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Phlebotomus papatasi and Leishmania major parasites express α-amylase and α-glucosidase Article in Acta Tropica · February 2001 DOI: 10.1016/S0001-706X(00)00164-9 · Source: PubMed
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Acta Tropica 78 (2001) 41 – 49 www.parasitology-online.com
Phlebotomus papatasi and Leishmania major parasites express a-amylase and a-glucosidase Raymond L. Jacobson *, Yosef Schlein Department of Parasitology, The Ku6in Center for the Study of Infectious and Tropical Diseases, The Hebrew Uni6ersity-Hadassah Medical School, 91120 Jerusalem, Israel Received 3 December 1999; received in revised form 4 September 2000; accepted 5 October 2000
Abstract Alpha-amylase and a-glucosidase activities were found in homogenates of young, unfed male and female Phlebotomus papatasi and in gut and salivary gland preparations. A significant increase in both enzyme activities in females and of a-amylase in males was recorded for flies that had fed overnight on a plant (Capparis spinosa). After plant feeding, a-amylase activity was relatively lower in female salivary glands and higher in guts, while in the males the activity in the salivary glands had increased. Alpha-glucosidase activity increased in guts of both sexes and in the salivary glands of the females. In addition, a-amylase activity was found in preparations of Leishmania major and L. infantum promastigotes, but not in those of L. dono6ani or L. tropica. Alpha-glucosidase activity was present in promastigote preparations of L. major, L. infantum, L. dono6ani, L. braziliensis, Crithidia fasciculata and Herpetomonas muscarum. It was lacking in similar preparations of L. tropica, Sauroleishmania agamae or Leptomonas seymouri. The growth rate of L. major promastigotes in medium supplemented with starch or with glucose was similar and it was significantly higher than in glucose poor medium. In this study, we demonstrate that P. papatasi and L. major possess the enzymes for hydrolyzing starch grains that are included in the plant tissue-diet of the sand flies. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Phlebotomus papatasi; Leishmania; Trypanosomatids; a-amylase; a-glucosidase
1. Introduction Phlebotomine sand flies obtain energy for their activities from a staple diet consisting of honeydews excreted by aphids (Killick-Kendrick and Killick-Kendrick, 1987; Moore et al., 1987; MacVicker et al., 1990; Wallbanks et al., 1991; * Corresponding author. Fax: +972-2-6757425. E-mail address:
[email protected] (R.L. Jacobson).
Molyneux, et al., 1991; Cameron et al., 1995) and ingested plant tissues from leaves and stems (Schlein and Jacobson, 1994; Schlein and Muller, 1995). These tissues contain various amounts of sucrose and starch that are the main products of photosynthesis. Sucrose is the principal carbohydrate exported from the leaves, while the excess accumulates temporarily as sucrose in the vacuoles and as starch granules in the chloroplasts (Taiz and Zeiger, 1991). In a recent study we have
0001-706X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 0 ) 0 0 1 6 4 - 9
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observed that the natural diet of Phlebotomus papatasi can include considerable amounts of starch grains (Schlein and Jacobson, 2000). We therefore investigated the possibility that starch hydrolyzing enzymes are present in Ph. papatasi and in their specific Leishmania major parasites. In addition, we compared the growth of L. major promastigotes in media supplemented with starch or with glucose. Starch is specifically hydrolyzed by a-amylase to maltose, which is then cleaved to glucose by a-glucosidase (Dixon and Webb, 1979) and activities of these enzymes have been reported for other sand flies. Alpha-amylase activity was detected in the salivary glands, crops and guts of Lutzomyia longipalpis (Charlab et al., 1999; Ribeiro et al., 2000), but a-glucosidase activity has only been found in the midguts of this sand fly (Gontijo et al., 1998) and Ph. langeroni (Dillon and El-Kordy, 1997).
2. Materials and methods
2.1. Feeding of sand flies on plant branches Laboratory reared Ph. papatasi were from a colony originating with flies from Kfar Adumim 15 km east of Jerusalem. The sand flies were routinely maintained at a temperature of 269 1°C and 80% relative humidity (RH). Branches of caper plants (Capparis spinosa) were freshly cut at sunset and their stems put in an Erlenmeyer flask with water. These branches were placed into 3.8 l cylindrical cardboard boxes covered with suitable netting into which series of 24 – 48 h old unfed males and females were introduced. A water soaked cotton wool swab was placed on the net cover. Other series were put into similar empty containers and received only water. All series were left overnight at 2491°C with 80% RH. In the morning, the flies were anesthetized with CO2 and preparations for enzyme assays were made. To estimate the number of sand flies that had fed on the C. spinosa branches, a random sample was tested for the presence of sugar by the cold anthrone test (Van Handel, 1972), modified for quantitative analysis of sand flies in a microplate
reader (Schlein and Jacobson, 1999). Using the modified method, the optical densities of the reaction fluids were converted to micrograms of sucrose from a standard curve. The reactions were classified as follows: Class 0, negative for sucrose; Class I, 0.5–1.5 mg sucrose; Class II, 1.6–5.0 mg sucrose, Class III, 5.1–15 mg sucrose; Class IV, \ 15 mg sucrose.
2.2. Trypanosomatid species and culti6ation of parasites The following parasites were obtained from the World Health Organization Leishmania Reference Center, Hebrew University Jerusalem. Leishmania (Leishmania) major isolates: MHOM/IL/67/Jericho II (LRC-L137), MHOM/ IL/90/LRC-L585, MHOM/IL/86/Blum (LRC-L509), IPAP/IL/84/ Uvda (LRC-L465) and IPAP/IL/98/Avivit (LRCL746); L. dono6ani MHOM/SD/??/Khartoum (LRC-L661); L. infantum MCAN/IL/97/Rex (LRC-L720); L. tropica, MHOM/IL/90/P283 (LRC-L590); L. (Viannia) braziliensis MHOM/ BR/75/M2903 Sauroleishmania agamae RAGA/ IL/8?/LRC-L409; Crithidia fasciculata, LRCL466; Herpetomonas muscarum, LRC-L523, NLB345; Leptomonas seymouri, LRC-L524, NLB 339. Strains of parasite were initially grown from frozen stabilates in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content (Biological Industries, Kibbutz Bet HaEmek, Israel) supplemented with 4 mM L-glutamine, penicillin, 200 mg ml − 1 streptomycin, 200 IU ml − 1 (Teva, Israel) and either 10% (v/v) inactivated fetal calf serum or serum free medium (Jacobson and Schlein, 1997). Parasites were passaged twice and grown to early logarithmic stage and washed three times in phosphate buffered saline (PBS) pH 7.2, before being used for experiments. The exponential growth of promastigotes was monitored in Panmede medium (Paines & Byrne, UK) (Gambarelli and Dumon, 1988), supplemented with 4.5 g l − 1 of extra pure starch, (B 0.5% maltose as the only reducing sugar present, BDH, UK), 25 mM glucose, or without any additive. Starting density of cells was 1 × 106 ml − 1 and cultures were gently agitated continuously at 279 1°C. Parasites were counted daily in quadruplicate and the experiments were repeated twice.
R.L. Jacobson, Y. Schlein / Acta Tropica 78 (2001) 41–49
2.3. Preparations for enzyme acti6ity assays Enzyme assays were carried out with preparations made from single whole sand flies; groups of dissected male or female guts or groups of dissected male or female salivary glands. The flies or the dissected organs were suspended and homogenized in either 110 mM Hepes buffer (pH 7.4) containing 100 mM NaCl and 10 mM MgCl2 for a-amylase activity (Ribeiro et al., 2000) or 50 mM citrate/phosphate buffer (pH 6.5) for a-glucosidase activity. The homogenates were lysed by snap freezing in liquid nitrogen and thawing three times, centrifuged at 800× g for 15 min at 4°C and the supernatants stored at − 70°C. Cultures of parasites at mid-log phase (3 days), at stationary phase (7 days) and uninoculated culture media were centrifuged at 800× g for 15 min at 4°C. Supernatants were collected and the protein fractions precipitated with 70% (of saturation at 0°C) ammonium sulphate (Fraction II), resuspended in PBS and stored as described by Schlein et al. (1991). The pellets of promastigotes were washed three times in PBS, diluted in PBS to 5×10 8 cells ml − 1 and lysed by snap freezing in liquid nitrogen and thawing three times. The lysates were centrifuged at 12 000× g for 10 min. and the supernatants stored at − 70°C. Protein concentrations in the cell lysates, supernatants or in gut homogenates were measured by the method of Bradford (1976), using a standard of bovine serum albumin.
2.4. Enzyme assays Enzyme assays were carried out with 25 mg of Fraction II proteins (containing the products of 5 × 106 parasites), lysed cells (5× 106 parasites) or with aliquots equivalent to a sand fly gut, single or pairs of salivary glands or individual whole fly homogenates. Sand fly preparations were adjusted to contain the equivalent of one gland or one gut or one whole fly in 8 ml for a-amylase assays. For a-glucosidase assays, sand fly preparations were adjusted to contain the equivalent of one gut or a whole fly in 10 ml and a pair of glands in 20 ml. Fraction II of uninoculated complete medium incubated with substrate or incubated substrate alone were used as controls in all the experiments.
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The substrate p-nitrophenyl-D-maltopentoside (Sigma, St. Louis, MO) was used for assaying a-amylase activity (David, 1982; Wallenfels et al., 1982). The reaction mixture was 110 mM Hepes buffer (pH 7.4) containing 100 mM NaCl and 10 mM MgCl2; 2 mM substrate; 2 U/ml a-glucosidase (Sigma, grade IV from yeast) as described by Ribeiro et al., 2000, and 8 ml of a sand fly sample or 25 mg of Fraction II protein or lysed cells, in a final volume of 100 ml. The reactions were incubated for 2 h at 37°C and terminated with an equal quantity of 0.45 M NaOH/glycine (pH 10.2). The reactions were centrifuged at 12 000× g for 2 min. The absorbance of the clear supernatant was measured at 405 nm in a plate reader (Bio-Tek Instruments, Winooski) against a substrate blank. To assay a-glucosidase activity, the substrate p-nitrophenol a-D-glucopyranoside (Sigma) diluted in 50 mM citrate/phosphate buffer (pH 6.5) to a final concentration of 12 mM, was used (Terra et al., 1979). The pH optima for the enzyme was pre-determined using 50 mM citrate/phosphate buffer in a range from pH 4 to 7, with the substrate and either a sand fly gut or Fraction II protein (data not shown). The reaction mixture was 10 ml of gut preparations, 20 ml of salivary gland preparations or 25 mg of Fraction II protein or lysed cells and 20 ml substrate in 50 mM citrate/phosphate buffer to give a final volume of 120 ml. The reaction mixtures were incubated at 37°C and monitored for increase in absorbance at 30 min intervals for 2 h. The reactions were terminated and processed, as described for the amylase assay. One enzyme unit of activity (U) is the amount of enzyme required to hydrolyze 1 mmol of substrate per minute under the conditions of the assay.
2.5. Statistical analysis The results of the assay series were tested for deviation from the Gaussian distribution by the Kolmogorov-Smirnov distance test. After all groups passed this test (P\ 0.1), unpaired t-test was used to test for differences in enzyme activity between assays with tissues of sand fly series that had been exposed to both C. spinosa and water or water only. (Zar, 1984).
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3. Results
3.1. Anthrone test for sand flies fed on C. spinosa A random sampling of female and male Ph. papatasi exposed to C. spinosa, were examined by the cold anthrone test for the presence of sugars. Of the females tested, most (20/23) had taken large sugar meals (range 1.6 – 23.7 mg sucrose/fly) and 18/24 of the males were positive for sugar (range 0.5–5.0 mg sucrose/fly).
rations of the other two species had no activity. Of the four species, only the promastigotes of L. major strains secreted a-amylase into the growth medium (Table 2). Control Fraction II proteins from the serum-free media were always negative.
3.4. Alpha-glucosidase acti6ity in Leishmania and other trypanosomatids Five species of Leishmania and four other trypanosomatid genera were tested for a-glucosidase
3.2. Alpha-amylase and a-glucosidase acti6ity in sand flies Alpha-amylase and a-glucosidase activities were higher in females than males in the whole fly homogenates of unfed Ph. papatasi. In a series that had been fed on plant branches these activities increased significantly, except for a-glucosidase in males: (female a-amylase, t = 4.969, df = 24, PB0.0001; male a-amylase, t = 5.578, df = 25, PB0.0001; female a-glucosidase, t = 6.346, df= 24, PB 0.0001) (Fig. 1(A – B)). Both types of activities were found in gut and salivary gland preparations of unfed flies and the changes following plant feeding were not in a uniform direction. The a-amylase activity decreased significantly in the salivary glands of females (t = 2.35, df = 23, P=0.027), but increased in male salivary glands (t=4.689, df = 23, P B0.0001). The amylase activity increased in the guts of females (t = 4.984, df= 26, P B0.0001), but not in those of males. The activity of a-glucosidase increased in the guts of both sexes (females, t = 10.38, df= 26, P B 0.0001; males, t = 6.94, df = 23, P B 0.0001) and in the salivary glands of the female flies (t = 21.98; df=23; P B0.0001) (Table 1).
3.3. Alpha-amylase acti6ity in Leishmania preparations The four species of Leishmania tested for aamylase activity were L. major, L. infantum, L. dono6ani and L. tropica. Cell lysates of the L. major strains had activities ranging from 2.1 to 4.3 U/mg protein, while that of L. infantum preparations were only 0.4 U/mg protein. Prepa-
Fig. 1. Box and whisker plots of a-amylase (A) and a-glucosidase (B) activity of whole fly homogenates of male and female Phlebotomus papatasi had been fed on Capparis spinosa (caper plant) as compared to unfed controls. (The box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median, the 50th percentile. Whiskers extend down to the smallest value and up to the largest.) (n = 13 – 15 per series).
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Table 1 Alpha-amylase and a-glucosidase activities in unfed Phlebotomus papatasi and in flies fed on Capparis spinosa (caper plant) and watera a-amylase mU/organ
a-glucosidase mU/organ
Test material
Unfed
Caper fed
Unfed
Female salivary glands Male salivary glands Female guts Male guts
0.99 9 0.1 (n=10) 0.65 90.1 (n= 10) 0.33 9 0.14 (n= 10) 0.21 9 0.04 (n= 10)
0.63 90.09b (n =15) 0.839 0.09b (n =15) 0.59 0.02b (n =18) 0.19 9 0.06 (n = 15)
3.1 90.11 1.290.09 14.191.6 10.991.2
a b
Caper fed (n =10) (n =10) (n =10) (n =10)
4.29 0.13b 0.9590.08 19.49 1.1b 14.39 1.2b
(n =15) (n =15) (n =18) (n =15)
The results represent the mean of the experiments and their standard deviation. Significantly different from unfed sand flies (PB0.05).
activity. Cell lysates and Fraction II proteins of L. major, L. dono6ani, L. infantum and L. braziliensis had divers activity levels but no activity was measured for L. tropica preparations. Alpha-glucosidase activity was also detected in cell lysates and Fraction II proteins of Crithidia fasciculata and Herpetomonas muscarum but not from Sauroleishmania agamae or Leptomonas seymouri (Table 3). The activity in cell lysates of L. major (1.3 and 1.2 U/mg protein) and L. dono6ani (1.9 U/mg protein) were significantly lower than those of L. infantum (7.9 U/mg protein) and L. braziliensis (13.3 U/mg protein). There were smaller differences between the activities of supernatant proteins (Fraction II) of these parasites and they were not congruent with the results from the cell lysates. The L. major strains LRC-L137 and LRC-L585 had the highest and lowest activities of 21.6 U/mg and 6.7 U/mg protein, respectively (Table 3). Fraction II protein control preparations from uninoculated culture media showed no activity.
3.5. Starch as a nutrient for L. major promastigotes The growth of L. major promastigotes was similar in Panmede media supplemented with either 25 mM glucose or 4.5 g l − 1 starch. In Panmede media without any supplement, exponential growth was significantly lower during the logphase and was 40% lower at the stationary phase (Fig. 2).
4. Discussion The demonstration of a-amylase and a-glucosidase activities in the digestive system of Ph. papatasi elucidates a new aspect in the nutrition of this species. These sand flies are widely distributed from Western Europe to North Africa, the Middle East and the Indian subcontinent and they transmit L. major in vast areas of savannah and desert (Ashford and Bettini, 1987). In a typical arid focus of L. major in the Jordan Valley, we observed that Ph. papatasi obtains carbohydrate meals by feeding directly on plants (Schlein and Table 2 a-Amylase activity promastigotesa,b
of
Parasites
a-Amylase U/mg protein
L. major L137 L. major L465 (3 days) L. major L465 L. infantum L. dono6ani L. tropica a
different
Leishmania
Cell lysatesc
Medium proteind
4.3 90.6 2.1 90.1
11.290.56 0.6 90.1
2.1 9 0.1 0.4 90.05 0 0
9.190.46 0 0 0
species
The results are the mean and standard deviation of two experiments run in triplicate. b Stationary phase cultures (7th day) grown in serum-free medium. c 5×106 lysed parasites. d Containing the Fraction II protein products of 5×106 parasites.
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Table 3 a-Glucosidase activity in different Leishmania species and trypanosomatid promastigotesa,b Parasites
a-Glucosidase U/mg protein Cell lysatesc
L. major L-L137 1.39 0.07 L. major L-465 (3 0.9 9 0.1 days) L. major L-465 1.2 9 0.015 L. infantum 7.99 0.1 L. dono6ani 1.99 0.08 L. tropica 0 L. braziliensis 13.3 9 0.7 Crithidia fasciculata 6.69 0.35 Herpetomonas 12.49 1.2 muscarum Sauroleishmania 0 agamae Leptomonas seymouri 0
Medium proteind 21.69 1.1 6.79 0.4 8.39 0.2 11.29 0.6 11.49 0.5 0 17.29 0.9 19.39 1.3 9.69 0.48 0 0
a The results are the mean and standard deviation of two experiments run in triplicate. b Stationary phase cultures (7th day) grown in serum-rich medium. c 5×106 lysed parasites. d Containing the Fraction II protein products of 5×106 parasites.
Jacobson, 1999). In that study, we evaluated the meals by testing the flies for sugar in the gut. However, sugars are not the only nutritive components in the plant tissue meals of Ph. papatasi. The photoassimilates that plants store during the day are sucrose and starch (Hopkins, 1995). Chenopods are a major component of the vegetation in deserts and belong to the C-4 plants that accumulate mostly starch (Lunn and Hatch, 1995). Plants of this family are also prevalent in the arid habitats in the Jordan Valley where Ph. papatasi feed on their tissue (Schlein and Jacobson, 1999). We have recently observed that plants tissue meals of Ph. papatasi include starch grains and such grains were found in 50% (26/32 females and 11/29 males) of fly series caught in the Jordan Valley (Schlein and Jacobson, 2000). This evidence implies that starch may be an important component in the food of Ph. papatasi and this supposition is substantiated by the presence of the enzyme activities that degrades starch. Additional
support for this assumption is the induction of a higher activity of both enzymes in males and female flies that had been allowed to feed on a plant. The wide variation between the activities measured for individual sand flies apparently reflects the differences observed in the tests for sugar. Some flies had taken large plant tissue meals while others refrained from feeding. This apparently is also the reason that changes in the enzymatic activities in guts and salivary glands of the plant fed series did not exhibit a uniform trend. There was a decrease in a-amylase activity in the salivary glands of the females and an increase in the midguts, while males had increased activity in the salivary glands and little change in the midguts. Alpha-glucosidase activity was slightly reduced in the male salivary glands and increased in the midguts while it increased significantly in female salivary glands and midguts. Another possible reason for the lack of uniformity is the time interval between actual feeding and processing which could vary between 1 and 17 h. Alpha-amylase has been described from Lu. longipalpis, the vector of L. chagasi, and following a blood meal it decreased in the salivary glands of the females (Charlab et al., 1999; Ribeiro et al., 2000). Low levels of amylase activity have been reported from whole body homogenates of other blood sucking Diptera (McGeachin et al., 1972; Sakai et al., 1976), and a cDNA homologous to amylase has been reported from Aedes aegypti salivary glands (Grossman and James, 1993; Grossman et al., 1997). Digestive glycosidase activity from Ph. papatasi has been assayed using high performance liquid chromatography (Samie et al., 1990). Alpha-glucosidase activity has been reported from both the midguts of Lu. longipalpis, (Gontijo et al., 1998) and Ph. langeroni, the vector of L. infantum, in which the enzyme activity increased following both sugar and blood meals (Dillon and El-Kordy, 1997). Alpha-glucosidase activity has been reported from other blood sucking Diptera, (Billingsley and Hecker, 1991; Marinotti and James, 1990; Marinotti et al., 1996). Different Leishmania parasites were assayed and a-amylase activity was detected in L. major
R.L. Jacobson, Y. Schlein / Acta Tropica 78 (2001) 41–49
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Fig. 2. Growth curve of Leishmania major promastigotes in culture media with 25 mM glucose (), 4.5 g l − 1 starch ( ) or without additive (). Each point represents the mean of two experiments and their standard deviations. Each time point was counted in quadruplicate, from duplicate cultures ( = eight estimates per time point).
and L. infantum promastigotes preparations and in their culture medium, where it increased 15fold during the growth from log to stationary phase. No enzymatic activity was recorded for preparations from cultures of L. tropica or L. dono6ani. Most of the Leishmania and other trypanosomatids secreted a-glucosidase into the media and this enzyme was detected in preparations of L. major, L. infantum and L. dono6ani. These observations add to the list of glycosidase activities that have been reported for Leishmania and other trypanosomatids including chitinase and Nacetylglucosaminadase (Schlein et al., 1991); sucrase in L. dono6ani (Blum and Opperdoes, 1994); N-acetylgalactosaminadase from L. amazonensis (Gontijo et al., 1996) and cellulase from L. major (Jacobson and Schlein, 1997). Leishmania promastigotes that reside exclusively in the gut depend on the diet of the sand fly to sustain their growth. Hence, after the infective blood meal is digested, the only source of nutrition for the parasites is the sugar meals that may include starch. Our experiments show that the amount of enzymes produced by cultured L. major are sufficient to promote the growth of the parasites in starch containing medium without
glucose. We suggest that the situation in the sand fly is similar and the parasites digest the starch in the natural system.
Acknowledgements This study was supported by NIH grant RO1 AI40926, the Center for the Study of Emerging Diseases and Deutsche Forschungsgemeinschaft for the German–Israeli–Palestinian cooperative project on leishmaniasis in Israel and the West Bank.
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