on heterologous of Trypanosoma
fine
chromatography
A simple of tetra-
Biochemistry
D’ALESANDRO,
The
vitro.
7, l-13.
R.,
of Protozoology SOBER, H. A., AND
G. 1964. estimation
polycephalum tarentolae,
heterologous
Quantitative studies ducing development mice. Experimental W.
H.
unsaturated
of Lipid 1958.
Temper-
in
16, 36-52.
Physarum Leishmania and Crithidia
the
1965.
C. L.,
of
R.
M.
L.
Selec-
enriettii
Analytical
Journal D.
P.
&i&mania
GREENBLATT,
LINCICOME,
RUDZINSKA,
139,
GLASER,
antibiotics.
zooflagellates soma lewisi
lewisi mental
1961.
in 196-197.
Parasitology
1965. Synthesis the slime mold
study. LINCICOME,
J. L.
tetracycline
AND
on
D.,
J.
in permeability intracellular para-
of
E., AND DU BUY, for quantitative
cycline 322-327.
SHOWACRE,
SHOWACRE,
Science
C. L.,
ature effect Experimental
KORN,
AND
fluorescence around
145, 163-165.
G., AND
GREENBLATT,
HAYES, J. method
F.,
Tetracycline of membranes
tive localization of living cells.
eration S.
LINCICOME
donovani and leishmania-leptomonad
REFERENCES CLARK,
AND
in
the
Journal Protein Fedof tetra-
TRYPANOSOME
MEMBRANE
cyclines by dog and human plasma. Proceedings of the Society for Experimental Biology and Medicine 105, 430-433. YORKE, W., ADAMS, A. R. D., AND MURGATFCOYD, F.
PERMEABILITY
243
1929. Studies in chemotherapy. I. A method for maintaining pathogenic trypanosomes alive in vitro at 37°C for 24 hours. Annals of Tropical Medicine and Parasitology 23, 501418.
PARASITOLOGY
18,
231-243
Regulation
(1966)
of
Cell
Membrane
Trypanosoma
Permeability
in
lewid
H. G. du Buy Laboratory
of
Biology
of
Viruses,
National Bethesda,
Institute Maryland
of Allergy
and
Infectious Diseases,
and
Metabolic
C. L. Greenblatt Laboratory
of Physical
Biology,
National Institute Bethesda, Maryland
of Arthritis
Diseases,
J. E. Hayes, Jr. Laboratory
of Technical
Development,
National
Heart
Institute,
Bethesda,
Maryland
and D. R. Lincicome Howard (Submitted
University,
Washington,
for publication,
14 June
D. C. 1965)
Du BUY, H. G., GREENBLATT, C. L., HAYES, J. E., JR., AND LINCICOME, D. R. 1966. Regulation of cell membrane permeability in Trypanosoma lewisi. Experimental Parasitology 18, 231-243. Fluorescent properties of tetracycline (TC) were used to study permeability changes of T. lewisi in various surroundings under phase and fluorescence microscopy. Mitocbondria and the mitochondrial elements of the kinetoplast fluoresced bright yellow under fluorescence microscopy when tetracycline entered the cells. Survival time of the organisms in saline was measured in hours, and in serum in days, either with or without TC. Survival time was proportionally shortened under conditions of fluorescence microscopy. This proportional shortening of the survival times was dependent on penetration of TC and the effect of the light necessary for fluorescence microscopy. The cells in saline became immediately visible and died rapidly, while those in serum became visible within an hour, followed by death. Serum serves as a protective permeability-regulating coat around the parasites. Thus, permeability changes which required hours or days under normal conditions could be measured in minutes with tetracycline. The protection afforded by different serum fractions of rabbit and rat sera was also measured. The beta globulins, and, to a lesser degree, the albumins, prevented the penetration of tetracycline. The presence of a serum barrier was also demonstrated by washing serum-coated organisms, by the use of fluorescein-labeled serum, and by leaching experiments. Anti-T. lewisi-antiserum does not prevent tetracycline penetration. The nature of the serum barrier and the probable role of the protective lipoproteincontaining serum fractions is discussed and their significance regarding chemotherapeutic studies pointed out.
culture, as well as the mitochondrial memTetracycline (TC) penetrates the cell mem- brane, and accumulates in the mitochondria brane of higher animals in vivo and in tissue (du Buy and Showacre, 1961). It is unable to penetrate the nuclear membrane of living 1 Supported in part by Research Grant No. AIcells, or the vacuolar wall surrounding intra03409 from the National Institutes of Health to D. R. cellular bacteria and certain intracellular Lincicome. INTRODUCTION
231
232
DU BUY,
GREENBLATT,
HAYES,
AND
LINCICOME
200 X g. The pellet was again suspended and centrifuged as before. The three supernatants, all rich in parasites, were combined. This suspension, which contained the Alsever-diluted serum of the first supernatant and the Alsever-Locke’s mixture of the two others, served as stock suspension. Immediately before use, a given amount of stock suspension was recentrifuged for one minute in 1.5 ml-centrifuge tubes in an International Model MB microcapillary centrifuge The pellets were resuspended in Locke’s solution with dextrose. Equal amounts of this suspension were dispensed into the small centrifuge tubes, recentrifuged for 1 minute, and the pellets resuspended in the various media under study to the volume of the original aliquot portion. For microscopic observation, samples were removed by capillary pipettes and placed on microscope slides with cover slips. Sera and serum proteins. Blood was obtained by heart puncture of anesthetized rats or rabbits, and the blood allowed to clot for a MATERIALS AND METHODS few hours at room temperature. The serum for 10 Test organism. Osborne-Mendel rats was collected after centrifugation (N.I.H. strain, randomly mated in closed minutes at 2000 X g, and either used fresh, or, if necessary, kept frozen (- 25” C) until colony) were inoculated intraperitoneally with 1 ml of a suspension of washed T. lewisi con- use. Only in the case of mice was blood obtaining approximately 3 X IO6 cells. One tained by decapitation. Rabbit serum fractions were obtained comweek later blood was collected from these rats by heart puncture in one-fifth its volume of mercially.4 The rat serum fractions consisted of (1) the albumins and globulins represented Alsever solution.” The suspension was centrifuged for 10 respectively by the supernatant and the pellet of serum treated with 50% (NH4)$04, and minutes at about 200 X g and the superna(2) the gamma globulins which were the first tant, containing the organisms, removed. The fraction obtained by passing serum through a pellet was resuspended to the original volume, DEAE-cellulose column (Sober and Peterson, with a one-to-one mixture of Alsever solution and Locke’s buffered glucose-salt solution3 1958). Tetracycline. A stock solution, made fresh and again centrifuged for 10 minutes at about daily of TC HCI (Lederle) , 1 mg per milli3 Alsever sol., per liter: dextrose, 20.5 gm; Na liter Locke’s solution, was adjusted to pH citrate 2H,O, 8 gm; NaCI, 4 gm; citric acid, O.SS 6.8-7.2 with KOH. gm. Tetracycline binding by serum. Measure8 Locke’s sol. with dextrose, per liter: NaCI, 8 gm; KCI, 0.2 gm ; Na2HP0,, 0.4 gm; KH,PO,, 0.3 gm; ments of binding of TC to serum proteins
parasites sufficiently to be observed by fluorescence microscopy (du Buy et al., 1964). The observation that Trypanosoma lewisi in serum did not fluoresce when TC (20-100 pg per milliliter) was added, whereas those in saline were almost immediately visible, suggested the use of TC and fluorescence microscopy as tools for a study on T. lewisi, where the host environment is serum, and the formation of a protective coating around a parasite could be studied in an extracellular environment (du Buy et al., 1963a,b). This paper presents an analysis of some factors in both homologous and heterologous sera, which, on the basis of observed differences in TC penetration, are responsible for the survival of the parasites outside the host. Preliminary accounts of this work were presented at the 1963 annual meetings of the American Society of Parasitologists (du Buy et al., 1963a) and the Third Annual Meeting of the American Society for Cell Biology (du Buy et al., 1963b).
CaC12, 0.2 gm; dextrose, sterilized separately.
2.5 gm;
CaCl,
and dextrose 4
The Mann
Research
Laboratories,
Inc.
TRYPANOSOME
MEMBRANE
PERMEABILITY
233
were done by dialysis equilibrium followed by 200 mercury-xenon arc. It was used with two analysis of the dialysand and dialysate for 3-mm Schott BG12 glass filters as primary TC. Two-ml portions of various dilutions of filter, and a l-mm Schott OG5 glass filter as sera in Locke-Ringer saline were placed in barrier filter. Visking casing and dialyzed for 12 hours Photography. Photographs were taken on against 40-ml portions of TC solution in Adox KB17 film or on Kodak Royal Pan (4 X 5 sheets), both developed in Acufine at Locke-Ringer saline in glass tubes with Teflon-lined caps on a rocking dialyzer. At the room temperature. end of this equilibration period, the dialysis RESULTS casings were removed, blotted dry, and opened. The TC contents of both the inner Preliminary studies for the establishmentof indices of the degree of viability of the orgaand outer solutions were determined by the nisms under varying conditions. By fluoresmethod of Hayes and du Buy (1964). The excess TC in the inner solution over the con- cence microscopy, T. lewisi suspended in centration present in the outer solution was serum with 200 ug TC per milliliter could be detected only by the motion of fluorescent regarded as due to the binding by the serum present. particles in their immediate surroundings, Dialyzed serum. Rat or mouse sera were causedby the movement of the parasites, In dialyzed for 24 hours at 4°C against 3-4 saline, 200 ug TC per milliliter caused suffichanges of either saline or Locke’s solution on cient accumulation of the antibiotic in the a magnetic stirrer, and kept frozen until used. organismsto make them almost immediately Leaching of intracellular material. Washed appear brightly yellow fluorescent against the T. lewisi cells were suspended in Locke’s solu- black or faintly yellow background. The irtion minus glucose, divided into equal ali- radiation itself increased the permeability of the drug as indicated by an increase in tluoquots, centrifuged, and the pellets resuspended in 2.5 ml of varying concentrations of rescent intensity. (For further discussion of normal serum, antiserum, or Locke’s solution. this point, seep. 239.) The fluorescent image The number of organisms was measured either showed no specific cell wall staining (Fig. 1, by counts in a Levy chamber, or volumetriA,). Yellow dots were seen, corresponding to cally by the use of the capillary centrifuge (a the granular elements observed under phase lo/o volume of organisms corresponds to about (Fig. 1, A1), and representing, by analogy 5 X 10’ organisms per milliliter). After variwith vertebrate studies, TC-stained mitoous time intervals, the organisms were centrichondria (du Buy and Showacre, 1961). A fuged in 70 X 7-mm test tubes in a capillary dark area indicated the location of the nucentrifuge and the UV absorption of the cleus. The region of the kinetoplast showedas supernatant at 260 mu measured with a Cary a dark vacuole with a yellow crescent, prespectrophotometer, Model 14. The material sumably representing the equivalent of its released to the medium was read as the optimitochondrial and its nucleic acid material cal density difference, 260-360 mu. (Clark and Wallace, 1960). Apparently the Microscopy. Microscopy was done with a TC penetrated through the cell membrane Zeiss W microscope, which could be used and accumulated in the mitochondria and in either with a tungsten light source for phase the mitochondrial elements of the kinetomicroscopy, or with a filtered mercury arc plasts. Even after death (Fig. 1, B1, B,) and light source for fluorescence microscopy as vacuolation (Fig. 1, B3) of the organism, described elsewhere (du Buy and Showacre, these elementscould still be recognized. 1961). Trypanosomes, suspendedin salt solutions The arc source used was an Osram HBO deteriorated in a few hours, but in serum they
234
DU
BUY,
GREENBLATT,
HAYES,
FIG. 1. Al, T. Zewisi in 200 pg of TC in saline observed fluorescence microscopy. Bl, dead organism in 200 pg of TC B3, vacuolation after death.
for days (Table I, column 2). The addition of TC in both cases did not alter these findings. However, when observed under the conditions of fluorescence microscopy, in the presence of TC their survival in both saline and serum was shortened, but proportionately so. In saline the organisms became
survived
AND
LINCICOME
by phase by phase;
microscopy; AZ, same organism by B2, same organism, by fluorescence;
visible in a few seconds, in serum within an hour. Apparently the considerable damage done by maintaining the organism in saline and the gradual deterioration in serum were proportionally accelerated by the photochemical damage. The photochemical damage, in its turn, appeared directly correlated with
TRYPANOSOME
and Tetracycline
Survival
MEMBRANE
TABLE I of Trypanosoma lewisi in Rat and Mouse
Fluorescence
Absence of TC
Seruma
Presence of TC
Phase microscopy 50% survival time Medium
235
PERMEABILITY
Fluorescence microscopy Intensity
37” (hr)
25”
I To
Tbnx
Movement at T, Trans.
1%ax
50% Survival time
Undul.
25”
(set)
100%
5 days
-
-
4+
30
2+
2+
Ifr
25
4+
2+
0
30
3+
0
1+ to4+
2.5
min
0
4+
35 min
to 4+
4+
12.5
to 4+
3+
RS 5 days
10%
RS 1% RS 0.1% RS 100% MS 10% MS 1% MS 0.1% MS Locke &D Locke Saline
24
hr
to 4+
min
6 min
4
hr
ca 8
3
days
-
-
4+
4+
>
12
min
ca 8
3
days
3.5
2+
2+ to4+
4+
>
10
min
hr
20
3+
0
3+
10
3+
0
2+
4
24
1
8 hr
0
ca 1
ca 6 hr
1.5
3+
0
1+
ca4hr ca 2 hr
10 10
2+ 3+
0 0
lf 1+
to 4+
5 min 90 set
to 2+
10 set 10 set 10 set
a RS: rat serum; MS: mouse serum; Locke & D: Locke’s solution with dextrose; ITo: intensity of fluorescence at zero time, graded from 0 to 4+; TImax: time of maximal fluorescence; Lr max: intensity of fluorescence at TImax; Transl.: translocation, and Undul.: undulation at zero time, graded from 0 to 4+. Last column: 50% survival time under continuous fluorescence irradiation. Each determination is based on at least two observations.
the degree of damage to the parasites under various conditions: (1) degree of translocation, scored from 4 + (maximum translocation, with wave motion of flagella) to 0 (no intensity constituted a measureof membrane translocation) ; (2) degree of mobility of the permeability to this drug. After onset of flagellum: undulation 4 + (whipping of death, the fluorescence decreased again. By flagellum) to 0 (immobile) ; (3) adherenceto this procedure, permeability changes due to a glasssurface and clumping; and (4) vacuchanges in the environment, which might olation (often sudden) and death (no moverequire hours or days to become apparent ment, increasein refractive index) (Fig. 3,B). Decreasing values of these indices paralunder ordinary conditions, were measuredin leled the degreeof increaseof permeability to minutes by fluorescencemicroscopy. The above observations led to the establish- TC, as observed by the intensity of fluoresment of arbitrary standards, by the scoring cence of the organisms,and thus served as an of four criteria which were used as indices of index for the permeability of other substances, the rate of accumulation of the antibiotic in the organism. As judged by fluorescent intensity, this rate of accumulation was rapid in saline, slow in serum. Thus, the fluorescent
236
DU
BUY,
GREENBLATT,
HAYES,
which possess permeability characteristics comparable to those of TC. By the above criteria, the effect of various sera and their fractions on TC penetration was studied. Effect of rat serum concentration on survival and fluorescence. In this and the subsequent experiments the organisms were prepared as described under Methods. The protective effect of 4 concentrations of rat serum was compared with that of Locke’s solution with and without dextrose. The results are summarized in Table I. The first two columns show that the 50% survival time decreases, both at 37’ and 25’C, with decreasing serum concentration. A comparison of the last column with the first two shows that protection against light damage in fluorescence microscopy decreases in the same order as protection against death on standing. From columns 3-7 in Table I, it can be seen that in decreasing serum concentrations in the salt solution the parasites show more and more fluorescence, concomitant with decreasing mobility, and death. The effect of 0.1% serum is about the same as that obtained in Locke’s solution with dextrose. Thus, the protective effect of rat serum decreases as the serum concentrations decrease.
.4ND
LINCICOME
Eflect of mouse serum. The effect of mouse serum was studied similarly to that of rat serum (Table I). Both sera supplied a similar permeability barrier against TC to the parasite. Thus, the factors for survival are present in heterologous as well as homologous sera. It should be remembered, however, that the factors for reproduction are presumably not present in mouse serum (Lincicome, 1958 ; Lincicome and Francis, 1961) . The effect of dialyzed rat and mouse serum. The protective effect of dialyzed rat and mouse serum was studied as described above. Normal as well as dialyzed serum, from either rat or mouse, increased the survival time under irradiation with filtered mercury arc light used for fluorescence microscopy. In these sera the organisms survived for more than 20 minutes, concomitant with the presence of a permeability barrier against TC, as compared with a survival time of 3 minutes in Locke’s solution. Thus, the length of the survival time in the dialyzed serum as well as the failure of TC to penetrate show that in this respect the difference between dialyzed and undialyzed serum is inapparent. From this it can be concluded that a considerable amount of the dialyzable small-molecule com-
60 t
PERCENT
FIG.
horse
2. Tetracycline serum. mouse,
n
(100 @g per A horse, 0
milliliter) rat.
RAT,
binding
MOUSE
AND
by
HORSE
different
SERUM
concentrations
of
rat,
mouse,
and
TRYPANOSOME
MEMBRANE
PERMEABILITY
237
FI G. 3. A , T. lewisi in fluorescein-labeled rat serum by phase microscopy. B, The organism appears as a dark shad ow against the fluorescent background of the tagged serum.
Panents of rat serum is nonessential to the 9x7rival the parasites. ‘he t?zect of serum dialysate. Organisms (31 < 10B I?er milliliter) washed in Locke’s solu We :re added to a dialysis bag containing 5 ml of Locke’s solution, and the bag sus-
pended in 5 ml of rat serum. One ml of the suspensionof organismswas added to a test tube containing the 24-hour dialysate in Locke’s solution from an equal, nonrenewed volume of rat serum. In either case, the survival time of the or-
238
DU
BUY,
GREENBLATT,
ganisms was increased considerably as compared with that in Locke’s solution. After 4 hours the controls were dead; those in the dialysis bag showed about 5% dead organisms, and those in the dialysate, 10%. After 24 hours those in the dialysis bag still showed 5% deaths, those in dialysate, 50%. It is apparent that the serum continues to supply necessary factors, whereas the dialysate becomes depleted in time. When studied for fluorescence, the organisms from the dialysis bag became visible after 90 seconds, those in dialysate after 30 seconds. The dead organisms were immediately visible in either case. Apparently, the dialyzable factors did not supply a permeability barrier to TC as did the nondialyzable portion. The increased survival time could be explained by assuming that factors from the dialysate temporarily counterbalanced the loss of these factors from the test organisms, brought about by the absence of the protein barrier. Serum protection. Two possibilities were considered in order to explain the absence of visible fluorescence of the trypanosomes in various sera: ( 1) The TC binding by serum was sufficient to lower the concentration of free TC available to the parasites to a point that fluorescence could not be observed. In that case, fluorescence should be observable, if the TC concentration were increased to a level at which free TC would be available. (2) The nature of the serum protection is such that TC cannot penetrate the organisms. A series of experiments was performed in order to choose between these possibilities: ( 1) Binding of tetracycline by serum. The first series was designed to determine whether the TC binding by serum was dependent on the concentration of TC. The binding of TC to rat, horse, and mouse serum was studied by the dialysis equilibrium technique. At concentrations between 100 and 300 ug TC per milliliter, it was found that the fraction of TC bound to horse serum was a function of the serum concentration but not of the TC concentration (Fig. 2). Accordingly, all sub-
HAYES,
AND
LINCICOME
sequent binding measurements were made at a total TC concentration of 100 ug per milliliter. Rat and horse serum were used at serum concentrations of 0, 10, 20, 50, and loo%, and mouse serum at 0, 20, and 100%. It can be seen from Fig. 2 that the TC binding increased with increasing serum concentration, with a maximum binding of 50-60s by the undiluted sera. Similar results were obtained by Wozniak (1960) for human and dog plasma. Thus, at a concentration of 100 ug per milliliter of TC, in undiluted sera, the concentration of unbound TC is about 40 yg per milliliter, or on the basis of the results presented in Fig. 2, 80 ug per milliliter when the TC concentration is 200 ug per milliliter. This, then, is the concentration of TC immediately available to the organisms. These concentrations are sufficiently high to observe fluorescence of the organisms, if it were taken up by them. Another series of experiments was performed to determine whether the TC concentration in serum could be increased to a point that the serum protection was no longer effective. To this end, organisms were washed two times in Locke’s solution without dextrose and resuspended in Locke’s solution with 40, 80, and 120 u.g of TC per milliliter, and in 100% rat serum, with 100, 200, and 300 and 600 pg of TC per milliliter containing about 40, 80, 120, and 240 ltg of unbound TC per milliliter (Fig. 2). The organisms were visible in Locke’s solution at all TC concentrations (even in 40 pg of TC, the visibility was f within 10 seconds). The organisms remained invisible in serum, except as black shadows against the fluorescence of the serum. Apparently, although sera bind a certain amount of TC, the remaining unbound TC, even at high concentration, is unable to penetrate the organisms protected by the serum. These results, thus, support the second alternative mentioned above (this page) : the nature of the serum protection is such that TC cannot penetrate the organisms.
TRYPANOSOME
MEMBRANE
(2) Serum coating. The second series of experiments was performed to determine whether serum could be shown to coat the cells, thus producing a TC barrier. A trypanosome suspension was twice washed with Locke’s solution with glucose to remove the serum, as indicated by stainability with TC. The organisms were then resuspended in normal rat serum conjugated with fluoresceinisothiocyanate (tagged serum) and exposed to it for 20 hours in order to allow a thorough coating with the tagged serum. In the first experiment, organisms suspended in the fluorescein-labeled serum could, under fluorescence microscopy, be recognized as dark shadows against the fluorescent background of the tagged serum (Fig. 3). In the second experiment, the organisms pretreated with tagged serum as above were washed once with Locke’s solution and immediately resuspended in nontagged serum, in order to prevent furt,her removal of tagged serum. Observation by fluorescence microscopy revealed the organisms as mobile fluorescent outlines against the nonfluorescent control serum (nontagged organisms remained invisible). In the third experiment, the pretreated and washed organisms were immediately resuspended in saline. These organisms were only slightly visible for a few seconds: the fluorescein serum was not retained sufficiently to show a clear fluorescent outline. Apparently the “coat” washed off, and the organisms behaved as those washed in saline: they adhered to the glass surfaces. Another experiment, not involving tagged serum, was performed to show that the serum coat was readily removable. Saline-washed trypanosomes were exposed for 60 minutes to whole rat serum with 200 ug of TC per milliliter added, and then washed twice in Locke’s solution with glucose and TC. They were observed immediately after the washes. Fluorescence microscopy showed that these organisms behaved as those in 1% serum, i.e., they were invisible for l-2 minutes, and slowly, while
PERMEABILITY
239
losing their mobility, they became more and more visible. Apparently, the serum pretreatment only temporarily prevented the penetration of the TC into the organisms, and this serum coat was readily removable by washing, which allowed the TC to enter, deteriorating the organisms. The nature of increasing fluorescence of T. lewisi during irradiation. The increase of fluerescence observed during irradiation, either in low concentrations of serum or in Locke’s solution, could be explained if the Locke’s solution in conjunction with the radiation damage increased the permeability of the organisms, gradually allowing the TC to enter. The following experiment was performed to test this explanation. The organisms were exposed for a few hours to TC in 1% rat serum in order to allow TC to be taken up. ‘They were then washed and resuspended in salineglucose devoid of TC. Placed under the fluorescence microscope, the initial fluorescence was seen to decrease, rather than to increase, following irradiation. Apparently, without an external supply of TC no increase of fluorescence can occur. The effect of serum fractions. In order to determine which fraction of the serum afforded most protection, some serum fractions were investigated. Experiments were performed with rabbit serum and its fractions, which were commercially available, namely, serum dialysate, serum albumin (fraction 5)) and alpha, beta, and gamma globulin fractions. Experiments were also performed with rat albumin, total globulins, and gamma globulin. As controls, rat serum, Locke’s solution with dextrose, and a bovine globin fraction were used. The organisms were prepared as described under Methods, and known aliquots resuspended in various fractions. As Table II shows, rabbit serum and rabbit beta globulin were indistinguishable from rat serum: the organisms remained motile. Under fluorescence microscopy the organisms remained in. . vrsrble for at least 10 minutes. In albumin they became only faintly fluorescent, and also
240
DU
BUY,
GREENBLATT,
HAYES,
AND
LINCICOME
lewisi
in Fractions
Fluorescence
microscopy
TABLE Survival
and Tetracycline
II
of Trypanosoma
Fluorescence
Fluorescence Medium
I -PO
Rat serum (100%) Rabbit (Rb) serum (100%) Rat albumin (30 mg/ml) Rat globulinsa (50 mg/ml) Rat y globulin (42 mg/ml) Immune rat serumb Dialyzed Rb serum (lOO’j%) Rb serum albumin (30 mg/ml) Rb u-globulin (30 mg/ml) Rb P-globulin (30 mg/ml) Rb y-globulin (30 mg/ml) Rb serum dialysate Locke’s Bovine globin Q 70% clumped at T,,. b Tighter clumping than Table I.
in normal
0 0 0 0 0 l-3+ 0 0 0 0 0 0 -c k rat
globulins;
intens.
T LICLX 8 min 45 set 75 min 0 3 set 20 set lo-15 set 35 set 10 set 10 set 35 set few
survived for more than 10 minutes. In the remaining fractions they adhered to the glass, became brightly fluorescent, and died in 15 240 seconds.Bovine globin proved to be toxic. Apparently, the lipoprotein-containing fractions (beta globulins) and the lipid-containing albumin protect better than the other fractions. The e8ect of rat anti-T. lewisi-antiserum. Organismsprepared as described under Methods were resuspendedin normal rat serum and in serum from rats infected 4-6 weeks previously with T. lewisi, both sera containing 200 ug of TC per milliliter. As reported above, the organismsin normal serum translocated freely when observed by phasemicroscopy, but were invisible by UV microscopy. The organisms in antiserum, under phase, clumped immediately and adhered to the glasssurfaces, while the undulation decreasedin time. A few freeswimming organisms,coming near the clump, were suddenly attracted by the clump, and remained stuck. By UV microscopy the organisms were visible immediately and then increased in visibility after 1-5 minutes and decreasedin undulation. In contrast to normal
free
I T,,X
3+ 4+ 1+ 2+
at T,
Transl.
Undul.
4-t 4+
4+ 4+
lf <1+ 0 0 0 4+ 4+ 4-t
4+ 4+
4f 3+ 3+ 3+ swimming
and Rat Serum
Movement
2+ lf 3+
of Rabbit
4+ 3+
2+
organisms.
< 75 set
3+ 3+ 4+ 4f 4+
u
2-t For
> 15 min > 10 min > 8 min > 9 min
2+
4f 4+ 4+
0
50% Survival time
further
5min 4 min > 10 min 4 min > 10 min 70 min 60 min 15-30 min 35 min legend,
see
serum, antiserum did not provide protection against TC penetration. Loss of intracellular medium in normal
material to the external and anti-T. lewisi-rat
serum. It has been reported previously that Leishmunia enriettii leaches intracellular material to the external medium, as indicated by an increaseof UV absorption at 260 mu when organisms are kept under unfavorable conditions (Greenblatt and Glaser, 1965). Similar experiments were performed with T. Zewisi (see Methods). Figure 4 presents a representative experiment in which the leaching in normal rat serum is compared with that in antiserum. Proteins, whether of the serum or organisms,are precipitated by perchloric acid. No distinction was made between small molecules that had leached out of intact organisms or those that were releasedby lysis. It can be seenthat in antiserum the leaching is more pronounced than in normal serum. This parallels the results obtained in experiments with TC penetration into organismsin antiserum, as measured by fluorescence microscopy. These results suggesta gradual breakdown
TRYPANOSOME
MEMBRANE
MINUTES
FIG.
4.
ultraviolet cipitation vs. time.
Leaching of organisms as measured by absorption at 260mp, after protein prewith 2% perchloric acid. Units: absorbance
of the protecting membrane in antiserum, which not only allowed the extracellular TC to enter, but also the intracellular material to leave the cells. The leaching of the organisms in Locke’s solution was variable. This was probably due to the variable amount of leachable material still present in the organisms after manipulation and to their disintegration to various degrees. DISCUSSION
Mouse and rabbit serum as well as rat serum permitted survival of T. Zedsi for at least 7 days in vitro, thus making it seem that survival factors involving both small nutritive molecules and permeability-determining proteins are present in homologous and heterologous sera. However, the factors related to reproduction and infectivity are either absent in the mouse or are counteracted there (Lincicome, 1958; Lincicome and Francis, 1961). We have stressed the protein factors which lessen tetracycline penetration, and on several occasions have spoken of it as a “coat.” This does not imply that exogenous proteins are the only permeability barriers, Also, in the
PERMEABILITY
241
absence of external protein, some temporary barrier to TC exists, as indicated by the temporary protection afforded by serum dialysate or Locke’s solution. This protection seems of a metabolic nature (see below). The formation of a protective layer by the cytoplasm, either around a foreign element (thus protecting the host against the parasite and, ipso facto, also protecting the foreign element under certain conditions against the host) or around host elements which must be kept separate from their surroundings, seems to be a general phenomenon which is readily demonstrable by the presence or absence of tetracycline fluorescence. Du Buy et QJ. (1964) have demonstrated such effects with Escherichia co&, Salmonella typhosa, Bacillus cereus, Blenorrhea virus, and Toxoplasma. In each case, the extracellular parasite was permeable to tetracycline, but once within host cell cytoplasm, the antibiotic was excluded by the formation of a “capsule.” Such a capsule has also been reported by Desowitz (1954) and Desowitz and Watson (1953), and has recently been visualized by electron microscopy, as reported by Rudzinska et al. (1964), who showed encapsulated intracellular forms of Leishmania donovani. In the case of T. letisi, where the host environment is the serum, the nature of the “coating” phenomenon, which may resemble the encapsulation process, can be studied more effectively than in intracytoplasmic parasites. Perhaps the clearest expression of this hostserum protection is seen when, with the appearance of immunity, immune rat serum no longer provides permeability protection to the parasite and tetracycline enters the parasite. This occurs in spite of the presence of the full complement of rat serum proteins, and makes unlikely the explanation that serum protein per se provides protection. Instead, it seems that there is competition between the normal and immune globulins for the parasite surface. The stickiness to the glass, which occurs after removal of normal serum by washing in Locke’s solution or by addition of antiserum,
242
DU
BUY,
GREENBLATT,
HAYES,
suggestsalso that the surface charge of the parasites has changed, probably due to the exposure of new chemical groups to the external medium. The demonstration of a fluorescein-labeled serum coat does indicate that a protein layer adheres to the organisms. The association is obviously a weak one which can be disrupted simply by washing. Somedifference must exist between this coat on the living, nonpenetrable and on the dead, penetrable organism, so that maintenance of this coat can be thought of as depending on a dynamic state. Korn et al. (1965) have noted that T. lewisi does not make stearic acid, but is capable of removing it from exogenousproteins, esterifying it, and desaturating it further. This kind of reaction might well serve as a model for the transitory nature of a “protein coat.” At the moment of the stripping of the fat from the protein, the fat would serve to bind the protein to the protozoa1 surface. In this regard it is noteworthy that the fraction containing the richest source of fats, the beta globulins, is most protective. Other casesof small molecule requirements provided by macromolecules are also discussedin the above article. The results reported have implications in regard to chemotherapeutic studies. It seems obvious, as Yorke et al. (1929) have pointed out, that prospective chemotherapeutic substances must be studied for their effect in media which contain the serum of the host. Penetration of such substanceswhich might have a subsequent tidal effect when studied in, for example, Locke’s solution with glucose might be unable to penetrate in the presence of host serum, and thus be ineffective in vivo. ACKNOWLEDGMENT
The authorswish to expressappreciationto Mr. Fred Riley for technicalassistance. T.
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