- Email: [email protected]
Extracellular phosphorylation in Leishmania major and Leishmania mexicana during heat shock transformation
Acta Tropica, 56(1994)1-6
1
ACTROP 00338
Extracellular phosphorylation in Le&hmania major and Leishmania mexicana during heat shock transformation Tom,is H e r m o s o a'*', Jos6 Luis Prrez b, Lisbeth Cornivelli b a n d A n g e l G. H e r n a n d e z b alnstituto de Medicina Tropical, Universidad Central de Venezuela, Apartado postal 47019, Caracas 1040, Venezuela and bCentro de Biologia Celular, Universidad Central de Venezuela, Caracas, Venezuela (Received 2 August 1993; accepted 30 August 1993)
The extracellular phosphorylation of exogenous substrates have been studied in two Leishmania isolates, L. mexicana and L. major, that differ in their capacity to transform from promastigotes to amastigote-like cells when submitted to heat shock condition. When submitted to heat shock both parasites showed an increase in their extracellular phosphorylation activity. L. major promastigotes that do not transform to amastigote-like cells at 37°C, do not phosphorylate exogenous substrate when the culture is grown at 25°C. In contrast, L. mexicana promastigotes, that transform to amastigote-like cells at 37°C, showed a strong phosphorylation at this temperature. As response to heat shock exposure, the extracellular phosphorylation for L. major increases steadily reaching a maximum at 360 min. On the other hand, L. mexicana also show an increase in phosphorylation during transformation, but in this case the maximum was detected after 10 min. The results are discussed in relationship to the capacity of the parasite to survive once inside the vertebrate host. Key words: Leishmania; Phosphorylation; Heat shock
Introduction Leishmania are unicellular protozoan parasites which cause a spectrum of different diseases in man including the three main forms: cutaneous, mucocutaneous and visceral leishmaniasis. The parasites have a relatively simple digenic life cycle (Dwyer and Gottlieb, 1983; Pearson et al., 1983). The extracellular form, the promastigote, resides and multiplies in the sandfly vector. Upon transmission to an appropriate mammalian host, the promastigotes are ingested by phagocytes (Chang and Bray, 1985). An additional aspect of the developmental cycle of Leishmania concerns the sequential development of sandfly promastigotes from a noninfective stage to an infective stage that can be mimicked in vitro by logarithmic and metacyclic stationary phase promastigotes (Sacks et al., 1985). During axenic transformation to metacyclic form, protein kinase activity increases, and phosphorylation pattern changes in L. major (Mukhopadhyay et al., 1988; *Corresponding author. SSDI 0001-706X(93)E0071 -S
Hermoso, 1989). Like few other eukaryotic cells Leishmania major have been shown to possess an external oriented surface protein kinase (Das et al., 1986; Lester et al., 1990). However, very little is known concerning L. mexieana. It was shown that live promastigotes of L. major phosphorylate components of the complement cascade (Hermoso et al., 1991); this points to a key event since these parasites utilize complement receptors on the macrophage to gain entrance into the host cells, where they persist and replicate (Blackwell et al., 1986). Leishmania experience rapid changes in their external environment as they pass from the sandfly midgut to the host. One of these changes involves a temperature shift; when promastigotes growing axenically are subjected to heat shock they develop into amasti-gote-like cells (Van der Ploeg et al., 1985) which have similar properties as the tissue derived amastigotes (Rainey et al., 1991). It has been reported that different species of Leishmania respond similarly to heat shock conditions via the production of heat shock proteins (Carratu and Maresca, 1992). However, the process of morphological transformation differs among them (Shapira et al., 1988). In this paper we demonstrate an increased activity of extracellular phosphorylation of exogenous substrates by L. major and L. mexicana promastigotes during the heat shock transformation. The kinetics of the process differ between the two species. The possible relationship of this increase of the protein kinase activity with the biological behavior of Leishmania is discussed.
Materials and methods
Reagents [~,-32p]ATP (3000 Ci/mmol) was obtained from Amersham International. Calf thymus histones II-S, salmon sperm protamine sulfate, phenyl-methylsulfonyl fluoride (PMSF), were from Sigma Chemical Co. All the other chemicals were analytical reagent grade. Parasites Promastigotes (day 4 in culture) from L. major (P strain, Liverpool reference number LV39) was obtained from Dr. R. Neal (London School of Hygiene and Tropical Medicine, England). L. mexicana (9012) was initially isolated from a human patient from Tfichira state, Venezuela. The 9012 strain of parasites were grown at 25°C in Schneider's Drosophila medium (Jaffe et al., 1984) containing 10% fetal calf serum, penicillin (2 U/ml) and streptomycin (2 U/ml). The parasites of the P strain were grown in continuous culture at 32°C in Schneider's medium, these cells do not undergo morphological changes at this temperature. Promastigotes (3 x 10 7 cells) were transferred to heat shock conditions at 37°C for varying times. Parallel cultures were left at 25°C. Phosphorylation of exogenous proteins Logarithmic phase promastigotes of L. major were washed twice with 20 mM TrisHC1 pH 7.2, 150 mM NaC1 and 2 mM glucose (labeling buffer) and resuspended at
108 cells/100 ~tl in labeling buffer plus 10 m M magnesium and protease inhibitors (EGTA 10 m M and P M S F 1 mM). To induce heat shock, the cells were incubated at 37°C during various periods of time. At the end of the heat shock period, the preparations were supplemented with protamine sulfate or mixed histone (10 Ixg each), then [7-32p]ATP (10 gCi per tube) was added and incubation was resumed for a further 15 min period at 37°C. Cells were then pelleted at 4°C (5 min, 6000 rpm), the supernatant removed and recentrifuged for 10 min at 14 000 rpm in an Eppendorf microfuge. The supernatants were analyzed by SDS-PAGE and autoradiography. For the study of phosphorylation of proteins from the parasite the scheme was the same but the addition of exogenous substrates was avoided.
Results
Fig. l a shows that in L. major promastigotes the phosphate incorporation into protamine sulfate increased steadily with time of exposure to heat shock, from almost zero at 25°C to a m a x i m u m detected after 360 min at 37°C (maximum period tested). In contrast L. mexicana promastigotes (9012) phosphorylated the exogenous substrate at 25°C. However, as in the case of L. major, an increase in the phosphorylation of protamine by L. mexicana was observed during heat shock. Digitalization of the autoradiogram indicates that a value of 4.19 m m 2 is reached after 20 min of heat shock for L. major. L. mexicana promastigotes behave as L. major, but the change started as soon as after 1 min of incubation at 37°C (Fig. lb). The increase in phosphorylation activity was 100% after 10 min heat shock. The increased phosphorylation of exogenous substrates by L. major during heat shock transformation was also shown, using a mixed histone substrate (Fig. 2). Again, as with protamine, almost no phosphorylation was detected prior heat shock (time 0, 25°C). On the other hand, 20 min after heat shock exposure, the phosphorylation of two components of the mixed histones could be detected. In this case the phosphorylation increases with time until 360 min. Fig. 3 shows that not only exogenous substrates such as protamine and histones
0012
p
9012
O
20
120
240
360
0
(rain)
0
1
2.5
5
10
0
(rain)
Fig. 1. Analysis of extracellular protein kinase activity to phosphorylate protamine sulfate during heat shock transformation. Viable promastigotes of L. major (P) or L. mexicana (9012) were exposed to a heat shock temperature of 37°C for up to 360 min (a), whereas L. mexicanapromastigoteswere transferred to a heat shock from 25°C to 37°C for up to 10 min (b). In all cases the parasites were incubated for 15 min with [y-32p]ATPafter the indicated heat shock temperature, then sedimented and the supernatant collected and analyzed on SDS-PAGE.
kDa ii~ii~iiii!~ili~ii~ii~i
97.4
i!i:i!i~i!:i!~i~iii~i!i!~
662
45.0
!iiiiiiiiiiiiiiiiiiiiiiii!!i!iii!
31.0
21.5
iii::i~i !!iii!iiii!iiiiiiiiiiiiiiiill
!:~:iiiiiiiii!i!i;:iii!iiiiiii
14.4
iiii!i]iii!iii!ii!iiiiiil
0
20
60
120
360
(min)
Fig. 2. Change in the extracellular protein kinase activity in L. major (P). Phosphorylation of exogenous mixed histones by viable promastigotes. The parasites were incubated in presence of ['¢-32p]ATP after 20, 60, 120, and 360 min as described in Materials and methods.
P
9012 kDa 974 662 !i!!ii!!~iiii~iii~ii~!: !!i: 450 310
ilAi !!~L~i!~!!~i ~ ~ ~
215
~ii~:i!:i!,~ii~~!i~!i!:~i~ O
20
60
120
360
0
14 4
(min)
Fig. 3. Phosphorylation of released phosphoproteins from Leishmania major (P). Cells submitted to heat shock during the indicated time were incubated with [7-s2p]ATP, were sedimented and supernatant collected and analysed by SDS-PAGE (10%).
5 can be phosphorylated during heat shock exposure, since phosphorylation of three L. major peptides of 31, 48 and 50 kDa was observed after 120 min at 37°C.
Discussion Surface protein kinases have been identified in Leishmania donovani, the causative agent of visceral leishmaniasis (Das et al., 1986), and in L. major, responsible in some cases of causing cutaneous leishmaniasis (Lester et al., 1990). The localization of these enzymes in the Leishmania cell surface is unusual, as protein kinases are generally regarded as intracellular enzymes. Not much is known about the extracellular phosphorylation capacity of New World species such as Leishmania mexicana. Throughout their life these parasites encounter stressful environmental conditions, including dramatic temperature fluctuations. In certain species of Leishmania an increase in the temperature of the promastigote culture from 26°C to 36°C induces morphological changes, whereas in others it does not. In this regard, the New World and Old World Leishmania differ in their response; at 36°C cutaneous species of South American origin often lose motility and become rounded or elliptical (Stinson et al., 1989). In contrast, promastigotes of the Old World species such as L. major or L. donovani, upon heat shock, very often do not undergo such change and fail to survive under these conditions. The work presented in this paper has been carried out with a continuous culture of L. major promastigotes that fail to transform under conditions of heat shock (Shapira et al., 1988). The morphological changes observed are moderate with an incomplete retraction of the flagellum; however, at the molecular level they gave the heat shock response (Shapira et al., 1988). This group of parasites fail to phosphorylate exogenous substrates when grown at 25°C, but the phosphorylation process starts after 20 min of heat shock at 37°C and increases steadily up to 360 min for both histone and protamine sulfate. On the other hand, L. mexicana promastigotes transform very rapidly and appear as amastigote-like cells after 4 h under heat shock conditions. Also, these promastigotes at 25°C are able to phosphorylate protamine sulfate. The phosphorylation activity of L. mexicana promastigotes when submitted to heat shock has a steep increase reaching a maximum after 10 min. Although these results do not necessarily indicate a direct relation between phosphorylation and transformation, it is interesting that parasites with similar phosphorylation behavior as L major and L. donovani (Lester et al., 1990; Das et al., 1986) fail to transform or transform at very slow rate, in contrast to a rapid transformation of New World species of L. mexicana. These results and the fact that L. major virulent promastigotes selected by PNA- agglutination have an increased number of proteins phosphorylated (Hermoso, 1989) and that purified leishmanial kinase LPK-I is able to phosphorylate proteins of the complement cascade (C3, C5, and C9), suggest a potential involvement of phosphorylation during the invasion process, and in the capacity of the parasite to survive once inside the vertebrate host.
Acknowledgements This investigation received financial support from the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICIT) (S1-2561, to T.H.).
References Blackwell, J.M., Ezekowitz, R.A.B., Roberts, M.B., Channon, J.Y., Sim, R.B. and Gordon, S. (1985) Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum. J. Exp. Med. 162, 324 331. Carratu and Maresca (1992) The Biology of the heat shock response in parasites. Parasitol. Today 8 (8), 260-266. Chang, K.P. and Bray, R.S. (1985) Biology of Leishmania and Leishmaniasis, In: K.P. Chang and R.S. Bray (Eds.), Leishmaniasis, Vol. 1, Elsevier, Amsterdam, pp. 1-30. Das, S., Saha, A.K., Mukhopadhyay, N.K. and Glew, R.H. (1986) A cyclic nucleotide-independent protein kinase in Leishmania donovani. Biochem. J. 240, 641-649. Dwyer, D.M. and Gottlieb, M. (1983) The surface membrane chemistry of Leishmania. J. Cell. Biochem. 23, 35-47. Hermoso, T. (1989) Protein phosphorylation and dephosphorylation in the protozooan parasite Leishmania major. Thesis, Weizmann Institute of Science, Rehovot, Israel, p. 76. Hermoso, T., Fishelson, Z., Becker, S., Hirshberg, K. and Jaffe, C.L. (1991) Leishmanial protein kinases phosphorylate components of the complement system. EMBO J. 10, 4061 4067. Jaffe, C.L., Grimaldi, G. and McMahon-Pratt, D. (1984) The cultivation and cloning of Leishmania. In: C.M. Morel (Ed.), Genes and Antigens of Parasites, UNDP/World Bank/WHO, pp. 580. Lester, D., Hermoso, T. and Jeffe, C.L. (1990) Extracellular phosphorylation in the parasite, Leishmania major. Biochim. Biophys. Acta 1052, 293-298. Mukhopadhyay, N.K., Saha, A.K., Lovelace, J.K., Da Silva, R., Sacks, D.L. and Glew, R.H. (1988) Comparison of the protein kinases and acid phosphatase activity of five species of Leishmania. J. Protozool. 35, 601-607. Pearson, R., Wheeler, D., Harrison, L. and Kay, D. (1983) The inmunobiology of leishmaniasis. J. Infect. Dis. 5, 907-927. Rainey, P.M., Spithill, T.W., McMahon-Pratt, D. and Pan, A.A. (1991) Molecular characterization of Leishmania pifanoi amastigotes in continuos axenic culture. Mol. Biochem. Parasitol. 49, 111 116. Sacks, D.L., Hieny. S. and Sher, A. (1985) Identification of ceil surface carbohydrate and antigenic changes between noninfected and infected developmental stage of Leishmania major promastigotes. J. Immunol. 135, 564-569. Shapira, M., McEwen, J. and Jaffe, C.L. (1988) The temperature effects on molecular processes which lead to stage differentiation in Leishmania. EMBO J. 7, 2895-2901. Stinson, S., Sommer, J.R. and Blum, J.J. (1989) Morphology of Leishmania braziliensis: Changes during reversible heat-induced transformation from promastigote to an ellipsoidal form. J. Parasitol. 75, 431-440. Van der Ploeg, L.H.T., Giannini, S. and Cantor, C. (1985) Heat shock genes: Regulatory role for differentiation in parasitic protozoa. Science 228, 1143-1146.
1
ACTROP 00338
Extracellular phosphorylation in Le&hmania major and Leishmania mexicana during heat shock transformation Tom,is H e r m o s o a'*', Jos6 Luis Prrez b, Lisbeth Cornivelli b a n d A n g e l G. H e r n a n d e z b alnstituto de Medicina Tropical, Universidad Central de Venezuela, Apartado postal 47019, Caracas 1040, Venezuela and bCentro de Biologia Celular, Universidad Central de Venezuela, Caracas, Venezuela (Received 2 August 1993; accepted 30 August 1993)
The extracellular phosphorylation of exogenous substrates have been studied in two Leishmania isolates, L. mexicana and L. major, that differ in their capacity to transform from promastigotes to amastigote-like cells when submitted to heat shock condition. When submitted to heat shock both parasites showed an increase in their extracellular phosphorylation activity. L. major promastigotes that do not transform to amastigote-like cells at 37°C, do not phosphorylate exogenous substrate when the culture is grown at 25°C. In contrast, L. mexicana promastigotes, that transform to amastigote-like cells at 37°C, showed a strong phosphorylation at this temperature. As response to heat shock exposure, the extracellular phosphorylation for L. major increases steadily reaching a maximum at 360 min. On the other hand, L. mexicana also show an increase in phosphorylation during transformation, but in this case the maximum was detected after 10 min. The results are discussed in relationship to the capacity of the parasite to survive once inside the vertebrate host. Key words: Leishmania; Phosphorylation; Heat shock
Introduction Leishmania are unicellular protozoan parasites which cause a spectrum of different diseases in man including the three main forms: cutaneous, mucocutaneous and visceral leishmaniasis. The parasites have a relatively simple digenic life cycle (Dwyer and Gottlieb, 1983; Pearson et al., 1983). The extracellular form, the promastigote, resides and multiplies in the sandfly vector. Upon transmission to an appropriate mammalian host, the promastigotes are ingested by phagocytes (Chang and Bray, 1985). An additional aspect of the developmental cycle of Leishmania concerns the sequential development of sandfly promastigotes from a noninfective stage to an infective stage that can be mimicked in vitro by logarithmic and metacyclic stationary phase promastigotes (Sacks et al., 1985). During axenic transformation to metacyclic form, protein kinase activity increases, and phosphorylation pattern changes in L. major (Mukhopadhyay et al., 1988; *Corresponding author. SSDI 0001-706X(93)E0071 -S
Hermoso, 1989). Like few other eukaryotic cells Leishmania major have been shown to possess an external oriented surface protein kinase (Das et al., 1986; Lester et al., 1990). However, very little is known concerning L. mexieana. It was shown that live promastigotes of L. major phosphorylate components of the complement cascade (Hermoso et al., 1991); this points to a key event since these parasites utilize complement receptors on the macrophage to gain entrance into the host cells, where they persist and replicate (Blackwell et al., 1986). Leishmania experience rapid changes in their external environment as they pass from the sandfly midgut to the host. One of these changes involves a temperature shift; when promastigotes growing axenically are subjected to heat shock they develop into amasti-gote-like cells (Van der Ploeg et al., 1985) which have similar properties as the tissue derived amastigotes (Rainey et al., 1991). It has been reported that different species of Leishmania respond similarly to heat shock conditions via the production of heat shock proteins (Carratu and Maresca, 1992). However, the process of morphological transformation differs among them (Shapira et al., 1988). In this paper we demonstrate an increased activity of extracellular phosphorylation of exogenous substrates by L. major and L. mexicana promastigotes during the heat shock transformation. The kinetics of the process differ between the two species. The possible relationship of this increase of the protein kinase activity with the biological behavior of Leishmania is discussed.
Materials and methods
Reagents [~,-32p]ATP (3000 Ci/mmol) was obtained from Amersham International. Calf thymus histones II-S, salmon sperm protamine sulfate, phenyl-methylsulfonyl fluoride (PMSF), were from Sigma Chemical Co. All the other chemicals were analytical reagent grade. Parasites Promastigotes (day 4 in culture) from L. major (P strain, Liverpool reference number LV39) was obtained from Dr. R. Neal (London School of Hygiene and Tropical Medicine, England). L. mexicana (9012) was initially isolated from a human patient from Tfichira state, Venezuela. The 9012 strain of parasites were grown at 25°C in Schneider's Drosophila medium (Jaffe et al., 1984) containing 10% fetal calf serum, penicillin (2 U/ml) and streptomycin (2 U/ml). The parasites of the P strain were grown in continuous culture at 32°C in Schneider's medium, these cells do not undergo morphological changes at this temperature. Promastigotes (3 x 10 7 cells) were transferred to heat shock conditions at 37°C for varying times. Parallel cultures were left at 25°C. Phosphorylation of exogenous proteins Logarithmic phase promastigotes of L. major were washed twice with 20 mM TrisHC1 pH 7.2, 150 mM NaC1 and 2 mM glucose (labeling buffer) and resuspended at
108 cells/100 ~tl in labeling buffer plus 10 m M magnesium and protease inhibitors (EGTA 10 m M and P M S F 1 mM). To induce heat shock, the cells were incubated at 37°C during various periods of time. At the end of the heat shock period, the preparations were supplemented with protamine sulfate or mixed histone (10 Ixg each), then [7-32p]ATP (10 gCi per tube) was added and incubation was resumed for a further 15 min period at 37°C. Cells were then pelleted at 4°C (5 min, 6000 rpm), the supernatant removed and recentrifuged for 10 min at 14 000 rpm in an Eppendorf microfuge. The supernatants were analyzed by SDS-PAGE and autoradiography. For the study of phosphorylation of proteins from the parasite the scheme was the same but the addition of exogenous substrates was avoided.
Results
Fig. l a shows that in L. major promastigotes the phosphate incorporation into protamine sulfate increased steadily with time of exposure to heat shock, from almost zero at 25°C to a m a x i m u m detected after 360 min at 37°C (maximum period tested). In contrast L. mexicana promastigotes (9012) phosphorylated the exogenous substrate at 25°C. However, as in the case of L. major, an increase in the phosphorylation of protamine by L. mexicana was observed during heat shock. Digitalization of the autoradiogram indicates that a value of 4.19 m m 2 is reached after 20 min of heat shock for L. major. L. mexicana promastigotes behave as L. major, but the change started as soon as after 1 min of incubation at 37°C (Fig. lb). The increase in phosphorylation activity was 100% after 10 min heat shock. The increased phosphorylation of exogenous substrates by L. major during heat shock transformation was also shown, using a mixed histone substrate (Fig. 2). Again, as with protamine, almost no phosphorylation was detected prior heat shock (time 0, 25°C). On the other hand, 20 min after heat shock exposure, the phosphorylation of two components of the mixed histones could be detected. In this case the phosphorylation increases with time until 360 min. Fig. 3 shows that not only exogenous substrates such as protamine and histones
0012
p
9012
O
20
120
240
360
0
(rain)
0
1
2.5
5
10
0
(rain)
Fig. 1. Analysis of extracellular protein kinase activity to phosphorylate protamine sulfate during heat shock transformation. Viable promastigotes of L. major (P) or L. mexicana (9012) were exposed to a heat shock temperature of 37°C for up to 360 min (a), whereas L. mexicanapromastigoteswere transferred to a heat shock from 25°C to 37°C for up to 10 min (b). In all cases the parasites were incubated for 15 min with [y-32p]ATPafter the indicated heat shock temperature, then sedimented and the supernatant collected and analyzed on SDS-PAGE.
kDa ii~ii~iiii!~ili~ii~ii~i
97.4
i!i:i!i~i!:i!~i~iii~i!i!~
662
45.0
!iiiiiiiiiiiiiiiiiiiiiiii!!i!iii!
31.0
21.5
iii::i~i !!iii!iiii!iiiiiiiiiiiiiiiill
!:~:iiiiiiiii!i!i;:iii!iiiiiii
14.4
iiii!i]iii!iii!ii!iiiiiil
0
20
60
120
360
(min)
Fig. 2. Change in the extracellular protein kinase activity in L. major (P). Phosphorylation of exogenous mixed histones by viable promastigotes. The parasites were incubated in presence of ['¢-32p]ATP after 20, 60, 120, and 360 min as described in Materials and methods.
P
9012 kDa 974 662 !i!!ii!!~iiii~iii~ii~!: !!i: 450 310
ilAi !!~L~i!~!!~i ~ ~ ~
215
~ii~:i!:i!,~ii~~!i~!i!:~i~ O
20
60
120
360
0
14 4
(min)
Fig. 3. Phosphorylation of released phosphoproteins from Leishmania major (P). Cells submitted to heat shock during the indicated time were incubated with [7-s2p]ATP, were sedimented and supernatant collected and analysed by SDS-PAGE (10%).
5 can be phosphorylated during heat shock exposure, since phosphorylation of three L. major peptides of 31, 48 and 50 kDa was observed after 120 min at 37°C.
Discussion Surface protein kinases have been identified in Leishmania donovani, the causative agent of visceral leishmaniasis (Das et al., 1986), and in L. major, responsible in some cases of causing cutaneous leishmaniasis (Lester et al., 1990). The localization of these enzymes in the Leishmania cell surface is unusual, as protein kinases are generally regarded as intracellular enzymes. Not much is known about the extracellular phosphorylation capacity of New World species such as Leishmania mexicana. Throughout their life these parasites encounter stressful environmental conditions, including dramatic temperature fluctuations. In certain species of Leishmania an increase in the temperature of the promastigote culture from 26°C to 36°C induces morphological changes, whereas in others it does not. In this regard, the New World and Old World Leishmania differ in their response; at 36°C cutaneous species of South American origin often lose motility and become rounded or elliptical (Stinson et al., 1989). In contrast, promastigotes of the Old World species such as L. major or L. donovani, upon heat shock, very often do not undergo such change and fail to survive under these conditions. The work presented in this paper has been carried out with a continuous culture of L. major promastigotes that fail to transform under conditions of heat shock (Shapira et al., 1988). The morphological changes observed are moderate with an incomplete retraction of the flagellum; however, at the molecular level they gave the heat shock response (Shapira et al., 1988). This group of parasites fail to phosphorylate exogenous substrates when grown at 25°C, but the phosphorylation process starts after 20 min of heat shock at 37°C and increases steadily up to 360 min for both histone and protamine sulfate. On the other hand, L. mexicana promastigotes transform very rapidly and appear as amastigote-like cells after 4 h under heat shock conditions. Also, these promastigotes at 25°C are able to phosphorylate protamine sulfate. The phosphorylation activity of L. mexicana promastigotes when submitted to heat shock has a steep increase reaching a maximum after 10 min. Although these results do not necessarily indicate a direct relation between phosphorylation and transformation, it is interesting that parasites with similar phosphorylation behavior as L major and L. donovani (Lester et al., 1990; Das et al., 1986) fail to transform or transform at very slow rate, in contrast to a rapid transformation of New World species of L. mexicana. These results and the fact that L. major virulent promastigotes selected by PNA- agglutination have an increased number of proteins phosphorylated (Hermoso, 1989) and that purified leishmanial kinase LPK-I is able to phosphorylate proteins of the complement cascade (C3, C5, and C9), suggest a potential involvement of phosphorylation during the invasion process, and in the capacity of the parasite to survive once inside the vertebrate host.
Acknowledgements This investigation received financial support from the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICIT) (S1-2561, to T.H.).
References Blackwell, J.M., Ezekowitz, R.A.B., Roberts, M.B., Channon, J.Y., Sim, R.B. and Gordon, S. (1985) Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum. J. Exp. Med. 162, 324 331. Carratu and Maresca (1992) The Biology of the heat shock response in parasites. Parasitol. Today 8 (8), 260-266. Chang, K.P. and Bray, R.S. (1985) Biology of Leishmania and Leishmaniasis, In: K.P. Chang and R.S. Bray (Eds.), Leishmaniasis, Vol. 1, Elsevier, Amsterdam, pp. 1-30. Das, S., Saha, A.K., Mukhopadhyay, N.K. and Glew, R.H. (1986) A cyclic nucleotide-independent protein kinase in Leishmania donovani. Biochem. J. 240, 641-649. Dwyer, D.M. and Gottlieb, M. (1983) The surface membrane chemistry of Leishmania. J. Cell. Biochem. 23, 35-47. Hermoso, T. (1989) Protein phosphorylation and dephosphorylation in the protozooan parasite Leishmania major. Thesis, Weizmann Institute of Science, Rehovot, Israel, p. 76. Hermoso, T., Fishelson, Z., Becker, S., Hirshberg, K. and Jaffe, C.L. (1991) Leishmanial protein kinases phosphorylate components of the complement system. EMBO J. 10, 4061 4067. Jaffe, C.L., Grimaldi, G. and McMahon-Pratt, D. (1984) The cultivation and cloning of Leishmania. In: C.M. Morel (Ed.), Genes and Antigens of Parasites, UNDP/World Bank/WHO, pp. 580. Lester, D., Hermoso, T. and Jeffe, C.L. (1990) Extracellular phosphorylation in the parasite, Leishmania major. Biochim. Biophys. Acta 1052, 293-298. Mukhopadhyay, N.K., Saha, A.K., Lovelace, J.K., Da Silva, R., Sacks, D.L. and Glew, R.H. (1988) Comparison of the protein kinases and acid phosphatase activity of five species of Leishmania. J. Protozool. 35, 601-607. Pearson, R., Wheeler, D., Harrison, L. and Kay, D. (1983) The inmunobiology of leishmaniasis. J. Infect. Dis. 5, 907-927. Rainey, P.M., Spithill, T.W., McMahon-Pratt, D. and Pan, A.A. (1991) Molecular characterization of Leishmania pifanoi amastigotes in continuos axenic culture. Mol. Biochem. Parasitol. 49, 111 116. Sacks, D.L., Hieny. S. and Sher, A. (1985) Identification of ceil surface carbohydrate and antigenic changes between noninfected and infected developmental stage of Leishmania major promastigotes. J. Immunol. 135, 564-569. Shapira, M., McEwen, J. and Jaffe, C.L. (1988) The temperature effects on molecular processes which lead to stage differentiation in Leishmania. EMBO J. 7, 2895-2901. Stinson, S., Sommer, J.R. and Blum, J.J. (1989) Morphology of Leishmania braziliensis: Changes during reversible heat-induced transformation from promastigote to an ellipsoidal form. J. Parasitol. 75, 431-440. Van der Ploeg, L.H.T., Giannini, S. and Cantor, C. (1985) Heat shock genes: Regulatory role for differentiation in parasitic protozoa. Science 228, 1143-1146.