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Trypanosoma cruzi: Interaction with vertebrate cells in vitro
EXPERIMENTAL
36, 150-157 ( 1974)
PARASITOLOGY
Trypanosoma
cruzi: Interaction Cells in Vitro
IV. Environmental
Temperature
JAMES A. DVORAK AND Laboratoy
with Vertebrate Effects
CHARLES M. POORE
of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 (Submitted for publication September 24, 1973)
DVORAK, J. A., AND POORE, C. M. 1974. Typanosoma cruzi: Interaction with vertebrate cells in vitro. IV. Environmental temperature effects. Experimental Parasitology 36, 150-157. The effects of 4 environmental temperatures (29, 32, 35, and 38 C) on the interaction between Typanosoma cruzi and bovine embryo skeletal muscle cells were quantified. Three aspects of the interaction (penetration of host cells by trypomastigotes, the lag period prior to the reproductive phase, and the reproductive phase) were markedly affected by temperature. There was a linear increase in the number of trypomastigotes penetrating cells in the 29-35 C range. Temperatures above 35 C can be considered supraoptimal as no further increase in the rate of penetration occurred. The lag period decreased linearly as temperature increased in the 29-35 C range; at 38 C, the lag period was markedly shortened. The doubling time of amastigotes increased linearly as temperature increased in the 32-38 C range; at 29 C, the doubling time was markedly lengthened. At all temperatures, parasites reproduced for 9 generations before cell rupture. The changes in lag period and doubling time complemented each other in the 32-38 C range. Thus, there was essentially no change in the overall length of the intracellular cycle which lasted 6.1 to 6.5 days. At 29 C, however, the cycIe was lengthened to 8.9 days. Thermodynamic analysis revealed marked differences, characterized by a negative activation energy and negative enthalpy, between the reproductive phase of parasites within vertebrate cells and the vertebrate cells themselves. However, the thermodynamic parameters of parasites reproducing extracellularly in liquid medium and intracellularly were the same. INDEX DESCRIPTORS: Tlypanosome cruzi; Cell lines; Bovine embryo skeletal muscle; Life cycles; Culture, in &TO; Thermodynamic analysis; Enthalpy; Temperature; Entropy; Energy, activation; Bioenergetics.
IWI-RODIJ~TI~N
The effects of different environmental temperatures on the growth and development of Typanosma cruzi have been studied by several workers. Pizzi and Christen (1950) and Fernandes and Castellani (1966) described their effects on parasites maintained in liquid medium; Neva et al. (1961), Trejos et al. (1963)
and Ribeiro et ~2. (1969) described their effects on the growth and development of parasites maintained in tissue culture. The effects of environmental temperature on the in viva course of T. cmcxi infections in vertebrates were described by Kolodny ( 1940)) Franca-Rodriguez and Mackinnon (1962), Trejos et al. (1965), Amrein (1967) and Marinkelle and Rodriguez 150
6 1974 by Academic Press, of reproduction in any form
Inc. reserved.
T. C?'UZi: TEMPEBATIJIUZ ANDVFZTEBBATFi
(1968) and in invertebrates by Neves (1971). It was established, in all cases, that changes in the environmental temperature produced alterations in the growth characteristics or development of the parasite. However, elucidation of specific temperature sensitive stages of the parasite’s cycle as well as the interrelationship of temperature sensitive stages within the cycle was not achieved due to a lack of suitable quantitative techniques. This resulted in difficulties in interpreting temperature sensitive phenomena that occurred. The ability to observe and quantify the entire intracellular cycle of T. crU2i in vertebrate cells (Dvorak and Hyde 1973) as well as quantify the kinetics and stoichiometry of the penetration phase of the interaction (Hyde and Dvorak 1973) allows a quantitative study of the effects of physical and/or chemical parameters for all stages of the cycle at a level of precision previously ,unobtainable. This report presents the results of quantitative studies of the effects of different environmental temperatures on the cycle of T. cruzi in vertebrate cells utilizing these techniques. MATERIALS AND METHODS
Two experimental protocols were used to quantify the effects of different environmental temperatures (29, 32, 35, and 38 C) on the complete cycle of T. cruxi in vertebrate cells. The penetration of vertebrate cells by trypomastigotes was quantified by the methods of Hyde and Dvorak ( 1973). Vertebrate cells established on coverglasses 24 hr prior to use at a concentration of lo4 cells/ml were exposed to parasites for 0, 2, or 4 hr periods at parasite concentrations ranging from 5 X lo5 to 2 X lo6 motile trypomastigotes/ml. All parasite suspensions were equilibrated to temperature prior to use. Two thermostatically controlled water baths, maintained to a tolerance of less than -t-OS C of the required
CELLS
151
temperature, were employed. One water bath was set at 35 C and constituted the control temperature; the other water bath was adjusted to 29, 32, or 38 C and constituted the experimental temperature. Thus, 35 C was the reference temperature and the mean number of parasites/cell for each experimental temperature was corrected by calculating its value relative to the 35 C reference. The 35 C, 2 hr reference was assigned a value of unity and the individual penetration rates were corrected for each time-temperature pair. The corrected value for the mean number of parasites/ cell, which allowed the interrelation of all temperatures studied, was termed the “penetration index.” The “penetration index” for each temper,ature pair was determined for two time intervals on three separate occasions. The effects of environmental temperature on the course of the intracellular cycle were quantified in individual vertebrate cells using a controlled-environment culture system (Dvorak and Stotler 1971). Vertebrate cells were established at a concentration of 2 x lo4 cells/ml and temperature equilibrated on the microscope stage prior to the introduction of trypomastigotes. Temperature was maintained to a tolerance of kO.2 C of the required temperature by means of a proportional-controlled air stream incubator (Nicholson Precision Instruments, Model C-300). Parasites were inoculated into culture chambers at concentrations ranging from 5 X 106 to 4 x lo6 motile trypomastigotes/ml. Perfusion pumps were stopped for 15 min-2 hr to allow the penetration of the vertebrate cells by trypomastigotes. The entire intracellular cycle was observed in a minimum of 20 individual vertebrate cells at each environmental temperature. Secondary bovine embryo skeletal muscle cells (BESM) were used for these studies. The cells were obtained and cultured as described by Dvorak and Hyde ( 1973) ; cell dispersions were prepared as
152
DVORAK AND POORE
FIG. 1. Isometric projection of the interrelationship of time and temperature to the penetration of BESM cells by Tryyanosoma cruzi trypomastigotes. The two heavy lines depict the effect of temperature at 2 and 4 hr intervals; the 4 light lines depict the effect of time at 29, 32, 35, and 38 C.
Within the temperature range of 29 35 C, the time-dependent penetration rate [penetration index/hr] increased nonlinearly and the penetration rate at 4 hr was always greater than at 2 hr. At 38 C, however, the 2 hr penetration rate was identical to the 35 C 2 hr rate [0.50] and by 4 hr, it had decreased to [0.33]. The distribution of parasites among vertebrate cells did not change over the 2938 C temperature range and 4 hr time course used for these studies. In all cases, a negative binomial distribution occurred even though the mean number of parasites/cell varied from 0.01 to 21.91. No attempt was made to quantify the motility of trypomastigotes during the 4 hr time period of these studies. Qualitatively, I
I
29
32
I
I
35
38
described by Luban and Dvorak (1974) except that, for penetration studies, the serum supplement consisted of 10% heat inactivated fetal bovine serum (FBS). Trypomastigotes of the Ernestina strain of 2’. cruzi, grown in BESM cell culture, were used for these studies. The history of this strain and the preparative procedures required for its use were described by Dvorak and Schmunis ( 1972). RESULTS
The effects of time and temperature on the kinetics of penetration of BESM cells by trypomastigotes are shown in Fig. 1 where, by isometric projection, both parameters are related to penetration index. An increase in temperature from 29 to 35 C resulted in a nearly linear increase in the temperature-dependent penetration rate (penetration index/degree C) at both the 2 hr and 4 hr intervals. However, the 4 hr rate (0.39) was more than twofold greater than the 2 hr rate (0.16). From 35 to 38 C, the 2 hr rate remained constant (0.16) and the 4 hr rate decreased sharply ( -0.41).
TEMPt°C)
Fm. 2. Effect of temperature on the “lag period,” prior to reproduction, of Typan0som.a cnczi in BESM cells. The solid circles are the mean values obtained at each temperature. The bars represent the standard error of each mean. The open circles at 29, 32, and 35 C represent a leastsquares analysis over this temperature interval (slope = -2.26 2 0.76).
T. cruxi: TEMPERATURE AND VERTEBRATE CELLS it was established that motile trypomastigotes were present after 4 hr incubation at the 4 temperatures used for this study. The morphologic course of events of the intracellular cycle was essentially the same at 29, 32, and 38 C as described previously at 35 C. Following penetration of the host cell, trypomastigotes reorganized to amastigotes and, after a lag period, reproduced and differentiated to trypomastigotes which escaped from the dead host cell (cf. Dvorak and Hyde 1973, for details). The major effects of different environmental temperatures were changes in the time required by the parasite to traverse the lag period and reproductive phase. As environmental temperature increased from 29 to 35 C, the lag period, i.e., the time interval between the penetration of the host cell by a trypomastigote and the onset of reproduction by an amastigote, decreased linearly (Fig. 2). The lag period lasted for 48.4, 39.8, and 34.9 hr at 29, 32 and 35 C, respectively. At 38 C, it lasted only 18.4 hr.
TEMP. (‘C)
FIG. 3. Effect of temperature on the doubling time of Typanosomu cruzi in BESM cells. The solid circles are the mean values obtained at each temperature. The bars represent the standard error of each mean.
153
f (x 10-31 FIG. 4. Arrhenius plot of doubling time (open circles) and lag period (solid circles) of Typanosomu cruxi in BESM cells.
An increase in environmental temperature from 32 to 38 C resulted in a linear increase in the time required for amastigotes to reproduce (Fig. 3). The doubling time of amastigotes was 12.0 hr at 32 C, 13.8 hr at 35 C and 15.7 hr at 38 C. At 29 C, the doubling time increased to 18.3 hr. Amastigotes reproduced for 9 generations and, subsequently, differentiated to trypomastigotes which escaped from the dead host cell. Trypomastigotes produced at the 4 temperatures used for this study were capable of invading new vertebrate cells and completing another intracellular cycle. Arrhenius plots of lag period and doubling time data (Fig. 4) confirmed tbe linear temperature dependence of the lag period in the 29-35 C range and of the reproductive phase in the 32-38 C range. Calculations of thermodynamic parameters of the lag period and reproductive phase were based upon the assumption that the rate at which the parasite passed through these phases is inversely proportional to the time required to complete the phase. The resulting activation energy, enthalpy, en-
154
DVORAK AND POORE TABLE Comparison
of Thermodynamic
Parameters of Trypanosoma Compared with Other Cell Types
Cell type
Trypanosoma cruzi grown in LIT reproductive phase”
cruzi Life Cycle
Activation energy, EG (Cal/mole)
Activation enthalpy, AH* (Cal/mole)
11,279 19,387 - 10,174
10,107 18,191 - 11,394
-2.4 31 -64
11,385 8832 8242
10,079 -8334
8867 -9558
-5.9 -68
10,660 11,328
27,500
27,500
Activation Free energy entropy, of activation, AS* AG* (cal/deg mole) (Cal/mole)
medium,
(19-25 C) (25-31 C) (31-37 C) Trypanosoma
I
cru.zi grown in BESM
cellsb
lag period (29-35 C) phase (32-38 C) reproductive Vertebrate cells type L5178Y, reproductive phase” (31-37 C) D Derived from the data of Fernandes b Present study. c From Watanabe and Okada (1967).
and Castellani,
tropy and free energy of the two phases are presented in Table I together with values for other cell types. The net result of temperature mediated changes in the lag period and reproductive phase on the overall intracellular cycle is shown in Fig. 5. Although both the lag period and #doubling time changed, these changes complemented each other in the 32-38 C temperature range. Consequently, the length of the overall cycle remained relatively constant at 6.1-6.5 days. At 29 C, however, the overall cycle lengthened to 8.9 days. DISCUSSION
A study of the effects of environmental factors on the interaction of T. crud with vertebrate cells in vitro permits an insight to mechanisms operative in the basic biological processes of the parasite and an elucidation of comparative differences between the biological processes of the host cell and the parasite. This, hopefully, will provide a better understanding of the interaction as it occurs in nature.
-118
Fig. 2, (1966).
Three parameters of the parasite’s cycle ( penetr,ation of the host cell, the lag period prior to reproduction and the doubling time) changed as a result of changes in environmental temperature. The first question that arises is whether these changes are ‘due to the direct action of varying temperature on the physiology of the parasite or whether they reflect temperaturemediated alterations in host cell physiology affecting parasite physiology. Two types of observations support the hypothesis that the changes in the rates of these two phases of the parasite’s life cycle were a direct consequence of temperature variation. First, although environmental temperature does affect vertebrate cells directly, the effect on the reproductive phase, for example, is markedly different from that observed with T. cruxi (cf. Watanabe and Okada (1967) for details of the reproductive phase of vertebrate cells). Also, the intracellular reproductive phase of T. cmzi in the 32-38 C range is bioenergetically identical to that observed with parasites growing in liquid medium (Table I).
T.
ClUZi:
TEMPERATURE
Second, in other host-parasite systems it has been demonstrated that there is a temperature-modulated difference in the sensitivity of certain synthetic pathways present in both the host and parasite (cf. Stevens 1966). Consequently, parasites can and do respond directly to variations in environmental temperature. There are, in general, three categories of response of living organisms to changes in environmental temperature: ( 1) direct, (2) compensatory acclimation and (3) genetic (Prosser 1967). The first two responses may be considered as parts of the time course of the reaction of an organism to changes in temperature and are probably most relevant to phenomena reported here. Genetic response is random and nondirected and it is unlikely that small environmental changes can act ‘directly on genetic material to induce adaptive mutations. The linear relationship of penetration index to temperature ‘over the 6 degree range from 29 to 35 C implies that trypomastigotes respond directly to environmental stimulus. The increased temperature-dependent penetration rate at 4 hr compared to 2 hr could be due to factors such as a lag time prior to the onset of host cell penetration by trypomastigotes which would lower the apparent 2 hr rate or, a time‘dependent physiologic change in trypomastigotes increasing their .ability to penetrate cells. Data elucidating the mechanism responsible for this phenomenon are not available. However, the fact that the 2 hr, 38 C penetration index is identical to the 2 hr, 35 C value and the time-dependent penetration rate decreased at 38 C implies that a direct physiologic change in the trypomastigotes occurred and that the mechanism responsible for penetration is temperature sensitive. Temperatures above 35 C probably can be considered supraoptimal and, consequently, penetration rate limiting. These observations may be relevant to infection as it occurs in nature. For example, the surface temperature of
AND VERTEBRATE
j3-
I
155
CELLS
I
I
I
I
I
: I : / I- ^ _ UlZ3456769
I -
I
I
1
I
I
1
2-
TIME
I
I
(days)
FIG. 5. Overall cycle of TTypunosonw crwi in BESM cells at four different temperatures. -29 c; -32C;---35C;----38C.
susceptible host cells is greatly affected by ambient temperature. Penetration of these cells by trypomastigotes ‘deposited with insect feces would, as shown, be directly affected. Thus, it seems probable that infection occurs efficiently in a narrow temperature range only. The decrease in the length of the lag period prior to reproduction as temperature increased implies that this stage in the parasite’s intracellular cycle is not, physiologically, static. Consequently, the term “lag period” in the physiologic sense is a misnomer. There is probably a number of interrelated physiologic events involving the transition from a motile nonreproductive trypomastigote to a sessile reproductive amastigote which occur during this period. Thermodynamic parameters (Table I) support the premise that loosely ordered, energy requiring reactions are oc-
156
DVORAK AND POORE
curring at temperature
limiting
rates in the
29-35 C range. The sharp decrease in the length of the lag period between 35 C and 38 C infers that a different rate constant may be modulating the overall process at the higher temperature. The linear increase in the reproductive phase in the 32-38 C range implies that this stage in the parasite’s intracellular cycle contains a thermolabile rate limiting step. As stated previously, the similarity in the thermodynamic parameters of the intraand extracellular reproductive phase indicates that the overall bioenergetics are the same for both situations. The negative entropy, AS”, value confirms the premise that ordered structures are being formed from disordered precursors (e.g., nucleotides + DNA). The positive free energy, aG*, implies that the process is not spontaneous. The unusual thermodynamic values of a negative activation energy, E,, and enthalpy, AH*, imply that there is a net loss of energy involved in the reproductive process in the 32-38 C range. This is significantly different from the reproductive phase of vertebrate cells in the 3137 C range or T. cmczi in the 19-31 C range, both of which require substantial amounts of energy. It would be important to determine whether this aspect of the reproductive cycle is unique to T. cruzi or a general phenomenon of pokilothermic parasites growing in the core temperature range of their homiothermic hosts. Recently, Ray and Ghosh (1973) reported that in Leishmania donosani the incorporation of labeled precursors into RNA at 37 C was markedly lower than at 22 C, the optimum growth temperature for the parasite. They demonstrated that RNA polymerase activity was not affected at 37 C but there was an increased degradation rate of the RNA. possibly, this type of phenomenon, which would effectively inhibit growth of the organism, also may be occurring with T. cruzi
.
Several workers have noted that changes in environmental temperature influence the course of a T. cruzi infection in homiotherms. Lowering of environmental temperature, for example, results in a marked increase in parasitemia and decreased survival time of infected animals. It is impossible to say whether the core temperature of the animals was changed as a result of changes in environmental temperature. It is unlikely that it was markedly affected, however. Therefore, any change in in viva morbidity or mortality of infected animals may be due to secondary effects of temperature on the homiotherm and not on the parasite itself. ACKNOWLEDGMENTS The authors express appreciation to Mr. Albert Gam for technical assistance and Dr. David Alling for assistance with statistical analysis of the lag period data.
REFERENW AMREIN, Y. U. 1967. Effects of environmental temperature on T ypanosoma cruzi infection in mice. Journal of Parasitology 53, 1160. DVORAK, J. A,, AND STOTLER, W. 1971. A controlled-environment culture system for high resolution light microscopy. Experimental Cell Research 68, 144-148. DVORAK, J. A., AND SCXMUNIS, G. A. 1972. T ypunosomu cruzi: Interaction with mouse peritoneal macrophages. Erpem’mentul Pamsitology 32, 289300. DVORAK, J. A., AND HYDE, T. P. 1973. Typunosoma cruzi: Interaction with vertebrate cells in uitro. I. Individual host cell-parasite interactions at the cellular and subcellular level. Experimental Parasitology 34, 268-283. FERNANDES, J. F., AND CASTELLANI, 0. 1966. Growth characteristics and chemical composition of Typanosomu cruzi. Experimental Parasitology 18, 195-202. FRANCA-RODRIGUEZ, M. E., AND MACKINNON, J. E. 1962. Effect0 de la temperatura ambiental sobre la infection por Typanosomu cruzi. Anales de la Fact&ad de Medicinu, Montevideo 47, 319-313. HYDE, T. P., AND DVORAK, J. A. 1973. Typunosomu cruzi: Interaction with vertebrate cells
T.
crUZi:
TEMPERATURE
in u&o. II. Quantitative analysis of the penetration phase. Experimental Parasitology 34, 284-294. KOLODNY, M. H. 1940. The effect of environmental temperature upon experimental trypanosomiasis (T. cruzi) of rats. American Journal of Hygiene 32, 21-23. LUBAN, N. A., AND DVORAK, J. A. 1974. Typanosoma cruzi: Interaction with vertebrate cells in vitro. III. Selection for biological characteristics following intracellular passage. Experimental Parasitology 36, 143-149. MARINICELLE, C. J., AND RODRIGUEZ, E. 1968. The influence of environmental temperature on the pathogenicity of Typanosoma crud in mice. Experimental Parasitology 23, 260-263. NEVA, F. A., MALONE, M. F., AI~D MYERS, R. 1961. Factors influencing the intracellular growth of Typanosoma cwzi in u&o. American Jourd of Tropical Medicine and Hygiene 10, 146-154. NEVES, D. P. 1971. Influencia da temperatura na evolucao do Typanosoma cruzi em triatomineos. Reuista do In&i&to de Medicina Tropical de Sao Paul0 13, 155-161. PEZZI, T., AND CHRISTEN, R. 1950. Influencias de variaciones mantenidas de cultivo de temperatura y pH en medios de cultivo de Typanosoma cm&. Boletin de Informaciones Parasitarias Chibnas, Santiago de Chile 5, 5.
AND
VERTEBRATE
CELLS
PROSSER, C. L. 1967. In “Molecular
157
Mechanisms of Temperature Adaptation,” pp. 351376. AAAS, Washington, DC. RAY, D. K., AND GHOSH, D. K. 1973. Leishmania donouani: Effect of temperature on RNA metabolism. Experimental Parasitology 33, 147-154. RIBEIRO, L. V., KIMIJRA, E., AND FERNANDES, J. F. 1969. Factors affecting the density of intracellular infection of tissue culture cells by Typanosoma cruzi. Revista Bmsileira de Biologia, Rio de Janeiro 29, 295-308. STEVENS, J. G. 1966. Selective inhibition of Herpesvirus DNA synthesis at elevated temperature. Virology 29, 570-579. TREJOS, A., GODOY, G. A., GREENBLA~, C., AND CEDILLOS, R. 1963. Effects of temperature on morphologic variation of Schizotypanum crud in tissue culture. Experimental Parasitobgy 13, 211-218. TREJOS, A., URQUILLA, M. A., AND PAREDES, A. R. 1965. Influence of environmental temperature on the evolution of experimental Chagas’ disease in mice. In “Progress in Protozoology,” International Congress Series No. 91, p. 144. Excerpta Medica Foundation, New York. WATANABE, I., AND OKADA, S. 1967. Effects of temperature on growth rate of cultured mammalian cells (L5178Y). Journal of Cell Biology 32, 309-323.
36, 150-157 ( 1974)
PARASITOLOGY
Trypanosoma
cruzi: Interaction Cells in Vitro
IV. Environmental
Temperature
JAMES A. DVORAK AND Laboratoy
with Vertebrate Effects
CHARLES M. POORE
of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 (Submitted for publication September 24, 1973)
DVORAK, J. A., AND POORE, C. M. 1974. Typanosoma cruzi: Interaction with vertebrate cells in vitro. IV. Environmental temperature effects. Experimental Parasitology 36, 150-157. The effects of 4 environmental temperatures (29, 32, 35, and 38 C) on the interaction between Typanosoma cruzi and bovine embryo skeletal muscle cells were quantified. Three aspects of the interaction (penetration of host cells by trypomastigotes, the lag period prior to the reproductive phase, and the reproductive phase) were markedly affected by temperature. There was a linear increase in the number of trypomastigotes penetrating cells in the 29-35 C range. Temperatures above 35 C can be considered supraoptimal as no further increase in the rate of penetration occurred. The lag period decreased linearly as temperature increased in the 29-35 C range; at 38 C, the lag period was markedly shortened. The doubling time of amastigotes increased linearly as temperature increased in the 32-38 C range; at 29 C, the doubling time was markedly lengthened. At all temperatures, parasites reproduced for 9 generations before cell rupture. The changes in lag period and doubling time complemented each other in the 32-38 C range. Thus, there was essentially no change in the overall length of the intracellular cycle which lasted 6.1 to 6.5 days. At 29 C, however, the cycIe was lengthened to 8.9 days. Thermodynamic analysis revealed marked differences, characterized by a negative activation energy and negative enthalpy, between the reproductive phase of parasites within vertebrate cells and the vertebrate cells themselves. However, the thermodynamic parameters of parasites reproducing extracellularly in liquid medium and intracellularly were the same. INDEX DESCRIPTORS: Tlypanosome cruzi; Cell lines; Bovine embryo skeletal muscle; Life cycles; Culture, in &TO; Thermodynamic analysis; Enthalpy; Temperature; Entropy; Energy, activation; Bioenergetics.
IWI-RODIJ~TI~N
The effects of different environmental temperatures on the growth and development of Typanosma cruzi have been studied by several workers. Pizzi and Christen (1950) and Fernandes and Castellani (1966) described their effects on parasites maintained in liquid medium; Neva et al. (1961), Trejos et al. (1963)
and Ribeiro et ~2. (1969) described their effects on the growth and development of parasites maintained in tissue culture. The effects of environmental temperature on the in viva course of T. cmcxi infections in vertebrates were described by Kolodny ( 1940)) Franca-Rodriguez and Mackinnon (1962), Trejos et al. (1965), Amrein (1967) and Marinkelle and Rodriguez 150
6 1974 by Academic Press, of reproduction in any form
Inc. reserved.
T. C?'UZi: TEMPEBATIJIUZ ANDVFZTEBBATFi
(1968) and in invertebrates by Neves (1971). It was established, in all cases, that changes in the environmental temperature produced alterations in the growth characteristics or development of the parasite. However, elucidation of specific temperature sensitive stages of the parasite’s cycle as well as the interrelationship of temperature sensitive stages within the cycle was not achieved due to a lack of suitable quantitative techniques. This resulted in difficulties in interpreting temperature sensitive phenomena that occurred. The ability to observe and quantify the entire intracellular cycle of T. crU2i in vertebrate cells (Dvorak and Hyde 1973) as well as quantify the kinetics and stoichiometry of the penetration phase of the interaction (Hyde and Dvorak 1973) allows a quantitative study of the effects of physical and/or chemical parameters for all stages of the cycle at a level of precision previously ,unobtainable. This report presents the results of quantitative studies of the effects of different environmental temperatures on the cycle of T. cruzi in vertebrate cells utilizing these techniques. MATERIALS AND METHODS
Two experimental protocols were used to quantify the effects of different environmental temperatures (29, 32, 35, and 38 C) on the complete cycle of T. cruxi in vertebrate cells. The penetration of vertebrate cells by trypomastigotes was quantified by the methods of Hyde and Dvorak ( 1973). Vertebrate cells established on coverglasses 24 hr prior to use at a concentration of lo4 cells/ml were exposed to parasites for 0, 2, or 4 hr periods at parasite concentrations ranging from 5 X lo5 to 2 X lo6 motile trypomastigotes/ml. All parasite suspensions were equilibrated to temperature prior to use. Two thermostatically controlled water baths, maintained to a tolerance of less than -t-OS C of the required
CELLS
151
temperature, were employed. One water bath was set at 35 C and constituted the control temperature; the other water bath was adjusted to 29, 32, or 38 C and constituted the experimental temperature. Thus, 35 C was the reference temperature and the mean number of parasites/cell for each experimental temperature was corrected by calculating its value relative to the 35 C reference. The 35 C, 2 hr reference was assigned a value of unity and the individual penetration rates were corrected for each time-temperature pair. The corrected value for the mean number of parasites/ cell, which allowed the interrelation of all temperatures studied, was termed the “penetration index.” The “penetration index” for each temper,ature pair was determined for two time intervals on three separate occasions. The effects of environmental temperature on the course of the intracellular cycle were quantified in individual vertebrate cells using a controlled-environment culture system (Dvorak and Stotler 1971). Vertebrate cells were established at a concentration of 2 x lo4 cells/ml and temperature equilibrated on the microscope stage prior to the introduction of trypomastigotes. Temperature was maintained to a tolerance of kO.2 C of the required temperature by means of a proportional-controlled air stream incubator (Nicholson Precision Instruments, Model C-300). Parasites were inoculated into culture chambers at concentrations ranging from 5 X 106 to 4 x lo6 motile trypomastigotes/ml. Perfusion pumps were stopped for 15 min-2 hr to allow the penetration of the vertebrate cells by trypomastigotes. The entire intracellular cycle was observed in a minimum of 20 individual vertebrate cells at each environmental temperature. Secondary bovine embryo skeletal muscle cells (BESM) were used for these studies. The cells were obtained and cultured as described by Dvorak and Hyde ( 1973) ; cell dispersions were prepared as
152
DVORAK AND POORE
FIG. 1. Isometric projection of the interrelationship of time and temperature to the penetration of BESM cells by Tryyanosoma cruzi trypomastigotes. The two heavy lines depict the effect of temperature at 2 and 4 hr intervals; the 4 light lines depict the effect of time at 29, 32, 35, and 38 C.
Within the temperature range of 29 35 C, the time-dependent penetration rate [penetration index/hr] increased nonlinearly and the penetration rate at 4 hr was always greater than at 2 hr. At 38 C, however, the 2 hr penetration rate was identical to the 35 C 2 hr rate [0.50] and by 4 hr, it had decreased to [0.33]. The distribution of parasites among vertebrate cells did not change over the 2938 C temperature range and 4 hr time course used for these studies. In all cases, a negative binomial distribution occurred even though the mean number of parasites/cell varied from 0.01 to 21.91. No attempt was made to quantify the motility of trypomastigotes during the 4 hr time period of these studies. Qualitatively, I
I
29
32
I
I
35
38
described by Luban and Dvorak (1974) except that, for penetration studies, the serum supplement consisted of 10% heat inactivated fetal bovine serum (FBS). Trypomastigotes of the Ernestina strain of 2’. cruzi, grown in BESM cell culture, were used for these studies. The history of this strain and the preparative procedures required for its use were described by Dvorak and Schmunis ( 1972). RESULTS
The effects of time and temperature on the kinetics of penetration of BESM cells by trypomastigotes are shown in Fig. 1 where, by isometric projection, both parameters are related to penetration index. An increase in temperature from 29 to 35 C resulted in a nearly linear increase in the temperature-dependent penetration rate (penetration index/degree C) at both the 2 hr and 4 hr intervals. However, the 4 hr rate (0.39) was more than twofold greater than the 2 hr rate (0.16). From 35 to 38 C, the 2 hr rate remained constant (0.16) and the 4 hr rate decreased sharply ( -0.41).
TEMPt°C)
Fm. 2. Effect of temperature on the “lag period,” prior to reproduction, of Typan0som.a cnczi in BESM cells. The solid circles are the mean values obtained at each temperature. The bars represent the standard error of each mean. The open circles at 29, 32, and 35 C represent a leastsquares analysis over this temperature interval (slope = -2.26 2 0.76).
T. cruxi: TEMPERATURE AND VERTEBRATE CELLS it was established that motile trypomastigotes were present after 4 hr incubation at the 4 temperatures used for this study. The morphologic course of events of the intracellular cycle was essentially the same at 29, 32, and 38 C as described previously at 35 C. Following penetration of the host cell, trypomastigotes reorganized to amastigotes and, after a lag period, reproduced and differentiated to trypomastigotes which escaped from the dead host cell (cf. Dvorak and Hyde 1973, for details). The major effects of different environmental temperatures were changes in the time required by the parasite to traverse the lag period and reproductive phase. As environmental temperature increased from 29 to 35 C, the lag period, i.e., the time interval between the penetration of the host cell by a trypomastigote and the onset of reproduction by an amastigote, decreased linearly (Fig. 2). The lag period lasted for 48.4, 39.8, and 34.9 hr at 29, 32 and 35 C, respectively. At 38 C, it lasted only 18.4 hr.
TEMP. (‘C)
FIG. 3. Effect of temperature on the doubling time of Typanosomu cruzi in BESM cells. The solid circles are the mean values obtained at each temperature. The bars represent the standard error of each mean.
153
f (x 10-31 FIG. 4. Arrhenius plot of doubling time (open circles) and lag period (solid circles) of Typanosomu cruxi in BESM cells.
An increase in environmental temperature from 32 to 38 C resulted in a linear increase in the time required for amastigotes to reproduce (Fig. 3). The doubling time of amastigotes was 12.0 hr at 32 C, 13.8 hr at 35 C and 15.7 hr at 38 C. At 29 C, the doubling time increased to 18.3 hr. Amastigotes reproduced for 9 generations and, subsequently, differentiated to trypomastigotes which escaped from the dead host cell. Trypomastigotes produced at the 4 temperatures used for this study were capable of invading new vertebrate cells and completing another intracellular cycle. Arrhenius plots of lag period and doubling time data (Fig. 4) confirmed tbe linear temperature dependence of the lag period in the 29-35 C range and of the reproductive phase in the 32-38 C range. Calculations of thermodynamic parameters of the lag period and reproductive phase were based upon the assumption that the rate at which the parasite passed through these phases is inversely proportional to the time required to complete the phase. The resulting activation energy, enthalpy, en-
154
DVORAK AND POORE TABLE Comparison
of Thermodynamic
Parameters of Trypanosoma Compared with Other Cell Types
Cell type
Trypanosoma cruzi grown in LIT reproductive phase”
cruzi Life Cycle
Activation energy, EG (Cal/mole)
Activation enthalpy, AH* (Cal/mole)
11,279 19,387 - 10,174
10,107 18,191 - 11,394
-2.4 31 -64
11,385 8832 8242
10,079 -8334
8867 -9558
-5.9 -68
10,660 11,328
27,500
27,500
Activation Free energy entropy, of activation, AS* AG* (cal/deg mole) (Cal/mole)
medium,
(19-25 C) (25-31 C) (31-37 C) Trypanosoma
I
cru.zi grown in BESM
cellsb
lag period (29-35 C) phase (32-38 C) reproductive Vertebrate cells type L5178Y, reproductive phase” (31-37 C) D Derived from the data of Fernandes b Present study. c From Watanabe and Okada (1967).
and Castellani,
tropy and free energy of the two phases are presented in Table I together with values for other cell types. The net result of temperature mediated changes in the lag period and reproductive phase on the overall intracellular cycle is shown in Fig. 5. Although both the lag period and #doubling time changed, these changes complemented each other in the 32-38 C temperature range. Consequently, the length of the overall cycle remained relatively constant at 6.1-6.5 days. At 29 C, however, the overall cycle lengthened to 8.9 days. DISCUSSION
A study of the effects of environmental factors on the interaction of T. crud with vertebrate cells in vitro permits an insight to mechanisms operative in the basic biological processes of the parasite and an elucidation of comparative differences between the biological processes of the host cell and the parasite. This, hopefully, will provide a better understanding of the interaction as it occurs in nature.
-118
Fig. 2, (1966).
Three parameters of the parasite’s cycle ( penetr,ation of the host cell, the lag period prior to reproduction and the doubling time) changed as a result of changes in environmental temperature. The first question that arises is whether these changes are ‘due to the direct action of varying temperature on the physiology of the parasite or whether they reflect temperaturemediated alterations in host cell physiology affecting parasite physiology. Two types of observations support the hypothesis that the changes in the rates of these two phases of the parasite’s life cycle were a direct consequence of temperature variation. First, although environmental temperature does affect vertebrate cells directly, the effect on the reproductive phase, for example, is markedly different from that observed with T. cruxi (cf. Watanabe and Okada (1967) for details of the reproductive phase of vertebrate cells). Also, the intracellular reproductive phase of T. cmzi in the 32-38 C range is bioenergetically identical to that observed with parasites growing in liquid medium (Table I).
T.
ClUZi:
TEMPERATURE
Second, in other host-parasite systems it has been demonstrated that there is a temperature-modulated difference in the sensitivity of certain synthetic pathways present in both the host and parasite (cf. Stevens 1966). Consequently, parasites can and do respond directly to variations in environmental temperature. There are, in general, three categories of response of living organisms to changes in environmental temperature: ( 1) direct, (2) compensatory acclimation and (3) genetic (Prosser 1967). The first two responses may be considered as parts of the time course of the reaction of an organism to changes in temperature and are probably most relevant to phenomena reported here. Genetic response is random and nondirected and it is unlikely that small environmental changes can act ‘directly on genetic material to induce adaptive mutations. The linear relationship of penetration index to temperature ‘over the 6 degree range from 29 to 35 C implies that trypomastigotes respond directly to environmental stimulus. The increased temperature-dependent penetration rate at 4 hr compared to 2 hr could be due to factors such as a lag time prior to the onset of host cell penetration by trypomastigotes which would lower the apparent 2 hr rate or, a time‘dependent physiologic change in trypomastigotes increasing their .ability to penetrate cells. Data elucidating the mechanism responsible for this phenomenon are not available. However, the fact that the 2 hr, 38 C penetration index is identical to the 2 hr, 35 C value and the time-dependent penetration rate decreased at 38 C implies that a direct physiologic change in the trypomastigotes occurred and that the mechanism responsible for penetration is temperature sensitive. Temperatures above 35 C probably can be considered supraoptimal and, consequently, penetration rate limiting. These observations may be relevant to infection as it occurs in nature. For example, the surface temperature of
AND VERTEBRATE
j3-
I
155
CELLS
I
I
I
I
I
: I : / I- ^ _ UlZ3456769
I -
I
I
1
I
I
1
2-
TIME
I
I
(days)
FIG. 5. Overall cycle of TTypunosonw crwi in BESM cells at four different temperatures. -29 c; -32C;---35C;----38C.
susceptible host cells is greatly affected by ambient temperature. Penetration of these cells by trypomastigotes ‘deposited with insect feces would, as shown, be directly affected. Thus, it seems probable that infection occurs efficiently in a narrow temperature range only. The decrease in the length of the lag period prior to reproduction as temperature increased implies that this stage in the parasite’s intracellular cycle is not, physiologically, static. Consequently, the term “lag period” in the physiologic sense is a misnomer. There is probably a number of interrelated physiologic events involving the transition from a motile nonreproductive trypomastigote to a sessile reproductive amastigote which occur during this period. Thermodynamic parameters (Table I) support the premise that loosely ordered, energy requiring reactions are oc-
156
DVORAK AND POORE
curring at temperature
limiting
rates in the
29-35 C range. The sharp decrease in the length of the lag period between 35 C and 38 C infers that a different rate constant may be modulating the overall process at the higher temperature. The linear increase in the reproductive phase in the 32-38 C range implies that this stage in the parasite’s intracellular cycle contains a thermolabile rate limiting step. As stated previously, the similarity in the thermodynamic parameters of the intraand extracellular reproductive phase indicates that the overall bioenergetics are the same for both situations. The negative entropy, AS”, value confirms the premise that ordered structures are being formed from disordered precursors (e.g., nucleotides + DNA). The positive free energy, aG*, implies that the process is not spontaneous. The unusual thermodynamic values of a negative activation energy, E,, and enthalpy, AH*, imply that there is a net loss of energy involved in the reproductive process in the 32-38 C range. This is significantly different from the reproductive phase of vertebrate cells in the 3137 C range or T. cmczi in the 19-31 C range, both of which require substantial amounts of energy. It would be important to determine whether this aspect of the reproductive cycle is unique to T. cruzi or a general phenomenon of pokilothermic parasites growing in the core temperature range of their homiothermic hosts. Recently, Ray and Ghosh (1973) reported that in Leishmania donosani the incorporation of labeled precursors into RNA at 37 C was markedly lower than at 22 C, the optimum growth temperature for the parasite. They demonstrated that RNA polymerase activity was not affected at 37 C but there was an increased degradation rate of the RNA. possibly, this type of phenomenon, which would effectively inhibit growth of the organism, also may be occurring with T. cruzi
.
Several workers have noted that changes in environmental temperature influence the course of a T. cruzi infection in homiotherms. Lowering of environmental temperature, for example, results in a marked increase in parasitemia and decreased survival time of infected animals. It is impossible to say whether the core temperature of the animals was changed as a result of changes in environmental temperature. It is unlikely that it was markedly affected, however. Therefore, any change in in viva morbidity or mortality of infected animals may be due to secondary effects of temperature on the homiotherm and not on the parasite itself. ACKNOWLEDGMENTS The authors express appreciation to Mr. Albert Gam for technical assistance and Dr. David Alling for assistance with statistical analysis of the lag period data.
REFERENW AMREIN, Y. U. 1967. Effects of environmental temperature on T ypanosoma cruzi infection in mice. Journal of Parasitology 53, 1160. DVORAK, J. A,, AND STOTLER, W. 1971. A controlled-environment culture system for high resolution light microscopy. Experimental Cell Research 68, 144-148. DVORAK, J. A., AND SCXMUNIS, G. A. 1972. T ypunosomu cruzi: Interaction with mouse peritoneal macrophages. Erpem’mentul Pamsitology 32, 289300. DVORAK, J. A., AND HYDE, T. P. 1973. Typunosoma cruzi: Interaction with vertebrate cells in uitro. I. Individual host cell-parasite interactions at the cellular and subcellular level. Experimental Parasitology 34, 268-283. FERNANDES, J. F., AND CASTELLANI, 0. 1966. Growth characteristics and chemical composition of Typanosomu cruzi. Experimental Parasitology 18, 195-202. FRANCA-RODRIGUEZ, M. E., AND MACKINNON, J. E. 1962. Effect0 de la temperatura ambiental sobre la infection por Typanosomu cruzi. Anales de la Fact&ad de Medicinu, Montevideo 47, 319-313. HYDE, T. P., AND DVORAK, J. A. 1973. Typunosomu cruzi: Interaction with vertebrate cells
T.
crUZi:
TEMPERATURE
in u&o. II. Quantitative analysis of the penetration phase. Experimental Parasitology 34, 284-294. KOLODNY, M. H. 1940. The effect of environmental temperature upon experimental trypanosomiasis (T. cruzi) of rats. American Journal of Hygiene 32, 21-23. LUBAN, N. A., AND DVORAK, J. A. 1974. Typanosoma cruzi: Interaction with vertebrate cells in vitro. III. Selection for biological characteristics following intracellular passage. Experimental Parasitology 36, 143-149. MARINICELLE, C. J., AND RODRIGUEZ, E. 1968. The influence of environmental temperature on the pathogenicity of Typanosoma crud in mice. Experimental Parasitology 23, 260-263. NEVA, F. A., MALONE, M. F., AI~D MYERS, R. 1961. Factors influencing the intracellular growth of Typanosoma cwzi in u&o. American Jourd of Tropical Medicine and Hygiene 10, 146-154. NEVES, D. P. 1971. Influencia da temperatura na evolucao do Typanosoma cruzi em triatomineos. Reuista do In&i&to de Medicina Tropical de Sao Paul0 13, 155-161. PEZZI, T., AND CHRISTEN, R. 1950. Influencias de variaciones mantenidas de cultivo de temperatura y pH en medios de cultivo de Typanosoma cm&. Boletin de Informaciones Parasitarias Chibnas, Santiago de Chile 5, 5.
AND
VERTEBRATE
CELLS
PROSSER, C. L. 1967. In “Molecular
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Mechanisms of Temperature Adaptation,” pp. 351376. AAAS, Washington, DC. RAY, D. K., AND GHOSH, D. K. 1973. Leishmania donouani: Effect of temperature on RNA metabolism. Experimental Parasitology 33, 147-154. RIBEIRO, L. V., KIMIJRA, E., AND FERNANDES, J. F. 1969. Factors affecting the density of intracellular infection of tissue culture cells by Typanosoma cruzi. Revista Bmsileira de Biologia, Rio de Janeiro 29, 295-308. STEVENS, J. G. 1966. Selective inhibition of Herpesvirus DNA synthesis at elevated temperature. Virology 29, 570-579. TREJOS, A., GODOY, G. A., GREENBLA~, C., AND CEDILLOS, R. 1963. Effects of temperature on morphologic variation of Schizotypanum crud in tissue culture. Experimental Parasitobgy 13, 211-218. TREJOS, A., URQUILLA, M. A., AND PAREDES, A. R. 1965. Influence of environmental temperature on the evolution of experimental Chagas’ disease in mice. In “Progress in Protozoology,” International Congress Series No. 91, p. 144. Excerpta Medica Foundation, New York. WATANABE, I., AND OKADA, S. 1967. Effects of temperature on growth rate of cultured mammalian cells (L5178Y). Journal of Cell Biology 32, 309-323.