research article recombinant dense granular...

Research ArticleRecombinant Dense Granular Protein (GRA5) forDetection of Human Toxoplasmosis by Western Blot

Xiao Teng Ching Yee Ling Lau Mun Yik FongVeeranoot Nissapatorn and Hemah Andiappan

Department of Parasitology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Yee Ling Lau lauyeelingumedumy

Received 16 January 2014 Revised 14 May 2014 Accepted 15 May 2014 Published 29 May 2014

Academic Editor Nongyao Sawangjaroen

Copyright copy 2014 Xiao Teng Ching et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Toxoplasma gondii infects all warm-blooded animals including humans causing serious public health problems and great economicloss for the food industry Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysateantigens from tachyzoites Here we have evaluated an alternative method for antigen production which involved a prokaryoticexpression system Specifically we expressed T gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinitypurification The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysisagainst 212 human patient serum samples We found that rGRA5 protein was 100 specific for analysis of toxoplasmosis-negativehuman sera Also rGRA5 was able to detect acute and chronic T gondii infections (sensitivities of 468 and 612 resp)

1 Introduction

Toxoplasmosis is a parasitic disease caused by Toxoplasmagondii (T gondii) which belongs to phylum Apicomplexa [1]It is an obligate intracellular protozoan parasite capable ofinfecting all warm-blooded domestic animals as well ashuman beings [2] The infection is globally distributedaffecting up to one-third of the worldrsquos human population [3]Infection of T gondii involves the transmission within andbetween hosts by zoites [4] Three infectious stages of theparasite are tachyzoite bradyzoite and sporozoite [5]Humans get infected with such disease through congenitaltransmission consumption of raw or undercooked meatcontaminated with T gondii tissue cysts or uptake of watercontaminated with sporulated oocysts from the infected catfeces [6]

Toxoplasmosis in immunocompetent individuals is oftenasymptomatic [7] but can cause severe clinical outcome toimmunocompromised patients leading to multisystem organfailure or even death [2] Meanwhile primary infection inpregnant women will likely transmit the parasite to the fetusvertically causing congenital toxoplasmosis and might even-tually bring about miscarriage in pregnant women [8 9]

Besides pregnant women similar infection does occur insheep and goats [10] giving rise to similar consequenceAbortions in these animals contribute to great economic lossin livestock industry Therefore it is crucial to conduct arapid highly accurate and early diagnostic test for T gondiiinfected patientshosts for prevention or early treatment

In general there are fewmethods available for conductinglaboratory diagnosis of toxoplasmosis including serologicassays (antibody detection) polymerase chain reaction (PCRspecific gene detection) histologic examination and isola-tion of the parasite followed by inoculation into peritonealcavities of mice (in vivo) or tissue cultures (in vitro) frombiopsy tissue and bloodbody fluids respectively of theinfected patients [11] However the most commonly useddiagnostic test would be the serological test which relies onthe Toxoplasma lysate antigens (TLAs) from tachyzoitespropagated in vivo or in vitroThere are several disadvantagespertaining to the usage of antigens originating from tachy-zoites high production cost time consuming inconstantquality contamination with extraparasitic components andexposure of the staff to the harmful living parasites [12] Toovercome this recombinant DNA technology plays animportant role in producing a larger quantity of recombinant

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 690529 8 pageshttpdxdoiorg1011552014690529

2 BioMed Research International

antigenic proteins for serodiagnosis of T gondii infection in asafer manner with lower production cost Besides recombi-nant tachyzoite proteins production either through prokary-otic or eukaryotic systems can reduce the variation of qualityenabling the development of amore specific and standardizedserological assay

Previous studies have reported the potential of variousspecific antigens of T gondii such as the surface proteins [1314] microneme proteins [15] rhoptry proteins [16 17] anddense granule proteins [18 19] as seromarkers either as singleor multiantigen for detection of T gondii-specific serumantibodies against acute (recently acquired) orand chronic(distant past) Toxoplasma infection

Dense granule (GRA) proteins are proteins with highimmunogenicity [20] They are found abundantly in bothtachyzoites and bradyzoites [21] and make up most of thecirculating antigens in the blood stream of an infected hostwhich can be detected as early as a few hours postinfection(acute phase) [22] GRA proteins were also found during thechronic stage of T gondii infection [20] As a result theimmunogenicity and prolonged expression of GRA proteinsmake them one of the promising candidates for recombinantprotein production

A total of 12 GRA proteins with the molecular weightranging from 21 to 41 kDa have been identified [4 21 23ndash25]Diagnostic performance of GRA antigens such as GRA2GRA6 GRA7 and GRA8 has been investigated via ELISAfor discriminating acute from chronic Toxoplasma infections[18 19 26ndash28] Recombinant GRA7 was also shown to detectacute T gondii infection more strongly compared to chronicinfection [29] Meanwhile in our previous study sensitivityand specificity of recombinant GRA2 for serodiagnosis ofToxoplasma-infected patientsrsquo sera have also been evaluatedthrough western blot which is capable of discriminatingpresent from past infection [30] More diagnostic candidatescapable of detecting the early acquired phase of toxoplasmosisought to be determined to improve the efficacy of serodiagno-sis especially of pregnant women in order to reduce the risk oftransplacental transmission

Dense granule antigen 5 (GRA5) is a 21 kDa hydrophobicprotein consisting of a N-terminal hydrophobic signal pep-tide and a hydrophobic transmembrane domain [31] It wasreported that GRA5 appears in both soluble and hydrophobicforms [32] GRA5 is secreted into the parasitophorous vac-uole (PV) by T gondii as a soluble form during the hostcell invasion [33] followed by transmembrane insertion intothe parasitophorous vacuole membrane (PVM) with its N-terminal projecting into the host cell cytoplasm while C-terminus remains in the vacuole lumen [32] A yeast two-hybrid analysis with GRA5 [34] showed that this antigenbinds to calciummodulating ligand (CALMG) for regulationof intracellular calcium concentration which helps to inhibitapoptosis [35] and further allows for long-term survival of Tgondii Besides playing an important role in host cell invasionmaintenance of the PV and long-term survival of the parasiteGRA5 was found to exist in all life stages of the parasite [36]

However only limited studies were done on the evalua-tion of the potential of GRA5 as a diagnostic marker in Tox-oplasma infection thus making it a protein of interest to be

studied in this research Only one study has been conductedshowing the suitability of the full-length recombinant GRA5for use as a component of an antigen cocktail for the detectionof anti-T gondii IgG antibodies [37] This research studywas aimed at the production of recombinant GRA5 (desig-nated rGRA5) antigen in bacteria and at evaluation of itsimmunogenic properties as a potential single-antigenic diag-nostic candidate through western blot At the same time wewill also find out if GRA5 can detect the early acute stage ofhuman toxoplasmosis through this study

2 Materials and Methods

21 Parasite T gondii tachyzoites (RH strain) were main-tained by serial intraperitoneal passage in BALBc mice andwere harvested from the peritoneal fluids after 3 to 4 days ofinfection The tachyzoites were washed and subsequentlyresuspended in sterile phosphate buffered saline (PBS) priorto usage

22 Construction of Recombinant Plasmids The T gondiiGRA5 gene sequence (corresponding to nucleotides 76ndash360)which encodes the GRA5 antigen was obtained from Gen-bank (accession number EU9187331) DNA was extractedfrom tachyzoites of T gondii (RH strain) and used as thetemplate for PCR amplification of the GRA5 gene withforward (51015840-GCGGAATTCGGTTCAACGCGTGAC-31015840) andreverse (51015840-GACGAATTCCTCTTCCTCGGCAACTTC-31015840)primers which introduced EcoRI restriction sites (under-lined) to facilitate cloning The PCR product was purifiedand cloned into the pRSET B prokaryotic expression vector(Invitrogen USA) at the EcoRI site The resulting recombi-nant GRA5-pRSET B construct permitted expression of anN-terminally polyhistidine- (His-) tagged rGRA5 (aminoacid residues 26ndash120) lacking its putative N-terminal signalsequence Both the GRA5-pRSET B construct and the non-recombinant pRSET B plasmid were transformed intothe prokaryotic expression host Escherichia coli (E coli)BL21(DE3)pLysSThe recombinant clones were screened andsequenced for verification purposes

23 Optimization of Heterologous Protein Expression in E coliOptimal conditions for rGRA5 protein expression in E coliwere determined prior to scaling up the protein produc-tion protocol for further study A single GRA5-pRSET B-containing colony was picked and inoculated into 5mL ofLuria-Bertani (LB) broth supplemented with ampicillin(100 120583gmL) and chloramphenicol (34 120583gmL) The culturewas grown overnight at 37∘C (200 rpm) and then diluted to afinal volume of 10mL with LB broth to yield an opticaldensity of 01 at 600 nm (OD

600) The culture was then

grown at 37∘C (sim250 rpm) until reaching an OD600

of 05 atwhich point protein expression was induced by addition ofdifferent concentrations (01 05 and 10mM) of isopropyl120573-D-thiogalactopyranoside (IPTG Invitrogen USA) for var-ious incubation periods (0 2 and 4 h) The cells were har-vested every hour by centrifugation at 5000timesg for 10min

BioMed Research International 3

before assessing protein expression using dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

24 Expression and Purification of rGRA5 Large-scale pro-tein production was achieved by inducing the culture with1mM IPTG and incubating it for 2 h before harvesting bycentrifugationThe Probond Purification System (InvitrogenUSA) and nitrilotriacetic acid-nickel (Ni-NTA Qiagen Ger-many) resinwere then used to purify rGRA5 according to themanufacturersrsquo instructions Briefly cell lysate was preparedunder denaturing conditions prior to the purification stepsThe cell pellet was resuspended in guanidine lysis buffer (6Mguanidine hydrochloride 500mM sodium chloride and20mM sodium phosphate pH 78) and rocked slowly for 5 to10min at room temperature to ensure thorough cell lysis fol-lowed by sonication on ice with three 5-second pulses (highintensity) After sonication the lysate was separated from cel-lular debris by centrifugation at 3000 timesg for 15min added toa columnwith resin and allowed to bind for 30min Once theresin settled the supernatant was aspirated and the columnwas washed two times with each of the following denaturingbinding buffer (8M urea 500mM sodium chloride and20mM sodium phosphate pH 78) denaturing wash buffer(8M urea 500mM sodium chloride and 20mM sodiumphosphate pH 60) and denaturing wash buffer (8M urea500mM sodium chloride and 20mM sodium phosphatepH 53) The supernatant was aspirated after each washingstep After the last wash the rGRA5 protein was eluted fromthe Ni-NTA resin with denaturing elution buffer (8M urea500mM sodium chloride and 20mM sodiumphosphate pH40) E coli carrying the empty pRSET B vector was usedas a negative control for both expression and purificationThe concentration of purified rGRA5 protein was measuredwith the Bradford Assay Kit (Bio-Rad USA) The identityof the expressed and purified rGRA5 protein was confirmedby matrix-assisted laser desorptionionization-time-of-flight(MALDI-TOF) mass spectrometry (MS)

25 In-Gel Tryptic Digestion of rGRA5 Affinity purifiedrGRA5was resolved by SDS-PAGEusing 12polyacrylamidegels which were stained with Coomassie Brilliant Blue R-250(Bio-Rad USA) for 2 h and then incubated with destainingsolution (7 acetic acid 5 methanol) overnight at roomtemperature The rGRA5 protein band was then excisedfrom the Coomassie-stained gel (based on size) and furtherdestained with 50 120583L of 50 acetonitrile (ACN) in 50mMammonium bicarbonate (NH

4HCO3)This step was repeated

several times (15ndash20min washes discarding the destainingsolution after each wash) until the excised gel was com-pletely destained The rGRA5-containing gel plug was thenincubated with 150120583L of 10mM dithiothreitol (DTT) in100mM NH

4HCO3for 30min at 60∘C The gel was subse-

quently cooled to room temperature the DTT solution wasdiscarded and the band was incubated with 150 120583L of 55mMiodoacetamide (IAA) in 100mM NH

4HCO3in the dark for

20min The gel plug was then washed four times with 50ACN in 50mM NH

4HCO3(500 120583L washes 20min each)

dehydrated via incubation with 50 120583L of 100 ACN for

15min and subjected to speed vacuum for 15min at ambienttemperature to remove the ACNThe gel plug was then incu-bated with 25120583L of trypsin (6 ng120583L) in 50mMNH

4HCO3at

37∘C Following overnight digestion 50 120583L of 50 ACN wasadded to the gel plug and it was incubated for 15min in orderto disintegrate the trypsin enzyme and extract protein fromthe gel plug The resulting liquid (containing the digestedprotein) was transferred into a new tube (Tube A) and the gelplug which remained in the old tube was further incubatedwith 50 120583L of 100 ACN for 15min Subsequently thisliquid was also transferred to Tube AThe protein-containingsolution in Tube A was then dried completely via speed vac-uum Prior to MALDI-TOF MS analysis the protein samplewas reconstituted in 10 120583L of 01 formic acid and desaltedusing a Zip-Tip (Millipore USA) For this the Zip-Tipmembrane was wetted and equilibrated with 50 ACN and01 formic acid respectivelyThe protein sample was boundonto the Zip-Tip membrane which was then washed with01 formic acid Finally the protein was eluted with 01formic acid in 50 ACN and analyzed by MALDI-TOF MS

26 MALDI-TOF MS Analysis The Zip-Tip-eluted proteinsample was mixed at a 1 1 ratio The matrix was providedby UMCPR staff before spotting onto the MALDI plate Theanalysis was carried out by University Malaya Center forProteomics Research (UMCPR)

27 SDS-PAGE and Western Blot Analysis Purified rGRA5protein was resolved by SDS-PAGE on 12 polyacrylamidegels and transferred onto methanol-activated polyvinylidenedifluoride (PVDF Bio-Rad USA) membranes which werethen cut into vertical strips The membranes were incubatedwith blocking solution (5 nonfat skimmilk in Tris BufferedSaline (TBS)) for 2 h at room temperature with constantshaking and were subsequently probed with diluted humanserum samples (1 200) for 2 h The membrane strips werewashed and then incubated for 1 hwith biotinylated goat anti-human IgMIgG (KPL USA 1 2500) secondary antibodyLastly the membrane strips were washed and incubated withstreptavidin-alkaline phosphatase (KPL USA 1 2500) atroom temperature for 1 h followed by detection using 5-bromo-4-chloro-3-indolyphosphatenitro blue tetrazolium(BCIPNBT Sigma USA)

28 Evaluation of Sensitivity and Specificity of rGRA5Diagnostic sensitivity and specificity of rGRA5 protein wereevaluated by western blot analysis using sera from bothtoxoplasmosis-diagnosed patients and toxoplasmosis-negative individuals Toxoplasmosis cases were divided intothree groups (1) patients with early acute toxoplasmosis(119899 = 44 IgM positive IgG negative) (2) patients with acutetoxoplasmosis (119899 = 47 IgM positive IgG positive) and (3)patients with chronic toxoplasmosis (119899 = 85 IgM negativeIgG positive) A fourth group was comprised of toxopla-smosis-negative control patients (119899 = 24 IgM negative IgGnegative) These human serum samples were grouped basedon results obtained fromNovalisaToxoplasma gondii IgG andToxoplasma gondii IgMenzyme-linked immunosorbent assay


Table 1 Immunoreactivities (sensitivity and specificity) of the rGRA5 antigen to serum samples from toxoplasmosis-positive andtoxoplasmosis-negative patients

Serum samples group Number of human serum samplesImmunoreactivities

Positive NegativeNumber Number

1 (Early acute IgGminusve IgM+ve) 44 0 0 44 1002 (Acute IgG+ve IgM+ve) 47 22 468 25 5323 (Chronic IgG+ve IgMminusve) 85 52 612 33 3884 (Toxoplasmosis-negative IgGminusve IgMminusve) 24 0 0 24 100Other infections 12 1 83 11 917Amoebiasis 3 0 0 3 100Cysticercosis 3 0 0 3 100Filariasis 3 0 0 3 100Toxocariasis 3 1lowast 333 2 667lowastOne out of three toxocariasis-positive sera samples reacted with the rGRA5 antigen This particular toxocariasis-positive serum sample was shown to be IgGpositive for toxoplasmosis based on the commercial kits

(ELISA) kits (NovaTec Germany) In addition specificity ofrGRA5 was determined using serum samples from patientsdiagnosed with amoebiasis (3 samples) cysticercosis (3samples) filariasis (3 samples) and toxocariasis (3 samples)These sera had given positive results in serological tests fortheir respective infections All serum samples were obtainedfrom the Diagnostic Laboratory at the Department ofParasitology University of Malaya Sensitivity (number oftrue positives[number of true positives + number of falsenegatives]) and specificity (number of true negatives[number of true negatives + number of false positives]) werecalculated and tabulated in Table 1

3 Results

31 Cloning of the GRA5 Gene Fragment We PCR-amplifieda fragment of T gondii GRA5 gene which encoded aminoacids 26ndash120 of the GRA5 protein (excluding the putativehydrophobic signal peptide) The resulting sim285 bp productwas cloned into the pRSET B vector in order to permitprokaryotic expression of N-terminally His-tagged rGRA5which could be purified using a nickel resin columnSequence analysis confirmed that the insertwithin theGRA5-pRSET B plasmid shared 100 identity with the publishedGRA5 gene

32 Optimization of rGRA5 Expression in E coli Productionof rGRA5 protein was optimized by altering various param-eters and expression levels were analyzed by SDS-PAGE asshown in Figure 1Upon induction of rGRA5 expression fromGRA5-pRSET B-containing E coli we observed a 20 kDaband of increasing intensity which was absent in the neg-ative control (empty pRSET B) Expression of this proteinincreased up to two hours after induction and remained con-stant after four hours Three different IPTG concentrationswere tested and 10mM was found to result in maximumrGRA5 expression Taken together these data suggested thatoptimal rGRA5 expression was achieved following induction

3624

17

(kDa) 1 2 3 4 5 6 7 8 9 10

20kDa

Figure 1 SDS-PAGE analysis on the optimized expression of rGRA5protein in E coli BL21 pLysS (DE3) Coomassie blue stained Lane 5prestained broad range protein marker Lane 1 cell pellet fractionsof pRSET B clone as negative control before induction (0 hr) Lane2 cell pellet fractions of pRSET B clone after induction with 05mMIPTG (4 hr) Lanes 3 to 4 cell pellet fractions of pRSET B clone afterinduction with 10mM IPTG (2 4 hr) Lane 6 cell pellet fractionsof GRA5 clone before induction (0 hr) Lanes 7 to 8 cell pelletfractions of GRA5 clone after induction with 05mM IPTG (2 4 hr)Lanes 9 to 10 cell pellet fractions of GRA5 clone after inductionwith 10mM IPTG (2 4 hr) The GRA5 protein band of interest wasobserved at molecular weight of 20 kDa (arrow) compared to thenegative control The band intensity increased from 0 to 2 hr afterinduction and remained constant at the 4th hr with 10mM IPTGthe optimum condition for maximum expression of the protein

with 10mM IPTG for 2 hours The same conditions wereapplied to larger scale production of rGRA5

33 Expression and Purification of rGRA5 Protein Follow-ing optimization of rGRA5 expression in E coli a nickelresin column was used to purify the recombinant protein(Figure 2(a)) which could be detected by western blotanalysis using serum from a Toxoplasma-infected patient(Figure 2(b))This further suggested that the induced 20 kDaband observed prior to purification corresponded to rGRA5(Figure 1)


35

25

17

(kDa) 1 2 3

20kDa

(a)

35

25

17

(kDa) 1 2 3

20kDa

(b)

Figure 2 SDS-PAGE analysis on purified rGRA5 protein (a) Coomassie blue stained Lane 2 purified pRSET B Lane 3 purified rGRA5 and(b) western blot probed with toxoplasmosis-infected patientrsquos serum Lane 2 purified rGRA5 Lane 3 purified pRSET B Lane 1 (panel a andpanel b) is the prestained broad range protein marker The 20 kDa purified rGRA5 was detected (arrow)

34 Confirmation of rGRA5 Protein Next we confirmed theidentity of our expressed and purified recombinant protein byMALDI-TOF MS analysis Indeed the results indicated thatthe isolated protein was T gondii GRA5

35 Western Blot Analysis of rGRA5 Protein with HumanSerumSamples Thepurified rGRA5 proteinwas tested for itsdiagnostic sensitivity and specificity through western blotanalysis with serum samples from toxoplasmosis-positive(Groups 1 2 and 3) and toxoplasmosis-negative (Group 4)patients In addition specificity was tested using sera frompatients infected with other parasites including amoebiasiscysticercosis filariasis and toxocariasisWe observed that therGRA5 protein had sensitivities of 0 (0 out of 44 sera)468 (22 out of 47 sera) and 612 (52 out of 85 sera)for early acute acute and chronic infections respectively(Table 1) In contrast 0 out of 24 control sera from thetoxoplasmosis-negative patients reacted with rGRA5 (100specificity) In Figure 3 five example results are shown foreach group (positive results for Groups 2 and 3 negativeresults for Group 4) Also only 1 (toxocariasis) out of the12 sera from patients infected with other parasites (data notshown) reacted with the rGRA5 protein (917 specificity)

4 Discussion

A fragment of the T gondii GRA5 gene was successfullycloned into a prokaryotic expression vector and transformedinto E coli Full-length recombinant GRA5 protein (rGRA5)was subsequently expressed and purified yielding a 20 kDaprotein However the predicted molecular weight of GRA5is 16 kDa While this discrepancy between the calculated and

observed molecular weights can be partially explained by thepresence of the His-tag in rGRA5 it is also possible that thisdifference stems from common features of GRA proteinssuch as proline residue composition [4] Even though weobserved this size discrepancy the identity of our purifiedprotein was verified by immunoblotting with Toxoplasma-infected sera and MALDI-TOF MS analysis

Identification of rGRA5 via MALDI-TOF MS involvedcareful processing which allowed for reliable confirmationof the purified protein Briefly the rGRA5-containing bandwas excised from a stained SDS-PAGE gel followed by anin-gel digestion protocol that included seven major steps (1)destaining of the gel plug (2) reduction of the protein (3)alkylation of the protein (4) dehydration (5) tryptic diges-tion of the protein (6) extraction of the digested protein and(7) desalting of the digested protein using a Zip-Tip Reduc-tion and alkylation (aminocarboxymethylation) of the pro-tein at cysteine residues with dithiothreitol (DTT) andiodoacetamide (IAA) respectively were important for per-manent disruption of disulfide linkages enabling overnighttrypsin digestion

It was demonstrated that the expression of predictedimmunodominant epitopes of GRA5 failed to show anyimmunoreactivity with a pool of T gondii-positive humansera [13] Therefore full-length rGRA5 was constructed andproduced in this study Our evaluation of rGRA5 immunore-activity revealed high specificities when testing sera fromtoxoplasmosis-negative patients or from those infected withother parasites (1000 and 917 resp) In addition ourfindings indicate the sensitivities of 468 and 612 whenanalyzing serum samples from patients with acute andchronic Toxoplasma infections respectively However noneof the serum samples from the early acute phase patients


34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)

Figure 3 Western blots of purified rGRA5 protein with sera of toxoplasmosis and toxoplasmosis-negative patients Lane 1 (panels andashc) theprestained broad range proteinmarker Lanes 2 (a) to 6 (a) results of 5 sera from chronic-profile patients (Group 3 IgG +ve IgMminusve) Lanes 2(b) to 6 (b) results of 5 sera from acute-profile patients (Group 2 IgG +ve IgM +ve) Lanes 2 (c) to 6 (c) results of 5 sera from toxoplasmosis-negative patients (Group 4 IgG minusve IgM minusve) The 20 kDa purified rGRA5 was detected by toxoplasmosis-positive sera (arrow)

reactedwith the rGRA5 protein In fact data from the presentstudy are in agreement with previous results obtained fromanalysis of rGRA5 antigen-mediated detection of IgG anti-bodies using ELISA [37] Specificity of the aforementionedstudy was shown to be 1000 whereas sensitivities of 630and 750 were reported for sera from acute and chronicinfections respectively Thus it strongly suggested thatrGRA5 yields a much higher reactivity towards IgG antibod-ies in sera from chronically infected patients compared tothose with acute infection Notably this protein shows nosensitivity towards IgM antibodies found in sera from earlyacute stage patients Our study involved the same expres-sion host BL21(DE3)pLysS for the expression of full-lengthrGRA5 as the above mentioned study In contrast differentexpression vectors and evaluation techniques were used Dueto its higher specificity western blot was chosen to evaluaterGRA5 protein in this study instead of the commonly usedELISA Also the chances of obtaining false-positive resultsvia western blot are much lower compared to ELISA [38] Infact it has been reported that western blot analysis issuperior to ELISA for screening sera samples because thistechnique gives more information is less affected by sampledegradation produces results of high confidence with directvisualization of antibodies bound to specific diagnosticantigens and offers improved determination of diagnosticantigen purity [39]

With regard to the future development of diagnostictests for T gondii the western blot results obtained in thisstudy should be reliable for predicting the efficacy of usingrGRA5 antigen in immunochromatographic tests (ICT) due

to similarities between the two assays (ie western blot andICT are both immunoassays utilizing nitrocellulose mem-branes and direct visualization of results) Indeed ICT is abetter serological test for diagnosis of infections (includingtoxoplasmosis) compared to ELISA which is commonly useddue to its simplicity However ICT is a rapid test with highaccuracy but lower cost compared to ELISA which is timeconsuming and laborious [40] In addition ICT can be usedin field conditions [40] especially for the diagnosis of farmanimals

Based on our results (Table 1) cross-reactivity was notobserved in sera samples from patients infected with amoe-biasis cysticercosis and filariasis However one out of threetoxocariasis-positive sera samples reacted with the rGRA5antigen This particular toxocariasis-positive serum samplewas shown to be IgG positive but IgM negative for toxoplas-mosis based on findings from Novalisa Toxoplasma gondiiIgG and IgM antibodies ELISA kits This indicates that therewas probably a coinfection of T gondii and Toxocara spp inthis infected patient [41] Although T gondii (a protozoan)and Toxocara spp (helminths) are two different parasitesthey both can be acquired through soil ingestion Thereforethe chances of coinfection between these two parasites arehighly possible [41]

5 Conclusions

Our findings show that rGRA5 lacks sensitivity for detectingIgM antibodies and displays a much lower reactivity towards


IgG antibodies in sera from patients with acute infectioncompared to those with chronic toxoplasmosis (468 versus612) These data indicate that rGRA5 protein is unable todistinguish between current and past infections Neverthe-less this protein can be combined with other T gondiiantigens (cocktails) in order to improve its sensitivity againsttoxoplasmosis-positive serum samples [37] Last but not leastthese findings should contribute to the future development ofan ICT incorporating this antigen (either alone or in combi-nation with other potential ESA) for diagnosis of T gondiiinfection

Ethical Approval

The authors declared that the experiments comply with thecurrent laws of the country in which they were performed(Malaysia)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank the Diagnostic Laboratory (Para SEAD)Department of Parasitology University of Malaya and Uni-versity Malaya Medical Centre (UMMC) for providing theserum samplesThey also thank theUniversityMalayaCentrefor Proteomics Research (UMCPR) for carrying out theMALDI-TOFMS analysis This research project is supportedby University of Malaya under the Postgraduate ResearchGrant (PS197-2009C) and High Impact Research Fund UM-MOHE UMCHIRMOHEMED16 from the Ministry ofHigher Education in Malaysia

References

[1] M Igarashi F Kano K Tamekuni et al ldquoToxoplasma gondiievaluation of an intranasal vaccine using recombinant proteinsagainst brain cyst formation in BALBc micerdquo ExperimentalParasitology vol 118 no 3 pp 386ndash392 2008

[2] A B Ismael D Sekkai C Collin D Bout and M-N MevelecldquoMIC3 gene of Toxoplasma gondii is a novel potent vaccinecandidate against toxoplasmosisrdquo Infection and Immunity vol71 no 11 pp 6222ndash6228 2003

[3] A M Tenter A R Heckeroth and L M Weiss ldquoToxoplasmagondii from animals to humansrdquo International Journal forParasitology vol 30 no 12-13 pp 1217ndash1258 2000

[4] C Mercier K D Z Adjogble W Daubener and M-F DelauwldquoDense granules are they key organelles to help understandthe parasitophorous vacuole of all apicomplexa parasitesrdquoInternational Journal for Parasitology vol 35 no 8 pp 829ndash8492005

[5] J P Dubey D S Lindsay and C A Speer ldquoStructures of Tox-oplasma gondii tachyzoites bradyzoites and sporozoites andbiology and development of tissue cystsrdquo Clinical MicrobiologyReviews vol 11 no 2 pp 267ndash299 1998

[6] J P Dubey ldquoPathogenicity and infectivity of Toxoplasma gondiioocysts for ratsrdquo Journal of Parasitology vol 82 no 6 pp 951ndash956 1996

[7] R Chen S-H Lu Q-B Tong et al ldquoProtective effect of DNA-mediated immunization with liposome-encapsulated GRA4against infection of Toxoplasma gondiirdquo Journal of ZhejiangUniversity Science B vol 10 no 7 pp 512ndash521 2009

[8] S S Gagne ldquoToxoplasmosisrdquo Primary Care Update for OBGYNS vol 8 no 3 pp 122ndash126 2001

[9] J G Montoya and O Liesenfeld ldquoToxoplasmosisrdquo The Lancetvol 363 no 9425 pp 1965ndash1976 2004

[10] D Buxton ldquoProtozoan infections (Toxoplasma gondii Neosporacaninum and Sarcocystis spp) in sheep and goats recentadvancesrdquo Veterinary Research vol 29 no 3-4 pp 289ndash3101998

[11] J GMontoya ldquoLaboratory diagnosis ofToxoplasma gondii infe-ction and toxoplasmosisrdquo Journal of Infectious Diseases vol 185no 1 supplement pp S73ndashS82 2002

[12] J Gatkowska E Hiszczynska-Sawicka J Kur L Holec andH Dlugonska ldquoToxoplasma gondii an evaluation of diagnosticvalue of recombinant antigens in a murine modelrdquo Experimen-tal Parasitology vol 114 no 3 pp 220ndash227 2006

[13] J Dai M Jiang Y Wang L Qu R Gong and J Si ldquoEvaluationof a recombinant multiepitope peptide for serodiagnosis ofToxoplasma gondii infectionrdquoClinical and Vaccine Immunologyvol 19 no 3 pp 338ndash342 2012

[14] Y L Lau G Thiruvengadam W W Lee and M Y FongldquoImmunogenic characterization of the chimeric surface antigen1 and 2 (SAG12) of Toxoplasma gondii expressed in the yeastPichia pastorisrdquo Parasitology Research vol 109 no 3 pp 871ndash878 2011

[15] L Holec A Gasior A Brillowska-Dabrowska and J Kur ldquoTox-oplasma gondii enzyme-linked immunosorbent assay using dif-ferent fragments of recombinant microneme protein 1 (MIC1)for detection of immunoglobulin G antibodiesrdquo ExperimentalParasitology vol 119 no 1 pp 1ndash6 2008

[16] L Holec-Gsior J Kur and E Hiszczynska-Sawicka ldquoGRA2 andROP1 recombinant antigens as potential markers for detectionof Toxoplasma gondii-specific immunoglobulin G in humanswith acute toxoplasmosisrdquo Clinical and Vaccine Immunologyvol 16 no 4 pp 510ndash514 2009

[17] P van Gelder F Bosman F De Meuter H Van Heuverswynand P Herion ldquoSerodiagnosis of toxoplasmosis by using arecombinant form of the 54-kilodalton rhoptry antigenexpressed in Escherichia colirdquo Journal of Clinical Microbiologyvol 31 no 1 pp 9ndash15 1993

[18] M Golkar K Azadmanesh G Khalili et al ldquoSerodiagnosis ofrecently acquired Toxoplasma gondii infection in pregnantwomen using enzyme-linked immunosorbent assays with arecombinant dense granule GRA6 proteinrdquo Diagnostic Micro-biology and Infectious Disease vol 61 no 1 pp 31ndash39 2008

[19] M Golkar S Rafati M S Abdel-Latif et al ldquoThe dense granuleprotein GRA2 a newmarker for the serodiagnosis of acute Tox-oplasma infection comparison of sera collected in both Franceand Iran from pregnant womenrdquo Diagnostic Microbiology andInfectious Disease vol 58 no 4 pp 419ndash426 2007

[20] CMercierM F Cesbron-Delauw andD J P Ferguson ldquoDensegranules of the infectious stages of Toxoplasma gondii theircentral role in the hostparasite relationshiprdquo in ToxoplasmaMolecular and Cellular Biology D Soldati and J Ajioka EdsHorizon Scientific Press 2007


[21] M-F Cesbron-Delauw ldquoDense-granule organelles of Toxo-plasma gondii their role in the Host-Parasite relationshiprdquoParasitology Today vol 10 no 8 pp 293ndash296 1994

[22] H P AHughes and F vanKnapen ldquoCharacterisation of a secre-tory antigen from Toxoplasma gondii and its role in circulatingantigen productionrdquo International Journal for Parasitology vol12 no 5 pp 433ndash437 1982

[23] H-J Ahn S Kim andH-WNam ldquoHost cell binding ofGRA10a novel constitutively secreted dense granular protein fromToxoplasma gondiirdquo Biochemical and Biophysical Research Com-munications vol 331 no 2 pp 614ndash620 2005

[24] A Michelin A Bittame Y Bordat et al ldquoGRA12 a Toxoplasmadense granule protein associated with the intravacuolar mem-branous nanotubular networkrdquo International Journal for Para-sitology vol 39 no 3 pp 299ndash306 2009

[25] M E Rome J R Beck J M Turetzky P Webster and P JBradley ldquoIntervacuolar transport and unique topology ofGRA14 a novel dense granule protein in Toxoplasma gondiirdquoInfection and Immunity vol 76 no 11 pp 4865ndash4875 2008

[26] D JacobsM Vercammen and E Saman ldquoEvaluation of recom-binant dense granule antigen 7 (GRA7) of Toxoplasma gondiifor detection of immunoglobulin G antibodies and analysis ofa major antigenic domainrdquo Clinical and Diagnostic LaboratoryImmunology vol 6 no 1 pp 24ndash29 1999

[27] S Li G Maine Y Suzuki et al ldquoSerodiagnosis of recentlyacquired Toxoplasma gondii infection with a recombinant anti-genrdquo Journal of Clinical Microbiology vol 38 no 1 pp 179ndash1842000

[28] A Redlich and W A Muller ldquoSerodiagnosis of acute toxoplas-mosis using a recombinant form of the dense granule antigenGRA6 in an enzyme-linked immunosorbent assayrdquoParasitologyResearch vol 84 no 9 pp 700ndash706 1998

[29] G Sadeghiani M Zare J Babaie et al ldquoHeterologous pro-duction of dense granule gra7 antigen of Toxoplasma gondii inEscherichia colirdquo Southeast Asian Journal of Tropical Medicineand Public Health vol 40 no 4 pp 692ndash700 2009

[30] X T Ching Y L Lau M Y Fong and V Nissapatorn ldquoEval-uation of Toxoplasma gondii-recombinant dense granular pro-tein (GRA2) for serodiagnosis by western blotrdquo ParasitologyResearch vol 112 no 3 pp 1229ndash1236 2013

[31] H-W Nam ldquoGRA proteins of Toxoplasma gondii mainte-nance of host-parasite interactions across the parasitophorousvacuolar membranerdquo Korean Journal of Parasitology vol 47supplement pp S29ndashS37 2009

[32] L Lecordier C Mercier L David Sibley and M-F Cesbron-Delauwz ldquoTransmembrane insertion of the Toxoplasma gondiiGRA5 protein occurs after soluble secretion into the host cellrdquoMolecular Biology of the Cell vol 10 no 4 pp 1277ndash1287 1999

[33] L Lecordier C Mercier G Torpier et al ldquoMolecular structureof a Toxoplasma gondii dense granule antigen (GRA 5) associ-ated with the parasitophorous vacuole membranerdquo Molecularand Biochemical Parasitology vol 59 no 1 pp 143ndash153 1993

[34] H-J Ahn S Kim H-E Kim and H-W Nam ldquoInteractionsbetween secreted GRA proteins and host cell proteins acrossthe paratitophorous vacuolar membrane in the parasitism ofToxoplasma gondiirdquoThe Korean Journal of Parasitology vol 44no 4 pp 303ndash312 2006

[35] P Feng J Park B-S Lee S-H Lee R J Bram and J U JungldquoKaposirsquos sarcoma-associated herpesvirus mitochondrial K7protein targets a cellular calcium-modulating cyclophilin ligandto modulate intracellular calcium concentration and inhibit

apoptosisrdquo Journal of Virology vol 76 no 22 pp 11491ndash115042002

[36] M Tilley M E Fichera M K Jerome D S Roos and M WWhite ldquoToxoplasma gondii sporozoites form a transient para-sitophorous vacuole that is impermeable and contains only asubset of dense-granule proteinsrdquo Infection and Immunity vol65 no 11 pp 4598ndash4605 1997

[37] L Holec-Gasior and J Kur ldquoToxoplasma gondii recombinantGRA5 antigen for detection of immunoglobulin G antibodiesusing enzyme-linked immunosorbent assayrdquo Experimental Par-asitology vol 124 no 3 pp 272ndash278 2010

[38] K Nockler S Reckinger A Broglia A Mayer-Scholl and PBahn ldquoEvaluation of aWesternBlot andELISA for the detectionof anti-Trichinella-IgG in pig serardquo Veterinary Parasitology vol163 no 4 pp 341ndash347 2009

[39] T Anderson A DeJardin D K Howe J P Dubey and M LMichalski ldquoNeospora caninumantibodies detected inMidwest-ern white-tailed deer (Odocoileus virginianus) by Western blotand ELISArdquo Veterinary Parasitology vol 145 no 1-2 pp 152ndash155 2007

[40] X Huang X Xuan H Hirata et al ldquoRapid immunochro-matographic test using recombinant SAG2 for detection ofantibodies againstToxoplasma gondii in catsrdquo Journal of ClinicalMicrobiology vol 42 no 1 pp 351ndash353 2004

[41] J L Jones D Kruszon-Moran K Won M Wilson and P MSchantz ldquoToxoplasma gondii and Toxocara spp co-infectionrdquoAmerican Journal of Tropical Medicine and Hygiene vol 78 no1 pp 35ndash39 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


antigenic proteins for serodiagnosis of T gondii infection in asafer manner with lower production cost Besides recombi-nant tachyzoite proteins production either through prokary-otic or eukaryotic systems can reduce the variation of qualityenabling the development of amore specific and standardizedserological assay

Previous studies have reported the potential of variousspecific antigens of T gondii such as the surface proteins [1314] microneme proteins [15] rhoptry proteins [16 17] anddense granule proteins [18 19] as seromarkers either as singleor multiantigen for detection of T gondii-specific serumantibodies against acute (recently acquired) orand chronic(distant past) Toxoplasma infection

Dense granule (GRA) proteins are proteins with highimmunogenicity [20] They are found abundantly in bothtachyzoites and bradyzoites [21] and make up most of thecirculating antigens in the blood stream of an infected hostwhich can be detected as early as a few hours postinfection(acute phase) [22] GRA proteins were also found during thechronic stage of T gondii infection [20] As a result theimmunogenicity and prolonged expression of GRA proteinsmake them one of the promising candidates for recombinantprotein production

A total of 12 GRA proteins with the molecular weightranging from 21 to 41 kDa have been identified [4 21 23ndash25]Diagnostic performance of GRA antigens such as GRA2GRA6 GRA7 and GRA8 has been investigated via ELISAfor discriminating acute from chronic Toxoplasma infections[18 19 26ndash28] Recombinant GRA7 was also shown to detectacute T gondii infection more strongly compared to chronicinfection [29] Meanwhile in our previous study sensitivityand specificity of recombinant GRA2 for serodiagnosis ofToxoplasma-infected patientsrsquo sera have also been evaluatedthrough western blot which is capable of discriminatingpresent from past infection [30] More diagnostic candidatescapable of detecting the early acquired phase of toxoplasmosisought to be determined to improve the efficacy of serodiagno-sis especially of pregnant women in order to reduce the risk oftransplacental transmission

Dense granule antigen 5 (GRA5) is a 21 kDa hydrophobicprotein consisting of a N-terminal hydrophobic signal pep-tide and a hydrophobic transmembrane domain [31] It wasreported that GRA5 appears in both soluble and hydrophobicforms [32] GRA5 is secreted into the parasitophorous vac-uole (PV) by T gondii as a soluble form during the hostcell invasion [33] followed by transmembrane insertion intothe parasitophorous vacuole membrane (PVM) with its N-terminal projecting into the host cell cytoplasm while C-terminus remains in the vacuole lumen [32] A yeast two-hybrid analysis with GRA5 [34] showed that this antigenbinds to calciummodulating ligand (CALMG) for regulationof intracellular calcium concentration which helps to inhibitapoptosis [35] and further allows for long-term survival of Tgondii Besides playing an important role in host cell invasionmaintenance of the PV and long-term survival of the parasiteGRA5 was found to exist in all life stages of the parasite [36]

However only limited studies were done on the evalua-tion of the potential of GRA5 as a diagnostic marker in Tox-oplasma infection thus making it a protein of interest to be

studied in this research Only one study has been conductedshowing the suitability of the full-length recombinant GRA5for use as a component of an antigen cocktail for the detectionof anti-T gondii IgG antibodies [37] This research studywas aimed at the production of recombinant GRA5 (desig-nated rGRA5) antigen in bacteria and at evaluation of itsimmunogenic properties as a potential single-antigenic diag-nostic candidate through western blot At the same time wewill also find out if GRA5 can detect the early acute stage ofhuman toxoplasmosis through this study

2 Materials and Methods

21 Parasite T gondii tachyzoites (RH strain) were main-tained by serial intraperitoneal passage in BALBc mice andwere harvested from the peritoneal fluids after 3 to 4 days ofinfection The tachyzoites were washed and subsequentlyresuspended in sterile phosphate buffered saline (PBS) priorto usage

22 Construction of Recombinant Plasmids The T gondiiGRA5 gene sequence (corresponding to nucleotides 76ndash360)which encodes the GRA5 antigen was obtained from Gen-bank (accession number EU9187331) DNA was extractedfrom tachyzoites of T gondii (RH strain) and used as thetemplate for PCR amplification of the GRA5 gene withforward (51015840-GCGGAATTCGGTTCAACGCGTGAC-31015840) andreverse (51015840-GACGAATTCCTCTTCCTCGGCAACTTC-31015840)primers which introduced EcoRI restriction sites (under-lined) to facilitate cloning The PCR product was purifiedand cloned into the pRSET B prokaryotic expression vector(Invitrogen USA) at the EcoRI site The resulting recombi-nant GRA5-pRSET B construct permitted expression of anN-terminally polyhistidine- (His-) tagged rGRA5 (aminoacid residues 26ndash120) lacking its putative N-terminal signalsequence Both the GRA5-pRSET B construct and the non-recombinant pRSET B plasmid were transformed intothe prokaryotic expression host Escherichia coli (E coli)BL21(DE3)pLysSThe recombinant clones were screened andsequenced for verification purposes

23 Optimization of Heterologous Protein Expression in E coliOptimal conditions for rGRA5 protein expression in E coliwere determined prior to scaling up the protein produc-tion protocol for further study A single GRA5-pRSET B-containing colony was picked and inoculated into 5mL ofLuria-Bertani (LB) broth supplemented with ampicillin(100 120583gmL) and chloramphenicol (34 120583gmL) The culturewas grown overnight at 37∘C (200 rpm) and then diluted to afinal volume of 10mL with LB broth to yield an opticaldensity of 01 at 600 nm (OD

600) The culture was then

grown at 37∘C (sim250 rpm) until reaching an OD600

of 05 atwhich point protein expression was induced by addition ofdifferent concentrations (01 05 and 10mM) of isopropyl120573-D-thiogalactopyranoside (IPTG Invitrogen USA) for var-ious incubation periods (0 2 and 4 h) The cells were har-vested every hour by centrifugation at 5000timesg for 10min














4HCO3at











3 Results



3624

17

(kDa) 1 2 3 4 5 6 7 8 9 10

20kDa





35

25

17

(kDa) 1 2 3

20kDa

(a)

35

25

17

(kDa) 1 2 3

20kDa

(b)




4 Discussion






34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)






5 Conclusions




Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























4HCO3at











3 Results



3624

17

(kDa) 1 2 3 4 5 6 7 8 9 10

20kDa





35

25

17

(kDa) 1 2 3

20kDa

(a)

35

25

17

(kDa) 1 2 3

20kDa

(b)




4 Discussion






34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)






5 Conclusions




Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results



3624

17

(kDa) 1 2 3 4 5 6 7 8 9 10

20kDa





35

25

17

(kDa) 1 2 3

20kDa

(a)

35

25

17

(kDa) 1 2 3

20kDa

(b)




4 Discussion






34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)






5 Conclusions




Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















35

25

17

(kDa) 1 2 3

20kDa

(a)

35

25

17

(kDa) 1 2 3

20kDa

(b)




4 Discussion






34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)






5 Conclusions




Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















34

26

17

1 2 3 4 5 6(kDa)

20kDa

(a)

34

26

17

1 2 3 4 5 6(kDa)

20kDa

(b)

34

26

17

1 2 3 4 5 6(kDa)

(c)






5 Conclusions




Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Ethical Approval




Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article recombinant dense granular...

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