ORIGINAL PAPER

Immunolocalization of arginine kinase (AK) in Toxocaracanis, Toxocara vitulorum, and Ascaris lumbricoides

D. G. R. S. Kulathunga & Susiji Wickramasinghe &

R. P. V. J. Rajapakse & Lalani Yatawara &

W. R. Jayaweera & Takeshi Agatsuma

Received: 25 October 2011 /Accepted: 27 February 2012 /Published online: 8 March 2012# Springer-Verlag 2012

Abstract Arginine kinase (AK) is a member of the phos-phagen kinase family. AK plays a major role in cellularenergy metabolism in invertebrates including nematodes.In the present study, we performed the direct immunofluo-rescence test to determine the immunolocalization of AK indifferent stages of the life cycle (eggs, larvae, and adultworms) of Toxocara canis, Toxocara vitulorum, andAscaris lumbricoides. Our results indicated variable levelsof expression of AK in different stages. Moreover, strongfluorescence was observed in cleaving eggs than in dormanteggs. The highest activity of the enzyme was observed in thefully developed eggs. This may be due to high expression ofAK in embryonic development, which is associated withincreased energy demand due to cleavage and cellulardifferentiation. Surprisingly, expression of AK is signifi-cantly higher in the middle part and posterior end comparedto anterior end of the larvae. In addition, AK is highlyconcentrated in cellular and metabolically active parts ofthe body such as hypodermis, muscle, intestine, ovaries,

oviducts, and uterus, while it is absent in noncellular areaslike cuticle. The present study revealed the presence of AKin T. canis, A. lumbricoides, and T. vitulorum and that itplays a major role in energy metabolism of these nematodes.Interestingly, antiserum was prepared against the recombi-nant T. canis AK and reacts with the native AKs of T. canis,A. lumbricoides, and T. vitulorum. AK levels could vary inrelation to maximum potential rates of ATP turnover,oxidative capacity, and energy output. Further studies onsubcellular localization of AK in these important helminthsprovide new information for researchers to develop effectiveanthelmintics against the parasites of veterinary and ofpublic health importance.

Introduction

Phosphagens are phosphorylated guanidine compounds thatare linked to ATP by way of a reversible reaction catalyzedby phosphagen (guanidine) kinases (phosphagen+MgADP+H+ ↔ guanidine acceptor+MgATP) (Wickramasinghe etal. 2007). The traditional and conventional view is thatphosphagens function as ATP buffers, permitting mainte-nance of high ATP values during periods in which there isdisequilibrium of ATP supply and demand (Kammermeier1987, 1993). Phosphagens also play a number of otherroles, including regulation of glycogenolysis, protonbuffering, and intracellular energy transport (Ellington2001). In vertebrates, phosphocreatine is the sole phos-phagen whereas invertebrates have at least seven uniquephosphagens and their corresponding kinases in additionto phosphocreatine (Van Thoai 1968; Watts 1968;Morrison 1973; McLeish and Kenyon 2005). Nematodephosphagen kinases are thought to be arginine kinase(AK). AK catalyzes the reversible transphosphorylationbetween adenosine diphosphate and phosphoarginine,

D. G. R. S. Kulathunga : S. Wickramasinghe :R. P. V. J. Rajapakse (*) :W. R. JayaweeraDepartment of Veterinary Pathobiology, Faculty of VeterinaryMedicine and Animal Science, University of Peradeniya,Peradeniya, Sri Lankae-mail: jayanthar@pdn.ac.lk

L. YatawaraDepartment of Medical Laboratory Science, Faculty of AlliedHealth Science, University of Peradeniya,Peradeniya, Sri Lanka

T. AgatsumaDepartment of Environmental Health Sciences,Kochi Medical School, Kochi University,Oko-cho,Nankoku City, Kochi 783-8505, Japan

Parasitol Res (2012) 111:663–671DOI 10.1007/s00436-012-2884-z

which is involved in ATP buffering. Therefore, AKplays a major role in metabolic events such as glyco-genolysis, glycolysis, and oxidative phosphorylation ininvertebrates with an AP/AK system (Ellington 2001).

The first measurement of AK activity in Ascaris lumbri-coides was done by Livingstone et al. (1983). Another studyshowed in vivo exchange of phosphorus between phosphoar-ginine and adenosine triphosphate in the rhabditoid nematodeSteinernma carpocapsae (Thompson et al. 1992). Furthermore,Platzer et al. (1999) suggested that this enzyme is a significantcomponent of the energy metabolism in both fully developed(L3) larvae and adult worms, probably playing a major role incellular energy metabolism. In addition, the presence of AK inToxocara canis, Ascaris suum, Caenorhabditis elegans, andHeterodera glycines was reported (Wickramasinghe et al.2007; Nagataki et al. 2008; Uda et al. 2006; Matthews et al.2003). Moreover, it has been suggested that AK localizationsites of T. canis could be cytoplasmic or associated with theendoplasmic reticulum (ER) or it can be a part of a secretorypathway (Wickramasinghe et al. 2007). Furthermore, a studyhas shown intracellular localization (cytosol) of AK inarthropods. Immunocytochemical study has revealed thatAK is found in actin-containing regions of crayfishmyofibrils (Benzonana and Gabbiani 1978), whereas inDrosophila AK is associated with the Z-line region of flightmuscle and the A band in tubular muscle (Lang et al. 1980).In addition, a study showed that AK is expressed throughoutthe developing nervous system of the grasshoppers (Wanget al. 1998).

Because of drug resistance and adverse side effects, thereis a need to develop novel chemotherapeutic agents againstimportant nematodes like T. canis, Toxocara vitulorum, andA. lumbricoides, which cause significant health problems inanimals as well as in humans. The adult worms of T. canisinhabit in the small intestine of dogs and other canids, andhumans can get the infection by accidental ingestion of T.canis infective eggs in contaminated soil or environment.There are several forms of this disease in humans, namely,ocular larva migrans, visceral larva migrans, and a spinal orneurological form (Urqhuart et al. 1996; Gibbons et al.2001; Li et al. 2006; Minnaar et al. 2002; Oliveira-Sequeira et al. 2002; Habluetzel et al. 2003; Glickman andMagnawal 1993; Kazacos 2000; Umehara et al. 2006). T.vitulorum is found in the small intestine of cattle and waterbuffalo, particularly buffalo calves between 1 and 3 monthsof age causing anemia, diarrhea, weight loss, anorexia, andeven death. Moreover, A. lumbricoides remains a major inter-national public health concern (Williams-Blangero et al.1999). Roundworm is the most prevalent of the intestinalhelminth infections, affecting one-fourth of the world’s popu-lation (Tanowitz et al. 1994). Severe infection can causeserious morbidity and mortality due to intestinal blockage(Tanowitz et al. 1994; Savioli et al. 1992).

Effective drug targets for these parasitic infections can begenes or proteins of parasites that are absent or quite differentfrom those in the mammalian host, and also, these targets mustbe playing a crucial role in the metabolism of the parasite(Jarilla and Agatsuma 2010). AK plays a significant role inenergy metabolism in invertebrates, and it is absent in mam-malian tissues. Therefore, it might be a potential drug targetagainst the nematodes like T. canis, A. lumbricoides and T.vitulorum. Further studies on immunolocalization of AK inthese important helminths provide new information forresearchers to develop effective anthelmintics against thenematodes of veterinary and of public health importance.However, no reports on immunolocalization of AK in variousstages of the life cycle of T. canis, A. lumbricoides, and T.vitulorum exist thus far. Therefore, in the present study, wehave demonstrated the expression of AK in different stages ofthe development of T. canis, T. vitulorum, and A. lumbricoidesby the fluorescent antibody technique.

Materials and methods

Expression and purification of T. canis recombinant AK

As previously described by Wickramasinghe et al. (2008), T.canis AK was amplified using polymerase chain reactions.The open-reading frame of T. canis AK was cloned into theBamH1/SalI site of pMAL-c2. The ligated product wastransformed into Escherichia coli JM109 cells. E. coliJM109 cells with pMAL plasmid, which expresses the T.canis AK as a fusion protein with maltose-binding protein(MBP), were cultured in Luria-Bertani medium. Expressionof the recombinant T. canis AK in E. coli cells was inducedwith 1 mM isopropyl β-D-1-thiogalactopyranoside at 25°Cfor 24 h.

The cells were solubilized in extraction buffer containing10% glycerol, 1.0 M Tris–HCl (pH 6.8), 2.3% sodiumdodecyl sulfate (SDS), 5% 2-mercaptoethanol. Then, itwas subjected to SDS-polyacrylamide gel electrophoresis(PAGE) in a 10% gel containing a molecular weight marker.SDS-PAGE analysis of T. canis AK showed a band with amolecular mass of approximately 85 kDa (see Fig. 1). It wasseparated and diluted in phosphate buffered saline (PBS).Protein concentration was measured (0.165 mg/ml) andstored at −20°C until use.

Preparation of antiserum against the recombinant T. canisAK

Antigen (recombinant TCAK) was injected to BALB/c miceat 3-day interval subcutaneously. Blood was collected after3 weeks from both immunized and control groups. Serawere separated and stored at −20°C until use.

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Fixation of adult worms

Adult worms of T. canis, T. vitulorum, and A. lumbricoideswere fixed in buffered formalin (10%), and 10-μm thicknesstissue sections were obtained using Thermo Shandon Cryotome(UK) from anterior and posterior end and middle part of theworms. Sections were deparaffinized and rehydrated.

Collection of eggs and larvae

T. canis eggs were obtained from the Toxocara-infected pups.A. lumbricoides eggs were collected from the fecal samples ofinfected humans. Eggs were washed thrice in PBS. Then, eggswere incubated in room temperature until they were embryo-nated. Eggs were collected at days 0, 5, and 10, and a directimmunofluorescence antibody (IFA) test was performed(Kolodziejczyk 1999). The fully embryonated eggs contain-ing L3 larvae (days 8 to 10 of incubation) (Kolodziejczyk1999) were suspended in PBS and decoated in 6–7% sodiumhypochlorite (NaOCl) solution for 20 min at 37°C as de-scribed by Stevenson and Jacobs (1977) and Smith et al.(1983). Decoated eggs were washed in PBS, until no chlorineodor could be detected, and CO2 was bubbled through thesuspension at 39°C until the larvae hatched (15–20 min).Hatched T. canis larvae were used to determine the immuno-localization of AK.

Immunofluorescence for fixed worm sections

Deparaffinized worm sections were washed in PBS (Na2HPO41.15 g, K2H2PO4 0.2 g, NaCl 8 g in 1 l of distilled water(pH 7.2)) and kept in a humidified chamber and incubated at37°C for 1 h with antisera prepared against the recombinant T.

85 kDa T. canis + MBP (45 + 40 kDa)

Fig. 1 SDS-polyacrylamide gel electrophoresis (PAGE) of the recom-binant T. canis AK expressed as a fusion protein with MBP. (A) Proteinladder, (B) recombinant T. canis AK with MBP

Fig. 2 Immunolocalization of arginine kinase in eggs and larvae of T.canis. a T. canis embryonated eggs (×40). b Negative control (eggs),no fluorescence (×40). c T. canis larvae (×40). d Negative control(larvae), no fluorescence (×40). e Cleaving egg, and f strong fluores-cence appeared in the cleaving egg (days 2–3) (×40). i Developed egg,

light microscopic view (×40). j Strong fluorescence appeared in fullydeveloped eggs (×40). g, k Light microscopic view of T. canis larvae(×40). h, l Strong fluorescence was observed in the middle part andposterior ends and no fluorescence in the anterior ends of the larvae(×40). a anterior end, p posterior end. Scale bars 50 μm

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canisAK (primary antibody) at a 1:50 dilution with IFA serumdilution buffer (NaN3 0.1 g, gamma globulin-free BSA 1.0 g,heat-inactivated normal (goat) serum 1 ml in 100 ml of PBS(pH 7.38)). Thereafter, sections were washed three times inPBS (5 min each time) and incubated with fluorescent conju-gate (affinity-purified antibody fluorescein-labeled goat anti-mouse IgG (H+L), Cat: 172-1806, Kierkegaard and PerryLaboratories) (1:50 diluted) for 30 min. Then, samples werewashed again three times in PBS as described previously. In thefinal washing, a drop of eriochrome black T was added to thewashing buffer. Finally, fluorescence-stained samples weredried and a drop of “Dabco” mounting medium was added toeach section and observed under N-800 M fluorescencemicroscope.

Immunofluorescence for eggs and larvae of T. canisand A. lumbricoides

T. canis and A. lumbricoides eggs were incubated separatelyat room temperature, and microscopic observations wereperformed to check the condition. Eggs were collected atdays 0, 5, and 10 and subjected to IFA test. Larvae were

collected as previously described and subjected to the IFAtest. The eggs and larvae were washed in PBS at 20°C andadjusted to a final suspension of 100 eggs/larvae per 50 μlof PBS. All samples were incubated with diluted (1:50)antiserum at 37°C for 1 h. Then, samples were centrifugedat 3,000 rpm for 5 min. The supernatant was removed, andeggs or larvae were washed three times in PBS. Then, eggsor larvae were resuspended in diluted (1:50) fluorescentconjugate and incubated at 37°C for 30 min. After incuba-tion, samples were washed three times as previously de-scribed and observed under the fluorescent microscope.

Results and discussion

Immunolocalization of AK in eggs and larvaeof T. canis and A. lumbricoides

A direct immunofluorescence test was performed to deter-mine the cellular localization of AK in eggs and larvae.Interestingly, antiserum was prepared against the recombi-nant T. canis AK and reacts with the native AK of T. canis,

Fig. 3 Immunolocalization of arginine kinase in eggs and larvae of A.lumbricoides. a A. lumbricoides eggs (×10). b Ascaris larvae (×40). cNegative control (eggs), no fluorescence (×40). d Light microscopicview of a developing egg (×40). e Strong fluorescence appeared in thedeveloping egg (×40). h Undeveloped egg (×40). i Weak fluorescencewas observed in the undeveloped egg (×40). f Light microscopic view

of larvae (×40). g, j Strong fluorescence was seen in the middle partand posterior end and no fluorescence in the anterior end of the larvae(×40). k Negative control, no fluorescence appeared in A. lumbricoideslarvae (×40). a anterior end, e embryo, p posterior end. Scale bars50 μm

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A. lumbricoides, and T. vitulorum. Similarly, Cesari et al.(2010) have shown the presence of a common membraneantigen between African schistosome species and species-specific antigens in Schistosoma mansoni BE that could beuseful to discriminate between species and/or to detectSchistosoma infections. Moreover, immunolocalization oftwo novel genes, lactate dehydrogenase A and B, on thetegument of Taenia solium was determined recently by Duet al. (2011). Western blot analysis showed cross-reactionswith other common human tapeworms or helminthes.

Weak fluorescence was observed in 0-day eggs (undevel-oped) of T. canis and A. lumbricoides. However, strongfluorescence was observed in the cleaving eggs (days 2–3)and eggs containing larvae (see Figs. 2 and 3). A previousstudy on the epimastigote of Trypanosome cruzi suggestedthat AK activity increases continuously (by sevenfold) overthe course of parasite growth curve, and there is a correla-tion between growth rate, enzyme-specific activity, andenzyme protein (Alonso et al. 2001). Similarly, the presentstudy shows that gradual increase of the AK activity duringthe embryonic development of T. canis and A. lumbricoides.

The highest activity of the enzyme was observed in the fullydeveloped eggs. This may be due to high expression of AK inembryonic development, which is associated with increasedenergy demand due to cleavage and cellular differentiation.Furthermore, low AK concentration in the undeveloped eggs(0-day eggs) suggests that it is not actively involved in theinitial stage of energy metabolism or it might be due to lowexpression of AK in inactive stage.

Moreover, our study revealed the presence of strongfluorescence in the posterior and middle parts of the bodywhereas no fluorescence was observed in the anterior end ofboth T. canis and A. lumbricoides larvae (see Figs. 2 and 3).It is indicated that AK is highly concentrated in both poste-rior end and middle part of the body than at the anterior endof larvae. This may be due to strong sinusoidal movementsof the posterior and middle part of the body than of theanterior end. In addition, increased expression of AK inlarvae might be due to oxidative stress triggered by highenergy demand. AP/AK is present in the musculature of allgroups possessing this system (Beis and Newsholme 1975;Grieshaber et al. 1994), and in the case of muscle fibers, this

Fig. 4 Immunolocalization of arginine kinase in A. lumbricoides(adult worm). a Microscopic section of the anterior end, hematoxylinand eosin stain (×10). b Anterior end of the worm. Strong fluorescencewas observed in the longitudinal muscles (×10). c No fluorescence wasseen in the cuticle (arrow), while strong fluorescence appeared in thehypodermis (h) and longitudinal muscles (m) (×40). d A longitudinalsection of female A. lumbricoides at midgut level. o oviduct, i flattenedintestine, u uterus filled with eggs, H&E stained (×10). e Transverse

section of an oviduct, H&E stain (×40). h Fluorescence was seen in thecells of the oviduct. f Transverse section of the intestine, H&E stains(×40). i Strong fluorescence was observed in the intestinal cells (×40).g Eggs in uterus, H&E stain (×40). j After immunofluorescence (×40).b basal lamina, c epithelium, d columnar-type epithelioid cells, eepidermis, l lumen, m muscles, o oviducts, i flattened intestine, t thinepithelia, u eggs in uterus. Scale bars 50 μm

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is correlated with energy output. In the case of both CP andAP, phosphagen levels in burst muscles are typically higherthan corresponding levels in muscles that exhibit moresustained modes of contractile activity (Ellington 2001).Therefore, we suggest that muscles in the posterior endand middle part of the body of the viable nematode larvaeffect powerful burst, escape responses that are short induration. In contrast, muscles in the anterior end of thelarvae perform tonic, sustained contractile activities.

Furthermore, a previous study has shown a time-dependentincrease of arginine kinase expression in T. cruzi epimasti-gotes when treated with hydrogen peroxide, suggesting theparticipation of this phosphagen system in oxidative stressresponses (Miranda et al. 2006). Possession of the phosphagensystems like that of AP/AK might be advantageous in inver-tebrates that live in stressful environments where hypoxia andfrequent intracellular acidosis prevailed (Grieshaber et al.1994; Ellington 2001). Nevertheless, more studies should becarried out to determine the subcellular localization of AK in

larvae and also to verify the actual reasons for the difference inAK concentration in various parts of the body.

Immunolocalization of AK in adult worms of T. canis, A.lumbricoides and T. vitulorum

The direct immunofluorescence test was performed for thetissue sections of T. canis, A. lumbricoides, and T. vitulorumworms to determine the immunolocalization of AK. Strongfluorescence was observed in the longitudinal muscles, epi-dermis, oviducts, flattened intestine uterus, and eggs, whileno fluorescence was seen in the cuticle (see Fig. 4) and intissue sections of A. lumbricoides. Similarly, tissue sectionsof T. canis showed strong fluorescence in the epidermis,longitudinal muscles, intestine, uterus, and eggs (see Fig. 5).Furthermore, similar results were obtained for T. vitulorum(Fig. 6). Thus, metabolically active organs or tissues includ-ing epidermis, muscle, intestine, ovaries, oviducts, and uter-us of T. canis, T. vitulorum, and A. lumbricoides have shown

Fig. 5 Immunolocalization of arginine kinase enzyme in T. canis(adult worm). a Microscopic section of anterior end, H&E stain(×10). b Anterior end of the adult worm before visualization from thefluorescent microscope (×10). c After immunofluorescence (×10). dTransverse section of the anterior end (×40), H&E stain. h Immunoflu-orescence of the transverse section of the anterior end. No fluorescencewas seen in the cuticle (arrow); however, strong fluorescence wasobserved in the epidermis (e) and longitudinal muscles (m) (×40). e

Longitudinal section of female worm at midgut level showing flattenedintestine (s) and eggs in uterus (u), H&E stained (×10). i Strongfluorescence was seen in the intestines (×10). f Transverse section ofthe intestine, H&E stain (×40). j Strong fluorescence was observed inthe intestinal tissues (×40). g Eggs in uterus, H&E stain (×40). jImmunofluorescence of the eggs (×40). b basal lamina, c epithelium,l lumen. Scale bars 50 μm

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fluorescence; however, no fluorescence was observed innoncellular areas like the cuticle.

RT-PCR and immunolocalization analysis confirmed thatNAD+-dependent glycerol-3-phosphate dehydrogenaseexpressed both at the stage of adult worm and metacercariaof Clonorchis sinensis and immunolocated at the tegument ofadult worm and tegumentary cells of metacercaria (Fan et al.2011). The presence of cytoplasmic and mitochondrial CKs incells is well established in vertebrates and invertebrate animals(Ellington 2001; Wyss et al. 1992). The existence of truemitochondrial AKs remains controversial, but a recent studyhas shown the presence of true mitochondrial AKs inDrosophila AK2 and Caenorhabditis AK4 (Uda et al.

2006). Other research has predicted the protein localizationsite of T. canisAK to be cytoplasmic or associated with ER orassociated with the secretory pathway (Wickramasinghe et al.2007). The present study revealed the presence of AK in T.canis, A. lumbricoides, and T. vitulorum and that it plays amajor role in energy metabolism of these nematodes. Ourstudy clearly indicates that AK is localized in metabolicallyhighly active cellular compartments such as epidermis,muscles, oviduct, uterus, and intestines. In addition, we ob-served the presence of stronger fluorescence in muscles thanin the epidermis during this study. Furthermore, Ellington(2001) has shown that phosphagen levels can differ in relationto cell type, and available data suggest that tissue levels of

Fig. 6 Immunolocalization of arginine kinase in T. vitulorum (adultworm). a Microscopic section of the anterior end H&E stain (×10). bImmunofluorescence of the anterior end. Strong fluorescence wasobserved in the longitudinal muscles (m); however, no fluorescencewas seen in the cuticle (arrow) (×10). c Negative control (×40). dPosterior end of the adult worm, light microscopic view (×10). e Strongfluorescence was observed in the muscles (m) and hypodermis (h) of

the posterior end (×10). f Microscopic section of body wall of theanterior end, H&E stain (×40). j Immunofluorescence of the body wall.Strong fluorescence was observed in the longitudinal muscles (m) andepidermis (e) whereas no fluorescence was seen in the cuticle (arrow)(×40). g Transverse section of the intestine, H&E stain (×40). h, iStrong fluorescence was observed in the intestinal epithelium (c) andbasal lamina (b) (×40). Scale bars 50 μm

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phosphagens are related to maximum potential rates of ATPturnover, oxidative capacity, and energy output. In the case ofmuscle fibers, they have a greater power output than the epi-dermis as they have contractile activity indicating high levels ofAK in muscle cells than in the hypodermis. Consequently,muscle cells exhibit stronger fluorescence than the epidermis.

Conclusion

T. canis, T. vitulorum, and A. lumbricoides are the mostimportant nematodes that cause significant health problemsin animals as well as in humans. There are various types ofdrugs for these parasitic infestations. Nevertheless, there is anopportunity for developing novel chemotherapeutic agents toovercome drug resistance based on the AP/AK system. AKplays an important role in metabolic pathways of these nem-atodes, and it is absent in mammalian tissues. In the presentstudy, we determined the immunolocalization of AK in T.canis, T. vitulorum, and A. lumbricoides by the direct immu-nofluorescence technique. Our results have shown the immu-nolocalization of AK in different stages of their life cycle.Stronger fluorescence was observed in cleaving eggs than indormant eggs. Surprisingly, expression of AK is very high inthe middle part and posterior ends compared to the anteriorend. Furthermore, AK is highly concentrated in cellular andmetabolically active parts of the body such as the hypodermis,muscle, intestine, ovaries, oviducts, and uterus, while it isabsent in noncellular areas like the cuticle. Therefore, wesuggest that there is a correlation or direct relationship be-tween cellular expression of AK and energy output.

Acknowledgments We would like to extend our thanks to Mr. N.A.N.D. Perera and Mr. K.BA.T. Bandara of Department of Veterinary Patho-biology, Faculty of Veterinary Medicine and Animal Science, Universityof Peradeniya, Sri Lanka for invaluable support during laboratory work.This researchwas financially supported by the National Research Councilof Sri Lanka (Research Grant No. 05-23 and 09-05).

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