ORIGINAL PAPER

Silencing of xylose isomerase and cellulose synthase by siRNAinhibits encystation in Acanthamoeba castellanii

Yousuf Aqeel & Ruqaiyyah Siddiqui & Naveed Ahmed Khan

Received: 26 September 2012 /Accepted: 12 December 2012 /Published online: 28 December 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract A key challenge in the successful treatment ofAcanthamoeba infections is its ability to transform into a dor-mant cyst form that is resistant to physiological conditions andpharmacological therapies, resulting in recurrent infections. Thecarbohydrate linkage analysis of cyst walls of Acanthamoebacastellanii showed variously linked sugar residues, includingxylofuranose/xylopyranose, glucopyranose, mannopyranose,and galactopyranose. Here, it is shown that exogenous xylosesignificantly reduced A. castellanii differentiation in encystationassays (P<0.05 using paired t test, one-tailed distribution).Using small interfering RNA (siRNA) probes against xyloseisomerase and cellulose synthase, as well as specific inhibitors,the findings revealed that xylose isomerase and cellulose syn-thase activities are crucial in the differentiation of A. castellanii.Inhibition of both enzymes using siRNA against xylose isom-erase and cellulose synthase but not scrambled siRNA attenu-ated A. castellaniimetamorphosis, as demonstrated by the arrestof encystation of A. castellanii. Neither inhibitor nor siRNAprobes had any effect on the viability and extracellular proteo-lytic activities of A. castellanii.

Introduction

Pathogenic Acanthamoeba are now well-recognized as thecausative agent of a blinding keratitis and fatal granulomatousamoebic encephalitis. It exists in two life forms: a vegetativetrophozoite form and a dormant cyst form. The transformationof Acanthamoeba from so-called “living” (metabolically ac-tive) organisms to “dead” (metabolically inactive or minimalmetabolic activity) impedes successful treatment. How can wekill something that is already dead? To date, the majority of

available drugs are targeted against functional aspects of apathogen (e.g., protein synthesis, DNA/RNA synthesis, cellwall synthesis, etc.), as it is easier to raze function than todemolish a structure. Thus, disrupting the encystation processby attacking the protective wall assembly or its precursorsynthesis should be a feasible chemotherapeutic strategyagainst Acanthamoeba as (1) blocking encystation could resultin a fatal outcome for Acanthamoeba or at least (2) render themsusceptible to available drugs. Thus, a complete understandingof mechanisms associated with encystation and cyst structureis a viable chemotherapeutic approach that would undoubtedlybe valuable in determining appropriate treatment.

The sturdy nature of Acanthamoeba cysts is attributed, inpart, to cellulose (Potter and Weisman 1972), but likely thepresence of other polysaccharides (containing xylose, galac-tose, and mannose in the linkage analysis) has recently beendocumented (Dudley et al. 2009). For cellulose biosynthe-sis, the cellular glycogen is converted into glucose viaglycogen phosphorylase (Lorenzo-Morales et al. 2008).Based on these findings, the presence of an enzyme systemfor xylose, in addition to glucose, incorporation into cellwalls is hypothesized in the present study.

Materials and methods

Acanthamoeba cultures

Acanthamoeba castellanii (keratitis isolate belonging to theT4 genotype) was obtained from the American Type CultureCollection (ATCC 50492). Amoebae were routinely grown in10 ml PYG medium [proteose peptone, 0.75 % (w/v); yeastextract, 0.75 % (w/v); and glucose, 1.5 % (w/v)] in T-75 tissueculture flask without shaking at 30 °C as previously described(Sissons et al. 2005). Media were refreshed 15–20 h prior toexperimentation. Amoebae adherent to the flask representedthe trophozoite form and were used for all assays.

Y. Aqeel : R. Siddiqui :N. A. Khan (*)Department of Biological and Biomedical Sciences, Aga KhanUniversity, Stadium Road,Karachi, Pakistane-mail: naveed5438@gmail.com

Parasitol Res (2013) 112:1221–1227DOI 10.1007/s00436-012-3254-6

Synthesis of small interfering RNA (siRNA) probes

For xylose isomerase, protein sequences from representativeorganisms, plant (Arabidopsis thaliana, AED96932.1;Physcomitrel la patens , XP_001774702.1), fungi(Phytophthora infestans, XP_002904118.1), sea squirt (Cionaintestinalis, XP_002125575.1), and bacteria (Escherichia coliK-12, AAN82821.1) were obtained from the PubMed database.The protein sequences were used for Basic Local AlignmentSearch Tool (BLAST) searches at the Baylor College ofMedicine website (http://www.hgsc.bcm.tmc.edu/microbial-detail.xsp?project_id=163) for any homologous putative xyloseisomerase of Acanthamoeba. Protein sequences of all organismstested including Acanthamoeba (obtained from BLAST searchanalysis) was aligned using ClustalW software and putativecatalytic site for xylose isomerase was identified on the basisof sequence homology. The corresponding nucleotide sequenceof the putative xylose isomerase catalytic domain ofAcanthamoeba was submitted to the siRNATarget Finder soft-ware. The xylose isomerase siRNA probes were as follows:sense strand 5′-UGUUGAACUGGUCCGUAUCUU-3′ andantisense strand 5′-GAUACGGACCAGUUCAACAUU-3′.

For cellulose synthase siRNA probes, protein sequences forthree possible orthologs for cellulose synthase ofAcanthamoebawere obtained: accession numbers EDCBI66TR, EDCBS53TR,and EDCCQ83TF (Anderson et al. 2005). The protein sequenceof each ortholog was determined using the ExPASy proteintranslate tool (http://web.expasy.org/translate/) and submittedto the Protein BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The protein frame exhibiting ho-mology to cellulose synthase was selected. The EDCBI66TRand EDCCQ83TF protein frames exhibited homology withcellulose synthase of Pythium arrhenomanes, Pythium violae,and Pythium coloratum, hence; their nucleotide sequences werealigned using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and siRNA probes were designed using the siRNATarget Finder software (http://jura.wi.mit.edu/bioc/siRNAext/).The sequence of siRNA probes were as follows: sense strand 5′-GAUCUACGAGAAGAACCGCUU-3′ and antisense strand5′-GCGGUUCUUCUCGUAGAUCUU-3′. For the controls, ascrambled siRNA with the following oligoribonucleotidesequences: sense 5′-CAAGCUGACCCUGAAGUUCUU-3′and anti-sense 5′-GAACUUCAGGGUCAGCUUGUU-3′ wasused as described previously (Lorenzo-Morales et al. 2008). Alloligoribonucleotides were purchased from Integrated DNATechnologies (Coralville, IA, USA). The siRNA probes wereresuspended in sterile distilled water to yield a 20-μM workingstock.

Encystation assays

Encystation assays were performed as described previously(Dudley et al. 2005). Briefly, 2×106 amoebae were incubated

in phosphate-buffered saline (PBS) in the presence of 50 mMMgCl2 and 10 % glucose (i.e., encystation trigger) in 24-welltissue culture plates without shaking at 30 °C for 72 h. Afterthis incubation, amoebae viability was quantified using ahemocytometer using Trypan blue exclusion assay. Next,sodium dodecyl sulfate (SDS, 0.5 % final concentration)was added for 10 min to solubilize trophozoites (cysts areresistant to SDS at 0.5 % concentration and they will remainintact) and cysts were enumerated using a hemocytometer.The percentage encystation was determined as follows: num-ber of amoebae, post-SDS treatment/number of amoebae, pre-SDS treatment×100=% encystation. Data are presented as themean±standard error (SE) of three independent experiments.

To determine the effects of saccharides on amoebae differ-entiation, encystation assays were performed in the presenceof exogenous sugars, including D-galactose, D-xylose, and D-mannose as previously described (Siddiqui et al. 2009).Briefly, 2×106 amoebae were incubated in PBS with 10 to100 μM concentrations of sugars in the presence of 50 mMMgCl2 and left at room temperature for 1 h. Following this,10% glucose was added as a trigger for encystation and plateswere incubated at 30 °C for 72 h. Finally, amoebae viabilitywas determined using Trypan blue and percentage amoebaeencystation was determined as previously described. In someexperiments, the effects of D-sorbitol, an inhibitor of xyloseisomerase (Henrick et al. 1989), on Acanthamoeba encysta-tion were determined. Encystation in control wells (withoutsaccharides) was considered 100 %, and the effect of siRNArepresented as relative change.

siRNA silencing assays

To elucidate the role of xylose isomerase and cellulosesynthase, siRNA probes against xylose isomerase and cel-lulose synthase were tested using encystation assays aspreviously described (Dudley et al. 2008). Briefly, 2×106

amoebae were incubated for 1 h in PBS with various con-centrations of siRNA primers ranging from 100 to 200 nM.The scrambled primers were used as controls. Next, 10 %glucose was added as an encystation trigger and plates wereincubated at 30 °C for 72 h; percentage encystation wasdetermined as previously. Encystation in control wells(without siRNA probes) was considered 100 %, and theeffect of siRNA represented as relative change.

Zymographic assays

To elucidate the effects of siRNA against xylose isomeraseand cellulose synthase on the extracellular proteolytic activi-ties ofAcanthamoeba, zymographic assays were performed aspreviously described (Matin et al. 2006). Briefly, encystationassays were performed in the presence of siRNA probes asdescribed previously. After 72 h, cell-free supernatants were

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collected by centrifugation at 3,000×g for 5 min. Equal quan-tity of cell-free conditioned medium was electrophoresed onSDS polyacrylamide gels containing 2 mg/ml gelatin.Following electrophoresis, gels were washed twice in 2.5 %Triton X-100 (w/v) for 1 h to remove residual detergent andleft to incubate overnight in developing buffer (50 mM Tris–Cl, pH 8.0 and 10 mM CaCl2). The next day, gels werewashed twice in distilled water before staining in CoomassieBrilliant Blue. Gelatin being the proteolytic substrate thatbinds to the dye, the unstained regions in the gel indicatedextracellular proteases.

Results

Synthesis of siRNA probes

For cellulose synthase, both EDCBI66TR and EDCCQ83TForthologs were aligned and the homologous region was usedto design the siRNA probe. For xylose isomerase, stop codonswere omitted, sequence alignment was undertaken withknown xylose isomerase, and the homologous sequences wereidentified, which contained a catalytic domain. The conservedregion in the alignment was used to design the siRNA probes.

Fig. 1 The addition of exogenous xylose inhibited encystation in A.castellanii. a To determine the effect of exogenous sugars, galactose,xylose, and mannose on the encystation of A. castellanii, encystationassays were performed. Briefly, 2×106 amoebae were incubated withdifferent concentrations of sugars and plates incubated for 1 h. Afterthis, 10 % glucose was added, which serves as a trigger for encystation,and was left for 3 days. Following this incubation, 0.5 % SDS wasadded in each well and percent encystation was determined by hemo-cytometer counting. Encystation in control wells (without saccharides)was considered 100 %, and the effect of siRNA was represented as

relative change. The results revealed that xylose inhibited d A. castel-lanii encystation in a concentration-dependent manner (50 % inhibitionwith 100 μM) (P<0.01, using paired t test, one-tailed distribution),whereas other sugars had no effect. b To identify the effects of sugarson the viability of A. castellanii, amoebae were enumerated before theaddition of 0.5 % SDS using Trypan blue staining. Exogenous sugarshad no effect on the viability of A. castellanii. The results are repre-sentative of three independent experiments performed in duplicate.Data are presented as the mean±SE

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Exogenous xylose but no other saccharides tested inhibitedencystation of A. castellanii

To determine the effect of exogenous sugars on A.castellanii encystation, galactose, xylose, and mannosewere used at concentrations of 10, 50, and 100 μM.The addition of xylose inhibited A. castellanii encysta-tion in a concentration-dependent manner (50 % inhibi-tion with 100 μM) (P<0.01, using paired t test, one-tailed distribution) (Fig. 1a). In contrast, the addition ofgalactose and mannose had no effect on the encystationof A. castellanii at the concentrations tested. None ofthe sugars had any effect on the viability of A. castel-lanii (Fig. 1b).

The addition of D-sorbitol, an inhibitor of xylose isomer-ase (Henrick et al. 1989), blocked encystation in A. castel-lanii in a concentration-dependent manner (>50 %inhibition at 100 μM concentration, P<0.01) and at levels

similar to exogenous xylose-induced inhibition (Fig. 2a),but again showed no effect on the viability of amoebae(Fig. 2b). The addition of D-sorbitol in the presence ofxylose did not exhibit any synergistic effects on A. castella-nii encystation (Fig. 2a).

Targeting xylose isomerase and cellulose synthase using siRNAprobes inhibited encystation in A. castellanii

siRNA probes against xylose isomerase and cellulosesynthase were tested in Acanthamoeba encystationassays. The findings revealed that inhibition of xyloseisomerase and cellulose synthase using siRNA probes atnanomolar range significantly reduced A. castellaniiencystation (>50 % inhibition with xylose isomerasesiRNA, P<0.01 and >50 % inhibition with cellulosesynthase siRNA, P<0.01) (Fig. 3a). On the contrary,the scrambled siRNA had no effect on A. castellanii

Fig. 2 Xylose isomerase playsan important role in A.castellanii encystation. aEncystation assays wereperformed in the presence ofsiRNA against xyloseisomerase, as well as itsinhibitor, sorbitol, as describedin the “Materials and methods”section. Encystation in controlwells (without saccharides orsorbitol) was considered 100 %,and the effect of sorbitol orxylose was represented asrelative change. The addition ofsorbitol alone resulted in >50 %reduced encystation ascompared with the control (P<0.01, using paired t test, one-tailed distribution), while xy-lose had no synergistic effect onA. castellanii encystation. b Todetermine effects of sorbitoland xylose on the survival of A.castellanii, amoebae were enu-merated before the addition of0.5 % SDS. The resultsrevealed that xylose alone or incombination with sorbitol hadno effect on the survival of A.castellanii. The results are rep-resentative of three independentexperiments performed in du-plicate. Data are presented asthe mean±SE

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encystation (Fig. 3a). None of the siRNA probes hadany effect on the viability of A. castellani (Fig. 3b).

Targeting xylose isomerase and cellulose synthaseusing siRNA had no effect on extracellular proteolyticactivities of A. castellanii

To determine the effects of siRNA against xylose isomeraseand cellulose synthase on extracellular proteases of A. cas-tellanii, zymographic assays were performed. The cell-freeconditioned media were collected and tested on sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels containing gelatin. The results revealed novisible effects of either siRNA probes on the extracellularproteolytic activities of A. castellanii. The supernatants fromA. castellanii incubated with siRNA against xylose isomer-ase, cellulose synthase, or scrambled siRNA exhibited sim-ilar protease bands of similar intensity (Fig. 4).

Discussion

The precise mechanisms of encystation in Acanthamoebaremain incompletely understood; however, it is accompa-nied with termination of cell growth, changes in cellularlevels of RNA, cyclic adenosine monophosphate, proteins,triacylglycerides, glycogen, isocitrate lyase, glycolate, ma-leate, acid phosphatase, deoxyribonuclease, and acid ribo-nuclease, expression of genes encoding proteins withhomology to protein kinase C, proteasome, heat shockprotein, xylose isomerase, Na P-type ATPase, subtilisin-like serine protease, cullin 4, autophage protein 8, andubiquitin-conjugating enzymes, and the appearance of adouble-walled structure, containing cellulose, surround-ing the cell, resulting in decreased cellular volume anddry weight (Martin and Byers 1976; Potter andWeisman 1976; Achar and Weisman 1980; Mehdi andGarg 1987; Moon et al. 2007, 2008, 2011).

Fig. 3 siRNA sequencesagainst xylose isomerase andcellulose synthase inhibitedencystation in A. castellanii. aEncystation assays wereperformed using siRNA probesagainst xylose isomerase andcellulose synthase or siRNA ofscrambled sequence.Encystation in control wells(without siRNA probes) wasconsidered 100 %, and theeffect of siRNAwas representedas relative change. The siRNAprobes against xylose isomeraseand cellulose synthase inhibitedA. castellanii encystation by 52and 59 %, respectively (P<0.01, using paired t test, one-tailed distribution), comparedwith the control, while scram-bled siRNA or transfectionreagent alone had no effect. bThe siRNA had no effect on theviability of A. castellanii, asdetermined by amoebae stain-ing with Trypan blue. Theresults are representative ofthree independent experimentsperformed in duplicate. Dataare presented as the mean±SE

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Acanthamoeba cyst walls protect the amoebae in avariety of hostile environmental settings, which lead totheir exposure and/or transmission to the susceptiblehosts and enable them to resist antimicrobial chemother-apy, leading to recurrence of infection (reviewed inSiddiqui and Khan 2012). Thus, an understanding ofthe cyst wall biochemical composition and associatedenzymatic pathways could identify potential targets fortherapeutic intervention. Our previous studies of theanalysis of cyst walls of A. castellanii, using gas chro-matography/mass spectrometry (GC/MS), revealed xy-lose (in addition to β-1,4-glucan-forming cellulose) asan important constituent (Dudley et al. 2009). In thepresent study, the siRNA probes against xylose isomer-ase and its exogenous inhibitor, sorbitol, blocked A.castellanii encystation, suggesting that xylose isomeraseplays an important role in amoebae differentiation.

Xylose isomerase, also known as glucose isomerase,catalyzes the reversible isomerization of xylose into xylu-lose and glucose into fructose (Lee et al. 1990), but how itinterferes with cyst wall biosynthesis leading to reducedencystation in A. castellanii is not clear. Notably, exogenousxylose but none of the other sugars tested reduced A. cas-tellanii encystation. This was surprising as we expected thatexogenous xylose would enhance xylose isomerase activityleading to increased encystation. A possible explanation ofour findings is that exogenous xylose replaces cellulose (i.e.,

SDS-resistant) biosynthesis with hemicellulose (i.e., SDS-labile) incorporation into the cyst wall, leading to immaturecysts. In support, the linkage analysis using GC/MS showedthat the cyst wall carbohydrates of A. castellanii possesslinear and branching saccharides. Hemicellulose is abranched heteropolymer polysaccharide that consists ofshorter chains forming amorphous structure that can behydrolyzed by acid or base or hemicellulose enzymes. Incomparison, cellulose is an unbranched homopolymer ofglucose, forming crystalline, strong microfibrils that areresistant to hydrolysis. Cellulose contains only anhydrousglucose; however, sugar monomers in hemicellulose caninclude xylose, mannose, galactose, rhamnose, and arabi-nose, in addition to glucose, but the xylose monomer ispresent in the largest amount. This is supported by thepresence of xylose in the linkage analysis in a significantamount of the dry weight of the cyst walls of A. castellanii(i.e., 7.0 %) (Dudley et al. 2009), suggesting the presence ofa mechanism which supplies a sufficient amount of uridinediphosphate (UDP)-xylose (facilitated by exogenous xy-lose) to the biosynthetic system of cyst walls. For example,in case of xylose isomerase from wheat germ, it is shownthat UDP-xylose is derived from UDP-glucose by the actionof UDP-glucose dehydrogenase (Strominger and Mapson1957) and UDP-glucuronate carboxylase (Ankel andFeingold 1965). This enzymatic pathway also contributesto the synthesis of the β-1,4-glucans backbone (i.e., cellu-lose), supported by findings that UDP-glucuronate carbox-ylases from wheat germ are activated by UDP-glucose andallosterically inhibited by UDP-xylose (John et al. 1977).These observations suggest that the enzyme system forUDP-xylose formation and incorporation into cell walls iscontrolled through activation by UDP-glucose and inhibi-tion by UDP-xylose, and if mechanisms are analogous inAcanthamoeba, they may offer potential targets in designingtherapies.

Additionally, the siRNA probes against cellulose syn-thase blocked A. castellanii encystation, confirming pre-vious findings that cellulose is an important constituentof Acanthamoeba cyst wall (Neff and Neff 1969; Potterand Weisman 1976). Because cellulose is absent inmammals, its biosynthetic pathway offers an importanttarget in the rationale development of therapeutic inter-ventions against Acanthamoeba infections. Overall,these findings show that xylose isomerase and cellulosesynthase are important mediators of encystation in A.castellanii and further highlighted the potential ofsiRNA technology for the development of amoebicidalagents to treat Acanthamoeba infections effectively.

Acknowledgments This work was partially supported by grantsfrom the Aga Khan University.

Fig. 4 The siRNA against xylose isomerase and cellulose synthasehad no effect on the proteolytic activities of A. castellanii. Zymo-graphic assays were performed to determine the effect of xyloseisomerase and cellulose synthase silencing on the extracellular pro-teases of A. castellanii. Briefly, A. castellanii 2×106 amoebae wereincubated with different concentrations of siRNA at 37 °C for 24 h.After 3 days, cell-free supernatant (conditioned medium) were collect-ed and equal volume was assayed on the SDS-PAGE gel containing2 mg/ml of gelatin. Following this, SDS was removed by washing in2.5 % Triton X-100 and gel was incubated overnight in developingbuffer. Note that siRNA against xylose isomerase, cellulose synthase,or scrambled siRNA had no effect on A. castellanii proteases, whichexhibited similar protease bands. The results are representative of threeindependent experiments

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