Is surface membrane acid phosphatase an essential requirement for the survival of Leishmania in macrophages ?

The occurrence of acid phosphatase activity amongst the leishmanias has been investigated with the aim of determining whether the enzymes are adaptations for survival and growth in insect or mammals. The results of the study suggest that there are no marked differences between the acid phosphatase activity of L.major mid log and stationary phase promastigotes. The activities of both, however, differed considerably from those of L.tropica and L.donovani . In contrast to L.tropica and L.donovani, is the apparent lack of surface acid phosphatase on L.major, and so it is suggested that the surface enzyme is not essential for survival of all leishmanias in macrophage. Introduction Acid phosphatase (phosphoric monoester hydrolase,( EC 3.1.3.2) occurs in most eukaryotic cells and is a normal constituent of lysosomes (Mullin et al, 2001,Chatterjea & Shinde 2005). The distribution of acid phosphatase activity in leishmanias, however, is unusual, especially the presence of surface –located enzymes( Mc Gwire et al, 2003 ; Paugam et al, 2003 ). Appearently there are three acid phosphatase complex and it has been reported that one inhibits the oxidative metabolism of neutrophils (Remaley et al ;1984 ,Hassan & Coombs1987 ,Spath et al, 2003). This finding led to the suggestion that surface acid phosphatase of leishmanias plays a crucial role in the parasite`s survival in macrophages (Mc Gwire et al 2002 ; Araujo Soares et al 2003;Yao et al, 2003). A second unusual feature of leishmania acid phosphatases is the large quantity of enzyme that is secreted (Mc Conville et al ,2002).The functional significance of this secreted activity is unclear, but again a role in invasion of macrophages is possible. It is now well established that L. major differs from all other leishmanias investigated in not secreting acid phosphatase; this suggests that either the way in which L . major survives in macrophages is different from the mechanisms used by other leishmanias or that secreted acid phosphatase is not related to survival in macrophages.The present study was undertaken to provide a fascinating insight in to the functional


Introduction
Acid phosphatase (phosphoric monoester hydrolase,( EC 3.1.3.2) occurs in most eukaryotic cells and is a normal constituent of lysosomes (Mullin et al, 2001,Chatterjea & Shinde 2005. The distribution of acid phosphatase activity in leishmanias, however, is unusual, especially the presence of surface -located enzymes ( Mc Gwire et al, 2003 ;Paugam et al, 2003 ). Appearently there are three acid phosphatase complex and it has been reported that one inhibits the oxidative metabolism of neutrophils (Remaley et al ;1984,Hassan & Coombs1987 ,Spath et al, 2003. This finding led to the suggestion that surface acid phosphatase of leishmanias plays a crucial role in the parasite`s survival in macrophages (Mc Gwire et al 2002 ;Araujo Soares et al 2003;Yao et al, 2003). A second unusual feature of leishmania acid phosphatases is the large quantity of enzyme that is secreted (Mc Conville et al ,2002).The functional significance of this secreted activity is unclear, but again a role in invasion of macrophages is possible. It is now well established that L. major differs from all other leishmanias investigated in not secreting acid phosphatase; this suggests that either the way in which L . major survives in macrophages is different from the mechanisms used by other leishmanias or that secreted acid phosphatase is not related to survival in macrophages.The present study was undertaken to provide a fascinating insight in to the functional significance of acid phosphatase by which leishmanias may be adapted for survival in macrophages and how this apparently varies with species .In an attempt to provide more information on acid phosphatase and the part it play ,I have investigated their occurrence in various forms of Iraqi leishmanias .

Acid phosphatase assay :
For enzyme assay , the cells were harvested by centrifugation at 5000xg and washed thrice with 0.9% NaCl in 50mM Tris buffer , pH 7.2 (TBS) , by similar centrifugation .The washed cells were suspended in TBS containing 1mM B.mercaptoethanol and homogenized by sonication using soniprep 150 . The homogenate was centrifuged at 105000xg for 60 min and the resultant pellets were resuspended in TBS to the volume of supernatant fraction . The 105000xg supernatant and pellet were used as the enzyme source . All these steps were carried out at 4C . Acid phosphatase was assayed according to the method described by (Glew et al ,1982) using P-nitrophenylpyrophosphate as substrate . Protein was estimated by the method of (Lowery et al ,1951) .Specific activity of the acid phosphatase is expressed as nmol /min / mg protein.

Isoelectric focusing:
Cells used for isoelectric focusing were washed 3 times (3000xg for 15 min at 4C° ) in 250mM sucrose , pelleted either used immediately or stored at -70C . Parasite homogenates were prepared by resuspending the pellets in 250mM sucrose and sonicating as described above.
Flat-bed isoelectric focusing (IEF) was performed using carrier ampholytes as outlined in the pharmacia guide to IEF . The gel contained 0.28g agarose in 25ml deionised, distilled water . The anode and cathode electrode strips were soaked in 0.05M H 2 SO 4 and 1M NaOH ,respectively . The samples applied contained equal amounts of protein (300-800 µg) unless indicated otherwise . A constant power supply set to deliver a maximum of 1500V , 50mA and 20W was applied and electrophoresis was continued for a total of 2.5h , by which time the human haemoglobin markers had focused .
Protein bands were stained using 0.2% (w/v) PAGE blue 53 dye . Acid phosphatase were stained using the methods described by (Harris & Hopkinson,1976). Approximate pIs of isoenzymes were determined by comparison to pI standards .

Results and Discussion
Each of the Leishmanias investigated, listed in Table 1,were found to contain relatively high levels of the acid phosphatase activities . The specific activites in the homogenates of L.major mid log phase and stationary promastigotes were quite similar to those investigated for other leishmanias .The activity of mid log phase promastigotes was slightly higher than that of stationary promastigotes and in preliminary experiments a similar situation was seen with L.tropica and L.donovani mid log phase and stationary phase promastigotes (Table 1).This is consistent with the report of (Hassan & Coombs,1987) that acid phosphatase activity in L.m.mexicana promastigotes was slightly higher than in amastigotes. The distribution of the acid phosphatase activity of Leishmanias investigated between the soluble and particulate fractions is shown also in Table 1. All activites except for L.major acid phosphatase were sedimentable more than 50% by centrifugation of cell homogenates at 105,000 Xg for 1 hour . In contrast ,only about 21% and 25% of the acid phosphatase activity of mid log phase and stationary promastigotes of L.major was sedimentable by centrifugation. Respectively.
In this respect , L.major differs from other species of Leishmania that have been investigated for which most of the acid phosphatase activity was found to be particulate (Gottlieb & Dwyer,1981, 1982Glewr et al, 1982, Hassan &Coombs,1987. Isoelectric focusing of homogenates of L.major consistently revealed just one isoenzyme with an apparent pI of 5.6 in mid log phase and stationray promastigotes (Fig.1) .This contrasts with L.donovani and L.tropica promastigotes ,which possess two isoenzymes as revealed by isoelectric focusing (Fig.2) As indicated isoenzymes of acid phosphatase of L. donovani(pIs 4.7 and 5.2 ) and L. tropica ( pIs 5.3 and 5.6) were at higher activity in mid log phase promastigotes than in stationary . Interestingly L.m.mexicana amastigotes contain different and fewer isoenzyme (Coombs et al ,1987) . Overall , these data show that mid log phase and stationry promastigotes of L.major possess very similar acid phosphatase activities and suggest that parasites do not acquire more or additional acid phosphatase activities concomitant with transforming to the metacyclic form .
Greater variation was found when the occurrence of acid phosphatase activities on the surface of the organisms was investigated.As shown in Fig.3 .acid phosphatase activity has occur on the surface membrane of mid log phase as wells as stationary promastigotes of L.donovani ; this was apparently the same as with L.m.mexicana (Hassan & Coombs,1988) and L.m. amazonensis ( Pimenta & de Souza, 1986) .In contrast ,however ,L.major promastigotes (mid log phase and stationary ) lacked surface acid phosphatase (Fig.3) under the conditions employed in this study.
The lack of surface bound activity in L.major correlate well with the finding that less of the acid phosphatase activity of this species is particulate compared to other mammalian leishmanias .It is possible that L.major possesses a surface acid phosphatase so different from that of other trypanosomatid that it was not detected by the method used. Nevertheless, the results show that with respect to surface acid phosphatase, L.major differs significantly from the other mammalian parasites and suggest that surface localized acid phosphatase is not essential for survival of all leishmanias in macrophages. The finding of the apparent lower activity of acid phosphatase present on the surface of L.m.mexicana amastigotes compared with promastigotes also point to a role for the enzyme in the insect host ( Hassan & Coombs, 1987) .Similarly ,the finding of high activity present on the surface of an insect trypanosomatid, H.m.muscarum, (Coombs et al ,1987) is especially intriguing and also provides strong evidence for a role of this enzyme in the invertebrate .At present ,there is no sufficient information to understand why only some insect trypanosomatids possess the enzyme or its role when present . These findings ,although providing no support for the proposal ,do not rule out the possibility that the surface acid phosphatase of L.donovani and L.mexicana plays some crucial part in survival of the parasites in macrophages ( Remaley et al ,1984( Remaley et al , ,1985Hassan & Coombs ,1987;Mottram et al ,2004) .Literatures ,however, suggested that other factors must also be involved and that the presence of a similar enzyme on the ancestral insect forms could have been a useful preadaptation for parasitism of mammals. The finding that C.fasciculata and L.donovani release acid phosphatase into the medium can be interpreted in a similar fashion ( Mottram et al, 2004) . A need for more information on the environmental condition of the various trypanosomatids in their insect hosts to enable us to understand the parts the enzymes play . The lack of detectable secretion of acid phosphatase by L.major also distinguishes this species from the other mammalian Leishmanias ( Dwyer & Gottlieb,1985) .Indeed the results of this study suggest that acid phosphatase is not central to the survival of L.major in mammals .It remain to be seen whether this is also true for other species , but currently available evidence indicates that different species of Leishmania may differ, at least quantitatively, in the ways in which they are adapted for survival in macrophages.
 Glew, R., Diven, W., Berens ,R. and Katsoulis,D.,(1982):  a. The activities given are those in parasite homogenates , are expressed in nmol/min/mg protein and are the means ± SD from three determinations . b. The activity sedimented in the 105,000xg pellet as a % of the total activity recovered .The figures are the means ±SD from replicate experiments