FEMS MicrobiologyLetters 94 (1992) 221-226 © 1992 Federation of European MicrobiologicalSocieties0378-I097/92/$05.00 Published by Elsevier
221
FEMSLE 04942
Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence N e e n a Singla ~', G.K. K h u i l e r i, and V.K. V i n a y a k ~' a Department of Experimental Medicine. and h Department of BiochemistB.', Po.~tgra,hmte ht~titute t~f Medical Education and Research, Chandigarh, bulia
Received 2 December 1901 Revision received 15 March 19t}2 Accepted 16 March It}92 Key words: Virulence; l,eishmania donocani; Promastigote
1. S U M M A R Y Seven cloned lines of promastigotes of Leishm a n i a d o n o c a n i ( U R 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 + 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 + 5.8 to 67.6 + 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree
Correspondence to: V.K. Vinayak, Division of Experimental
Parasitology and Parasitic Immunology,Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India.
of virulence. These data suggest that acid phosphata~e activity could be used as a marker tc differentiate avirulent from virulent populations of promastigotcs of L. d o n o c a n i . 2. I N T R O D U C T I O N Loss of virulence of wild uncloned strains of Leishraania species after prolonged in vitro main-
tenance has been suggested to be related to loss of an antigen associated with invasiveness of promastigotes" [1], changes in membrane glycosylation [2,3] or overgrowth of non-infective population in 'wild-type' culture [4]. Alternatively, permissiveness of macrophages for survival and multiplication of Leishmania may be related to failure of macrophage microbicidal mechanisms because of the presence of superoxide dismutase [5,6], trypanothione and trypanothione reductase [71 and membrane-bound acid phosphatase [8-10] in L e i s h m a n i a , providing resistance to toxic oxygen products of activated macrophages [57,10,11]. The latter has been suggested to enable the parasite to obtain necessary nutrients from
222 organic phosphates in the environment for its survival [11]. However, the precise role of acid phosphatase in virulence of the parasite still remains ill-defined. This study has thus been designed to assess membrane-bound acid phosphatase activity and its correlation with virulence potential of different clones of Leishmania donorani, a parasite responsible for visceral leishmaniasis.
3. MATERIALS AND METHODS
3.1. Cloned populations of promastigotes of L. donocani The cloning of stationary phase uncloned promastigotes of L. donot'ani (UR 6) was done by limiting dilution [12]. Brief!y, a 50-/.tl aliquot containing a single promastigote of L. donorani in each well of 96-wei| tissue culture plates (Costar, USA), diluted with 200 p.l of RPMI 1640 medium containing 10% fetal calf serum (FCS) was transferred to beef extract agar monophasic medium slants and incubated at 26°C for 7-10 days. A single discrete colony from each of the slants picked up with a sharp-edged Pasteur pipette was propagated in Brain Heart Infusion agar with Hanks' balanced salt solution as an overlay [14] and referred as 'cloned population'.
3.Z Determhlation of infecticity and cirulence of cloned and uncloned populations of promastigotes The stationary phase promastigotes from parent uncloned and cloned populations were interacted with 48-72-h-old mouse macrophages of line J774G8 as described by Chang [15] with a few modifications. Briefly, the macrophages: a promastigotes suspension, seeded on a glass coverslip in wells of a 24-well tissue culture plate (Costar, USA), was covered with 1-2 ml of RPM1 medium containing 10% FCS. Following incubation at 34°C for 3-7 days, the coverslips were removed and screened for number of infected macrophages and the number of amastigotes within infected macrophages. The percent infectivity and virulence indices were calculated as follows:
Percent infectivity No. infected macrophages No. examined macrophages
× 100
Virulence index No. amastigotes × No. infected macrophages =
No. examined macrophages Each experiment was repeated three times in duplicate.
3.3. Surface-associated acid phosphatase enzyme actirity of cloned and uncloned populations of promastigotes Acid phosphatase enzyme activity was determined according to Saito and Suter [16] with a few modifications [17]. In brief, washed promastigotes were suspended in 0.1 M acetate buffer (pH 5.2)containing 1 mM magnesium sulphate (1 mg protein/ml buffer) and lysed by repeated freezing and thawing. The reaction mixture in a 0.3-ml aliquot consisting of 25 /tg of promastigotes cell lysate protein and 2.6 mM of substrate, p-nitrophenyl phosphate in acetate buffer, was incubated at 37°C for 30 min. The reaction was stopped with 0.8 ml of 0.1 N NaOH and the release of p-nitrophenoi (PNP) was estimated spectrophotometrically (Kontron, Uvikon 860) at 400 nm. The enzyme activity was expressed as gM of p-nitrophenol released per mg protein of promastigotes cell lysate.
3.4. Attachment of cirulent and acirulent cloned populations of promastigotes to macrophages The stationary phase promastigotes of virulent and avirulent populations were added to 48-72h-old macrophages of line J774G8. Following incubation at 34°C in 5% CO 2 atmosphere for 4 h, the percent macrophages with attached promastigotes and the number of promastigotes attached per 100 macrophages were assessed.
4. RESULTS Seven cloned, isolated populations were labelled as UR6-C2, UR6-C4. UR6-CI3, UR6-CI9,
223 Table l Standardization of effector:target cell ratios Criteria
Day
Macrophages: promastigotes ralio 1 : I11
1:211
1:411
1:80
Percent infectivity
3 7
37.1 + 3.11 511.1-+ 13.8
511.6+ 111.2 (,0.(, -+ 18.11
72.11+ 2.8 711.0+ 5.(,
53.11+ 7.0 55.0± 4.2
Virulence index
3 7
I 1.9 -+I}.t~1 11,.4_+11.17
15.4 + 2.5 211.(,± 7.0
37.0+3.11 37.5 ± 1.4
311.7-+ 13.`4 42.4± 1.4
Results as mean _+SD of three sets of experiments done in duplicate.
UR6-C24, UR6-C25 and UR6-C26. T h e in vitro interaction of stationary p h a s e u n c l o n e d promastigotes of L. d o n o c a n i with mouse m a c r o p h a g e s o f line J774G8 at m a c r o p h a g e to a p r o m a s t i g o t e ratio of ! :411 for 3 days was f o u n d to be optimal with highest p e r c e n t infectivity (72%) a n d virulence index [37] (Table 11. K e e p i n g this e f f e e t o r : t a r g e t cell ratio constant, no cloned p o p u l a t i o n except UR6-C25, had significant diff e r e n c e s in the p e r c e n t infectivity, which r a n g e d f r o m 63.2 + 1.6 to 74 + 10.4. N o n e of the cloned p o p u l a t i o n s , except UR6-C25, s h o w e d a significant difference in the p e r c e n t infectivity as comp a r e d to p a r e n t u n c l o n e d p o p u l a t i o n (66.5 + 6.5) (Table 2). T h e clone UR6-C25 had a significantly low ( P < 0.0011 virulence index (0.10 + 0.161, while a n o t h e r clone U R 6 - C 2 4 had a significantly
h i g h e r ( P < 0.(X)I) virulence index (93.0 + 9.8) as c o m p a r e d to the p a r e n t u n c l o n e d p o p u l a t i o n (33 + 6.2). T h e cloned populations, UR6-C25 and UR6-C24, were labelled as avirulent and highly virulent, respectively, in p r o m a s t i g o t e s of L. d o n o r a n i UR6. T h e o t h e r five clones viz. UR6-C2, UR6-C4, U R 6 - C I 3 , U R 6 - C I 9 and UR6-C26, had virulence indices ranging from 46.4 + 5.8 to 67.6 + 3.5 (Table 2). P r o m a s t i g o t e s from virulent and avirulent cloned populations attached to m a c r o p h a g e s with almost identical d e g r e e (Table 3). However, the p r o m a s t i g o t e s from the virulent cloned line multiplied inside host cells leading to 163-365 a m a s t i g o t e s per 100 infected macrop h a g e s (Table 2). T h e acid p h o s p h a t a s e activity of the avirulent cloned p o p u l a t i o n of p r o m a s t i g o t e s of L. dono-
Table 2 Percent infectivity, virulence index and acid phosphatase activity of unc!oned and chined populations of L. domnani Culture
Percent i n f e c t ) vity
Virulence index
Average number of amastigotcs per II1(1macrophagc
Acid phosphatase activity "
Uncloned UR6 Cloned UR6-C2 Cloned UR(,-C4 Cloned UR6-CI3 Cloned UR6-CI9 Cloned U R(,-C24 Cloned UR6-C25 Cloned UR6-C26
66.5.+ 6.5 (,5.11_+ 9.(, (,7.0_+ 3.0 (,8.3 + 4.2 74.11.+ 111.4 71.0 _+ 3.5 3.(,_+ 5.(, (,3.2+_ 1.6
33.0 .+ 6.2 41,,8 + 111.2 5`4.0 _+ 7.5 67.6 _4- 3.5 4(,.4 .+ 5.8 `43.11 _+ '4.8 * 1).1|1.+ I1.11,** 52.9 _+ 2.9
149.+ 13 213±21 261 +_21 2811+_30 1'47_+_37 3(,5 _+22 33.+ 5.1 11,3.+ 7.11
521 .+38.8 ~172-+87.0 1225 +_511.11 1297 4-28.7 `438.+ 13.8 17311_+45.3 258.+58.11 48`4.+43.8
Results as means_+SD of three sets of experiments done in duplicate. a M of p-nitrophenol released/min/mg protein. * P < 0.001 compared to uncloned UR6. ** P < IL(X}Icompared to uncloned UR6.
224 Table 3 Attachment of avirulenl and virulent promastigotes to macrophages Clone
Virulent UR6-C24 Avirulent UR6-C25
Percent macrophages with attached promastigotes
Number of promastigotes attached
33.3+ 3.9
64.7_+10.8
30.3+4.6
68.2_+ 8.9
5. DISCUSSION
Values are means_+SD of seven observations.
l.ani was 258 /zM of p-nitrophenol (PNP) released per mg protein of promastigote cell lysate as compared to 1730 tzM PNP released by acid
2000
1800
i 1600
/
14oo
~/
7-/
12oo IOOO
800
phosphatase of the highly virulent clone. A positive correlation between acid phosphatase activity and the degree of virulence of promastigotes of L. donot'ani was observed (Fig. 1, Table 2).
/
600 400 200
10 20 30 40 50 60 70 80 90 100 Virulence index Fig. I. Correlation between acid phospbatase activity and degree of virulence of promastigotes of L. donot'ani.
The study indicated that the wild culture of L. donot'ani (UR6) consisted of several populations (clones) of promastigotes with varying degree of virulence potentials. The observed variations in the virulence of cloned promastigotes may be a reflection of the factors related to the parasite and the target cell. The phenomenon of metacyclogenesis has earlier been shown to be associated with an increased virulence of promastigotes because of enhanced expression of surface-associated acid phosphatase [18,19], and ligand surface lipophosphoglycan [20,21] and glycoproteins [22]. Since all the ,:loned promastigotes employed were taken from the stationary growth phase and in metacyclic form, the possibility of observed variations due to growth phase variations can be ruled out. We observed no significant difference in the degree of attachment of promastigotes from virulent or avirulent clones to macrophages, suggesting no difference in the type or expression of ligand molecules on both types of promastigote populations. Earlier studies by Handman et al. [12] reported no significant difference in the membrane glycosylation of surface proteins of cloned virulent or avirulent populations of promastigotes. Thus variations in virulence of promastigotes appear not to be related to surface ligand molecules. The observed variations in virulence also appear not to be related to different microbicidal functional potentials of macrophages, since these were taken from a permanent mouse macrophage cell line J774G8, a fully permissive cell line to phagocytose the promastigotes and had identical functional activities [15]. It appears that parasite-related factors that help the parasite to survive in hostile macrophages local milieu may be involved. The ability of Leishrnania parasites to withstand an acidic environment inside macrophages has led to the identification of acid phosphatases, namely (a) extracellularly
225 s e c r e t e d s o l u b l e acid p h o s p h a t a s e [23,24] a n d (b) m e m b r a n e - b o u n d acid p h o s p h a t a s e [8-10]. O u r d a t a indicate t h a t t h e m e m b r a n e - b o u n d acid p h o s p h a t a s e activity o f p r o m a s t i g o t e s f r o m t h e v i r u l e n t c l o n e w a s significantly h i g h e r as c o m p a r e d to t h a t f r o m t h e avirulent clone. In fact, a direct c o r r e l a t i o n b e t w e e n t h e d e g r e e o f virulence a n d t h e m e m b r a n e - b o u n d aci0 p h o s p h a t a s e activity o f p r o m a s t i g o t e s w a s o b s e r v e d . R e c e n t l y , a s t u d y by K a t a k u r a a n d K o b a y a s h i [17] i n d i c a t c d t h a t a strain o f Leishmania, w h e n p a s s a g e d t h r o u g h s u s c e p t i b l e B a l b / c mice, p o s s e s s e d h i g h e r acid p h o s p h a t a s e activity a s s o c i a t e d with i n c r e a s e d infectivity. It a p p e a r s t h a t e n h a n c e d acid p h o s p h a t a s e activity o f t h e p a r a s i t e ( b e i n g active at acidic p H ) m a y be h e l p i n g t h e p a r a s i t e to (i) a d a p t itself to acidic e n v i r o n m e n t [25], (ii) provide n u t r i e n t s by d e p h o s p h o r y l a t i n g h o s t cell p h o s p h o p r o t e i n s [l l] or (iii) resist toxic m e t a b o lites o f m a c r o p h a g e s so as to p e r m i t i n t r a c e l l u l a r e s t a b l i s h m e n t o f t h e p a r a s i t e [10,11]. N e v e r t h e less, t h e r e s u l t s o f t h e p r e s e n t s t u d y a n d t h a t o f K a t a k u r a a n d K o b a y a s h i [17] indicate t h a t acid p h o s p h a t a s e activity o f p a r a s i t e c o u l d be exp l o i t e d as a m a r k e r o f virulence.
ACKNOWLEDGEMENTS T h e financial a s s i s t a n c e p r o v i d e d by t h e C o u n cil o f Scientific a n d I n d u s t r i a l R e s e a r c h is greately acknowledged.
REFERENCES [1] Schneider, C.R. and Hertig. M. (10861 Exp. Parasitol. 18. 25-34. [2] Dawidowicz, K., Hernandez. A.G., Infante, R.B. and Convit, J. (1975) J. Parasitol. 61, 9511-953.
[3] Hernandez. A.G.. Rodriquez. N., Dagger. F. and Greenblatt. C.L. (198(I) Mol. Biochem. Parasitol. I. 143-149. [4] Grimaldi, G. Jr., Momen, H.. Soares, M.J. and Moriearty, P.L. (19821 Int. J. Parasitol. 12, 185--190. [5] Me'~hnick. S.R. and Eaton. J.W. (19811 Biochem. Biophys. Res. Commun. 102. 970-976. [6] Murray. H.W. (19811 J. Exp. Med. 153, 1302-1315. [7] Fairlamb, A.H. (19881 In: Leishmaniasis(D.T. Hart, Ed.). pp. 265-473. Plenum NATOASI Series, New York, NY. [8} Gottlieb, M. and Dx~,yer. D.M. (19811 Exp. Parasitol. 52, 117-128. [9] Gottlieb, M. and D~3, ~r, D.M. (It;8l) Science 212, 939941. [10] Ramley. A.T., Glew. R.H., Kuhns, b.B., Basford, R.E,, Waggoner, A.S., Ernsl L.A. and Pope, M. (19851 Exp. Parasitol. ~0, 331-341. [I I] Saha, A.K., Das, S., Glew, R.|i. and Gottlieb, M° 11985) J. Clin. Microbiol. 22, 329-332. [12] Handman, E.. Hocking, R.E.. Mitchell, G.F. and Spithill, T.W. ( 1083) Mol. Biochem. Parasitol. 7, I 11- 126. [131 Ray, J.C. (19321 Indian J. Med. Res. 21), 355-367. [14] Matin, F., Garcia de Lomas, J., Penarrubia, M.P. and Penalver, J. (10821 Ann. Trop. Med. Parasitol. 76, 6117613. [15] Chang. K.P. (198(11Science 2119, 124(I-1242. [16] Saito. K. and Suter, E. (19651J. Exp. Med. 121,727-739. [17] Katakura, K. and Kobayashi, A. (1988) Infect. Immun. 56. 2856-2860. [18] Bandyopadhyay, P., Ghosh, D.K., Ah)keDe. Ghosh. K.N., Chaudhari. P.P., Das, P. and Bhattacharya. A. (19911 J. Parasitol. 77. 411-416. [1O] Manionson. D.J. and Coombs. G.H. (19861 IRCS Med. Sci. 14, 557-558. [20] Grogl, M., Franke, E.D., McGreevy. P.B. and Kuhn, R.E. (19871 Exp. Parasitol. 63. 353-.359. [211 Sacks, D.I. and DaSilva, R,P. (1981) J. Immunol. 139. 31199-31116. [22] Kwelder. M,, Lemesre, J.L., Santoro. Kusnierz. J.P., Sadigursky, M. and Capron. A. (19891 Parasite immunol. II. 197 2119. [23] Lovelace, J.K., D~Tcr, D.M. and Tottlieb, M. ( 19861 Mol. Biochem. Parasitol. 21), 243-251. [24] Bates, P.A., Hermes, I. and Dwyer, D.M. (1989) Exp. Parasitol. 68, 335-346. [251 Glew, R.H., Saha, A.K., Das, S. and Ranley, A.T. (19881 Microbiol. Rev. 52, 412-432.
221
FEMSLE 04942
Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence N e e n a Singla ~', G.K. K h u i l e r i, and V.K. V i n a y a k ~' a Department of Experimental Medicine. and h Department of BiochemistB.', Po.~tgra,hmte ht~titute t~f Medical Education and Research, Chandigarh, bulia
Received 2 December 1901 Revision received 15 March 19t}2 Accepted 16 March It}92 Key words: Virulence; l,eishmania donocani; Promastigote
1. S U M M A R Y Seven cloned lines of promastigotes of Leishm a n i a d o n o c a n i ( U R 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 + 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 + 5.8 to 67.6 + 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree
Correspondence to: V.K. Vinayak, Division of Experimental
Parasitology and Parasitic Immunology,Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India.
of virulence. These data suggest that acid phosphata~e activity could be used as a marker tc differentiate avirulent from virulent populations of promastigotcs of L. d o n o c a n i . 2. I N T R O D U C T I O N Loss of virulence of wild uncloned strains of Leishraania species after prolonged in vitro main-
tenance has been suggested to be related to loss of an antigen associated with invasiveness of promastigotes" [1], changes in membrane glycosylation [2,3] or overgrowth of non-infective population in 'wild-type' culture [4]. Alternatively, permissiveness of macrophages for survival and multiplication of Leishmania may be related to failure of macrophage microbicidal mechanisms because of the presence of superoxide dismutase [5,6], trypanothione and trypanothione reductase [71 and membrane-bound acid phosphatase [8-10] in L e i s h m a n i a , providing resistance to toxic oxygen products of activated macrophages [57,10,11]. The latter has been suggested to enable the parasite to obtain necessary nutrients from
222 organic phosphates in the environment for its survival [11]. However, the precise role of acid phosphatase in virulence of the parasite still remains ill-defined. This study has thus been designed to assess membrane-bound acid phosphatase activity and its correlation with virulence potential of different clones of Leishmania donorani, a parasite responsible for visceral leishmaniasis.
3. MATERIALS AND METHODS
3.1. Cloned populations of promastigotes of L. donocani The cloning of stationary phase uncloned promastigotes of L. donot'ani (UR 6) was done by limiting dilution [12]. Brief!y, a 50-/.tl aliquot containing a single promastigote of L. donorani in each well of 96-wei| tissue culture plates (Costar, USA), diluted with 200 p.l of RPMI 1640 medium containing 10% fetal calf serum (FCS) was transferred to beef extract agar monophasic medium slants and incubated at 26°C for 7-10 days. A single discrete colony from each of the slants picked up with a sharp-edged Pasteur pipette was propagated in Brain Heart Infusion agar with Hanks' balanced salt solution as an overlay [14] and referred as 'cloned population'.
3.Z Determhlation of infecticity and cirulence of cloned and uncloned populations of promastigotes The stationary phase promastigotes from parent uncloned and cloned populations were interacted with 48-72-h-old mouse macrophages of line J774G8 as described by Chang [15] with a few modifications. Briefly, the macrophages: a promastigotes suspension, seeded on a glass coverslip in wells of a 24-well tissue culture plate (Costar, USA), was covered with 1-2 ml of RPM1 medium containing 10% FCS. Following incubation at 34°C for 3-7 days, the coverslips were removed and screened for number of infected macrophages and the number of amastigotes within infected macrophages. The percent infectivity and virulence indices were calculated as follows:
Percent infectivity No. infected macrophages No. examined macrophages
× 100
Virulence index No. amastigotes × No. infected macrophages =
No. examined macrophages Each experiment was repeated three times in duplicate.
3.3. Surface-associated acid phosphatase enzyme actirity of cloned and uncloned populations of promastigotes Acid phosphatase enzyme activity was determined according to Saito and Suter [16] with a few modifications [17]. In brief, washed promastigotes were suspended in 0.1 M acetate buffer (pH 5.2)containing 1 mM magnesium sulphate (1 mg protein/ml buffer) and lysed by repeated freezing and thawing. The reaction mixture in a 0.3-ml aliquot consisting of 25 /tg of promastigotes cell lysate protein and 2.6 mM of substrate, p-nitrophenyl phosphate in acetate buffer, was incubated at 37°C for 30 min. The reaction was stopped with 0.8 ml of 0.1 N NaOH and the release of p-nitrophenoi (PNP) was estimated spectrophotometrically (Kontron, Uvikon 860) at 400 nm. The enzyme activity was expressed as gM of p-nitrophenol released per mg protein of promastigotes cell lysate.
3.4. Attachment of cirulent and acirulent cloned populations of promastigotes to macrophages The stationary phase promastigotes of virulent and avirulent populations were added to 48-72h-old macrophages of line J774G8. Following incubation at 34°C in 5% CO 2 atmosphere for 4 h, the percent macrophages with attached promastigotes and the number of promastigotes attached per 100 macrophages were assessed.
4. RESULTS Seven cloned, isolated populations were labelled as UR6-C2, UR6-C4. UR6-CI3, UR6-CI9,
223 Table l Standardization of effector:target cell ratios Criteria
Day
Macrophages: promastigotes ralio 1 : I11
1:211
1:411
1:80
Percent infectivity
3 7
37.1 + 3.11 511.1-+ 13.8
511.6+ 111.2 (,0.(, -+ 18.11
72.11+ 2.8 711.0+ 5.(,
53.11+ 7.0 55.0± 4.2
Virulence index
3 7
I 1.9 -+I}.t~1 11,.4_+11.17
15.4 + 2.5 211.(,± 7.0
37.0+3.11 37.5 ± 1.4
311.7-+ 13.`4 42.4± 1.4
Results as mean _+SD of three sets of experiments done in duplicate.
UR6-C24, UR6-C25 and UR6-C26. T h e in vitro interaction of stationary p h a s e u n c l o n e d promastigotes of L. d o n o c a n i with mouse m a c r o p h a g e s o f line J774G8 at m a c r o p h a g e to a p r o m a s t i g o t e ratio of ! :411 for 3 days was f o u n d to be optimal with highest p e r c e n t infectivity (72%) a n d virulence index [37] (Table 11. K e e p i n g this e f f e e t o r : t a r g e t cell ratio constant, no cloned p o p u l a t i o n except UR6-C25, had significant diff e r e n c e s in the p e r c e n t infectivity, which r a n g e d f r o m 63.2 + 1.6 to 74 + 10.4. N o n e of the cloned p o p u l a t i o n s , except UR6-C25, s h o w e d a significant difference in the p e r c e n t infectivity as comp a r e d to p a r e n t u n c l o n e d p o p u l a t i o n (66.5 + 6.5) (Table 2). T h e clone UR6-C25 had a significantly low ( P < 0.0011 virulence index (0.10 + 0.161, while a n o t h e r clone U R 6 - C 2 4 had a significantly
h i g h e r ( P < 0.(X)I) virulence index (93.0 + 9.8) as c o m p a r e d to the p a r e n t u n c l o n e d p o p u l a t i o n (33 + 6.2). T h e cloned populations, UR6-C25 and UR6-C24, were labelled as avirulent and highly virulent, respectively, in p r o m a s t i g o t e s of L. d o n o r a n i UR6. T h e o t h e r five clones viz. UR6-C2, UR6-C4, U R 6 - C I 3 , U R 6 - C I 9 and UR6-C26, had virulence indices ranging from 46.4 + 5.8 to 67.6 + 3.5 (Table 2). P r o m a s t i g o t e s from virulent and avirulent cloned populations attached to m a c r o p h a g e s with almost identical d e g r e e (Table 3). However, the p r o m a s t i g o t e s from the virulent cloned line multiplied inside host cells leading to 163-365 a m a s t i g o t e s per 100 infected macrop h a g e s (Table 2). T h e acid p h o s p h a t a s e activity of the avirulent cloned p o p u l a t i o n of p r o m a s t i g o t e s of L. dono-
Table 2 Percent infectivity, virulence index and acid phosphatase activity of unc!oned and chined populations of L. domnani Culture
Percent i n f e c t ) vity
Virulence index
Average number of amastigotcs per II1(1macrophagc
Acid phosphatase activity "
Uncloned UR6 Cloned UR6-C2 Cloned UR(,-C4 Cloned UR6-CI3 Cloned UR6-CI9 Cloned U R(,-C24 Cloned UR6-C25 Cloned UR6-C26
66.5.+ 6.5 (,5.11_+ 9.(, (,7.0_+ 3.0 (,8.3 + 4.2 74.11.+ 111.4 71.0 _+ 3.5 3.(,_+ 5.(, (,3.2+_ 1.6
33.0 .+ 6.2 41,,8 + 111.2 5`4.0 _+ 7.5 67.6 _4- 3.5 4(,.4 .+ 5.8 `43.11 _+ '4.8 * 1).1|1.+ I1.11,** 52.9 _+ 2.9
149.+ 13 213±21 261 +_21 2811+_30 1'47_+_37 3(,5 _+22 33.+ 5.1 11,3.+ 7.11
521 .+38.8 ~172-+87.0 1225 +_511.11 1297 4-28.7 `438.+ 13.8 17311_+45.3 258.+58.11 48`4.+43.8
Results as means_+SD of three sets of experiments done in duplicate. a M of p-nitrophenol released/min/mg protein. * P < 0.001 compared to uncloned UR6. ** P < IL(X}Icompared to uncloned UR6.
224 Table 3 Attachment of avirulenl and virulent promastigotes to macrophages Clone
Virulent UR6-C24 Avirulent UR6-C25
Percent macrophages with attached promastigotes
Number of promastigotes attached
33.3+ 3.9
64.7_+10.8
30.3+4.6
68.2_+ 8.9
5. DISCUSSION
Values are means_+SD of seven observations.
l.ani was 258 /zM of p-nitrophenol (PNP) released per mg protein of promastigote cell lysate as compared to 1730 tzM PNP released by acid
2000
1800
i 1600
/
14oo
~/
7-/
12oo IOOO
800
phosphatase of the highly virulent clone. A positive correlation between acid phosphatase activity and the degree of virulence of promastigotes of L. donot'ani was observed (Fig. 1, Table 2).
/
600 400 200
10 20 30 40 50 60 70 80 90 100 Virulence index Fig. I. Correlation between acid phospbatase activity and degree of virulence of promastigotes of L. donot'ani.
The study indicated that the wild culture of L. donot'ani (UR6) consisted of several populations (clones) of promastigotes with varying degree of virulence potentials. The observed variations in the virulence of cloned promastigotes may be a reflection of the factors related to the parasite and the target cell. The phenomenon of metacyclogenesis has earlier been shown to be associated with an increased virulence of promastigotes because of enhanced expression of surface-associated acid phosphatase [18,19], and ligand surface lipophosphoglycan [20,21] and glycoproteins [22]. Since all the ,:loned promastigotes employed were taken from the stationary growth phase and in metacyclic form, the possibility of observed variations due to growth phase variations can be ruled out. We observed no significant difference in the degree of attachment of promastigotes from virulent or avirulent clones to macrophages, suggesting no difference in the type or expression of ligand molecules on both types of promastigote populations. Earlier studies by Handman et al. [12] reported no significant difference in the membrane glycosylation of surface proteins of cloned virulent or avirulent populations of promastigotes. Thus variations in virulence of promastigotes appear not to be related to surface ligand molecules. The observed variations in virulence also appear not to be related to different microbicidal functional potentials of macrophages, since these were taken from a permanent mouse macrophage cell line J774G8, a fully permissive cell line to phagocytose the promastigotes and had identical functional activities [15]. It appears that parasite-related factors that help the parasite to survive in hostile macrophages local milieu may be involved. The ability of Leishrnania parasites to withstand an acidic environment inside macrophages has led to the identification of acid phosphatases, namely (a) extracellularly
225 s e c r e t e d s o l u b l e acid p h o s p h a t a s e [23,24] a n d (b) m e m b r a n e - b o u n d acid p h o s p h a t a s e [8-10]. O u r d a t a indicate t h a t t h e m e m b r a n e - b o u n d acid p h o s p h a t a s e activity o f p r o m a s t i g o t e s f r o m t h e v i r u l e n t c l o n e w a s significantly h i g h e r as c o m p a r e d to t h a t f r o m t h e avirulent clone. In fact, a direct c o r r e l a t i o n b e t w e e n t h e d e g r e e o f virulence a n d t h e m e m b r a n e - b o u n d aci0 p h o s p h a t a s e activity o f p r o m a s t i g o t e s w a s o b s e r v e d . R e c e n t l y , a s t u d y by K a t a k u r a a n d K o b a y a s h i [17] i n d i c a t c d t h a t a strain o f Leishmania, w h e n p a s s a g e d t h r o u g h s u s c e p t i b l e B a l b / c mice, p o s s e s s e d h i g h e r acid p h o s p h a t a s e activity a s s o c i a t e d with i n c r e a s e d infectivity. It a p p e a r s t h a t e n h a n c e d acid p h o s p h a t a s e activity o f t h e p a r a s i t e ( b e i n g active at acidic p H ) m a y be h e l p i n g t h e p a r a s i t e to (i) a d a p t itself to acidic e n v i r o n m e n t [25], (ii) provide n u t r i e n t s by d e p h o s p h o r y l a t i n g h o s t cell p h o s p h o p r o t e i n s [l l] or (iii) resist toxic m e t a b o lites o f m a c r o p h a g e s so as to p e r m i t i n t r a c e l l u l a r e s t a b l i s h m e n t o f t h e p a r a s i t e [10,11]. N e v e r t h e less, t h e r e s u l t s o f t h e p r e s e n t s t u d y a n d t h a t o f K a t a k u r a a n d K o b a y a s h i [17] indicate t h a t acid p h o s p h a t a s e activity o f p a r a s i t e c o u l d be exp l o i t e d as a m a r k e r o f virulence.
ACKNOWLEDGEMENTS T h e financial a s s i s t a n c e p r o v i d e d by t h e C o u n cil o f Scientific a n d I n d u s t r i a l R e s e a r c h is greately acknowledged.
REFERENCES [1] Schneider, C.R. and Hertig. M. (10861 Exp. Parasitol. 18. 25-34. [2] Dawidowicz, K., Hernandez. A.G., Infante, R.B. and Convit, J. (1975) J. Parasitol. 61, 9511-953.
[3] Hernandez. A.G.. Rodriquez. N., Dagger. F. and Greenblatt. C.L. (198(I) Mol. Biochem. Parasitol. I. 143-149. [4] Grimaldi, G. Jr., Momen, H.. Soares, M.J. and Moriearty, P.L. (19821 Int. J. Parasitol. 12, 185--190. [5] Me'~hnick. S.R. and Eaton. J.W. (19811 Biochem. Biophys. Res. Commun. 102. 970-976. [6] Murray. H.W. (19811 J. Exp. Med. 153, 1302-1315. [7] Fairlamb, A.H. (19881 In: Leishmaniasis(D.T. Hart, Ed.). pp. 265-473. Plenum NATOASI Series, New York, NY. [8} Gottlieb, M. and Dx~,yer. D.M. (19811 Exp. Parasitol. 52, 117-128. [9] Gottlieb, M. and D~3, ~r, D.M. (It;8l) Science 212, 939941. [10] Ramley. A.T., Glew. R.H., Kuhns, b.B., Basford, R.E,, Waggoner, A.S., Ernsl L.A. and Pope, M. (19851 Exp. Parasitol. ~0, 331-341. [I I] Saha, A.K., Das, S., Glew, R.|i. and Gottlieb, M° 11985) J. Clin. Microbiol. 22, 329-332. [12] Handman, E.. Hocking, R.E.. Mitchell, G.F. and Spithill, T.W. ( 1083) Mol. Biochem. Parasitol. 7, I 11- 126. [131 Ray, J.C. (19321 Indian J. Med. Res. 21), 355-367. [14] Matin, F., Garcia de Lomas, J., Penarrubia, M.P. and Penalver, J. (10821 Ann. Trop. Med. Parasitol. 76, 6117613. [15] Chang. K.P. (198(11Science 2119, 124(I-1242. [16] Saito. K. and Suter, E. (19651J. Exp. Med. 121,727-739. [17] Katakura, K. and Kobayashi, A. (1988) Infect. Immun. 56. 2856-2860. [18] Bandyopadhyay, P., Ghosh, D.K., Ah)keDe. Ghosh. K.N., Chaudhari. P.P., Das, P. and Bhattacharya. A. (19911 J. Parasitol. 77. 411-416. [1O] Manionson. D.J. and Coombs. G.H. (19861 IRCS Med. Sci. 14, 557-558. [20] Grogl, M., Franke, E.D., McGreevy. P.B. and Kuhn, R.E. (19871 Exp. Parasitol. 63. 353-.359. [211 Sacks, D.I. and DaSilva, R,P. (1981) J. Immunol. 139. 31199-31116. [22] Kwelder. M,, Lemesre, J.L., Santoro. Kusnierz. J.P., Sadigursky, M. and Capron. A. (19891 Parasite immunol. II. 197 2119. [23] Lovelace, J.K., D~Tcr, D.M. and Tottlieb, M. ( 19861 Mol. Biochem. Parasitol. 21), 243-251. [24] Bates, P.A., Hermes, I. and Dwyer, D.M. (1989) Exp. Parasitol. 68, 335-346. [251 Glew, R.H., Saha, A.K., Das, S. and Ranley, A.T. (19881 Microbiol. Rev. 52, 412-432.
