International Journal for Parasitology 39 (2009) 693–702

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International Journal for Parasitology

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Entamoeba histolytica: Oxygen resistance and virulence

Espiridión Ramos-Martínez a, Alfonso Olivos-García a,*, Emma Saavedra b, Mario Nequiz a,Ernesto C. Sánchez a, Eusebio Tello a, Mohamed El-Hafidi b, Andrés Saralegui c, Erika Pineda b,José Delgado a, Irmgard Montfort a,�, Ruy Pérez-Tamayo a

a Departamento de Medicina Experimental Facultad de Medicina, Dr. Balmis 148 Colonia Doctores, Universidad Nacional Autónoma de México, 06720 México D.F., CP 04510, Mexicob Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F., CP 14080, Mexicoc Unidad de Microscopía Confocal, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, CP 62210, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 August 2008Received in revised form 7 November 2008Accepted 10 November 2008

Keywords:Entamoeba histolyticaVirulenceOxygen resistanceReactive oxygen speciesHyperbaric oxygen

0020-7519/$36.00 � 2008 Australian Society for Paradoi:10.1016/j.ijpara.2008.11.004

* Corresponding author. Tel.: +52 55 56232664; faxE-mail address: olivosa@yahoo.com (A. Olivos-Gar

� Deceased.

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins,amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this workwe compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E.histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemo-lytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O2. The only dif-ference observed was a higher in vitro susceptibility of nvEh to O2. The molecular mechanism of thatdifference was analyzed in both groups of amoebae after high O2 exposure. vEh O2 resistance correlatedwith: (i) higher O2 reduction (O��2 and H2O2 production); (ii) increased H2O2 resistance and thiol perox-idase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the highlevel of carbonylated proteins in nvEh after O2 exposure, membrane oxidation by reactive oxygen specieswas not observed. These results suggest that the virulent phenotype of E. histolytica is related to thegreater ability to reduce O2 and H2O2 as well as PFOR reactivation, whereas nvEh undergoes irreversiblePFOR inhibition resulting in metabolic failure and amoebic death.

� 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Entamoeba histolytica is the parasite responsible for humanamoebiasis, a disease characterized by extensive tissue destruction(Brandt and Pérez-Tamayo, 1970). This illness causes approxi-mately 100,000 deaths annually worldwide (World HealthOrganization, 1997). Microbial pathogenicity is the outcome ofthe host-parasite interaction that results in illness; furthermore,host damage can result from either direct microbial action, hostinflammatory and immune responses, or both (Casadevall andPirofski, 1999). It has been suggested that some E. histolytica mol-ecules, such as amoebapores (Andra et al., 1996; Bracha et al.,2003), cysteine proteases (Lushbaugh et al., 1985; Keene et al.,1986; Reed et al., 1989; Montfort et al., 1993; Tran et al., 1998),adhesins (Huston et al., 2003), phospholipases (Ravdin et al.,1985) and collagenases (Muñoz et al., 1984) could be responsiblefor tissue damage, but data supporting these proposals are eitherpartial or indirect. On the other hand, human susceptibility tovirulent E. histolytica (vEh) is variable (Walker and Sellards, 1913)and the intestinal flora could be involved through glycosidases,

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anti-oxidant molecules or proteases (Westphal, 1932; Mirelman,1987; Variyam, 1996). In experimental acute amoebic liver abscessin hamster (ALAH), it has been shown that the host inflammatoryresponse is primarily responsible for tissue destruction (Olivos-García et al., 2004, 2007), as originally proposed by Tsutsumiet al. (1984). Other variables that may influence the outcome of hu-man amoebiasis are the different degrees of virulence revealed byhuman isolates or axenic strains of E. histolytica (Bruckner, 1992).In addition, because the parasites are capable of living and replicat-ing in the microaerophilic environment of human intestine and E.histolytica O2 detoxification pathways decrease in mouse intestine(Gilchrist et al., 2006), other functions related to invasiveness, tis-sue adaptation and/or immune evasion could play an importantrole in E. histolytica virulence. When vEh is maintained in axenicculture for long periods of time, its virulence (as the ability toproduce ALAH) is lost (Phillips, 1973; Ghadirian et al., 1986;González-Garza et al., 2000; Olivos et al., 2005). Moreover, it hasbeen proposed that non-virulent E. histolytica (nvEh) might beeliminated from the hamster liver by reactive oxygen species(ROS) and nitric oxide species (NOS) (Olivos et al., 2005). The aboveresults suggest that E. histolytica virulence could be related to itsoxidant and/or anti-oxidant properties, as proposed many yearsago (Bracha and Mirelman, 1984; Mirelman, 1987; Kumar et al.,1992). In this work, using vEh and nvEh (both from the same

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694 E. Ramos-Martínez et al. / International Journal for Parasitology 39 (2009) 693–702

HM-1 IMSS strain) the following in vitro and in vivo functions werecompared: complement resistance, proteolytic activity, haemolyticactivity, erythrophagocytosis, cytotoxicity, survival in mouse cae-cum, and susceptibility to H2O2 and to O2. The main differences be-tween both amoebae were higher H2O2 and O2 susceptibilities ofnvEh; which were associated with low O2 reduction, significantlylow thiol peroxidase activity, and irreversible O2 inactivation ofpyruvate:ferredoxin oxidoreductase (PFOR) activity from the glyco-lytic pathway. Our results suggest that O2 reduction and anti-oxida-tive capacity are necessary conditions for E. histolytica virulence.

2. Materials and methods

2.1. Chemicals

Diphenylene iodonium (DPI), edaravone (MCI-186), EUK-8(manganese N,N0-bis(salicylidiene)ethylenediamine chloride),tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), dihy-droethidium (DHE) and dihydrorhodamine (DHR) were fromCalbiochem (San Diego, CA, USA). Gas cylinders containing99.99% O2 were obtained from INFRA (México). Azocasein,glycogen, H2O2, dimethyl sulfoxide (DMSO), paraformaldehyde,b-NADPH; 4,5-dihydroxy-1,3-benzene-disulfonic acid (tiron),2-thiobarbituric acid (TBA), menadione, 2,4 dinitrophenylhydra-zine (DNPH), horseradish peroxidase (HRP) and all other reagentsused were purchased from Sigma–Aldrich (St. Louis, MO, USA)unless otherwise indicated.

2.2. Parasites and amoebic liver abscess in hamster

Axenic cultures of vEh strain HM-1 IMSS were maintained inTYI-S-33 medium according to standard protocols (Diamondet al., 1978). Virulence was defined as the ability of 5 � 105 tropho-zoites to produce multiple liver abscesses in hamsters (100 g aver-age weight) 7 days after intraportal injection. Virulence wasmaintained by passing axenic amoebae through hamster liverstwice a month, recovering the parasites from 7 days old abscessesand growing them again axenically. The reason for this treatmentwas not because of a rapid loss of virulence (since this phenome-non occurs after variable but longer time periods), but for themaintenance of a uniform degree of virulence. nvEh were main-tained permanently (>4 years) in the same axenic culture condi-tions and were used when intraportal injection of 3 � 106

trophozoites failed to produce any lesion in the hamster’s liver.All animal experiments were conducted according to the directionsof the General Health Law of Mexico.

2.3. Sub-infective vEh in hamster liver

Different amounts of sub-infective vEh (2 � 105, 1 � 105 and0.5 � 105) were injected into the portal vein of 24 hamsters (fourper group) as described above. The animals were sacrificed 6 and24 h after parasite injection, their livers processed for histologyand stained with periodic acid–Schiff (PAS).

2.4. In vitro amoebic functions

2.4.1. ErythrophagocytosisErythrophagocytosis was evaluated according to Keller et al.

(1988). Briefly, 5 � 105 amoebae were incubated with 5 � 107 ham-ster erythrocytes in 1 ml of PBS for 15 min at 36 �C. The samples werecentrifuged at 500g for 3 min. The pellets were recovered, resus-pended in 0.4 ml of distilled water, followed by addition of 1 ml ofPBS and centrifuged. The final pellet was clarified with 0.5 ml of for-mic acid and diluted with 0.5 ml of PBS. The absorbance was furtherdetermined at 397 nm in a spectrophotometer.

2.4.2. Haemolytic activityHaemolytic activity was measured as previously described

(Ankri et al., 1998) by incubating 5 � 105 trophozoites with500 � 106 hamster erythrocytes in 1 ml of PBS at 36 �C for 1 h.The samples were centrifuged for 5 min at 4500g, the supernatantwas recovered and the absorbance was determined at 570 nm.

2.4.3. Proteolytic activityProteolytic activity was determined as previously described

(Pérez-Montfort et al., 1987). Briefly, a pellet of 5 � 105 trophozo-ites was lysed by freezing in liquid nitrogen, diluted with 0.2 ml ofPBS and 0.4 ml of an azocasein solution (2.5 mg/ml) was added.The mixture was incubated for 3 h at 36 �C and the reaction wasstopped with 0.6 ml of 10% (w/v) cold trichloroacetic acid (TCA).The mixture was centrifuged for 5 min at 4500g and absorbancewas determined in the supernatant at 366 nm.

2.4.4. Complement resistanceComplement resistance was determined by incubating tropho-

zoites in fresh hamster serum (5 � 105/ml) for 2 h at 36 �C. Amoe-bic viability was determined by Trypan blue exclusion (Olivos-García et al., 2004).

2.4.5. CytotoxicityCytotoxicity of axenic trophozoites was tested on polymorpho-

nuclear cells (PMNs) freshly obtained from the peritoneal cavity ofhamsters injected 3 h previously with 1 ml of 1% glycogen in PBS.The assay was performed by incubating 1 � 105 amoebae with2.5 � 106 PMNs in TYI-S-33 medium for 3 h at 36 �C, and thencounting viability of target cells by Trypan blue exclusion. Resultswere compared with control samples without parasites.

2.5. Amoebic survival in mouse cecum

Amoebic survival in mouse cecum was assessed as previouslydescribed (Houpt et al., 2002). Briefly, 10 C3H/HeJ mice of eithersex were divided in two groups, anesthetized with ketamine/xyla-zine and injected with either vEh or nvEh (5 � 106/0.2 ml PBS) intothe caeca, exposed by laparoscopic surgery. The animals were sac-rificed by pentobarbital overdose 3 days after infection. The pres-ence in each caecum of viable and motile parasites wasexamined by Trypan blue exclusion under light microscopy andthe percentage of infected animals was calculated.

2.6. O2 resistance

Hanks medium without phenol red was saturated with molecu-lar O2 (medicinal grade) for 30 min by constant bubbling and theO2 concentration determined with a Clark’s oxygen electrode. vEhor nvEh amoebae (1 � 106) were incubated in Hanks medium(1.2 ml) or Hanks medium saturated with O2 at 36 �C in 1.5 mlEppendorf tubes which were hermetically sealed. Amoebic survivalwas monitored at 30, 45 and 60 min using Trypan blue exclusion.Parasite viability was compared with controls incubated in TYI-S-33 medium.

2.7. O��2 and peroxidase-H2O2 in live parasites

vEh or nvEh (1 � 106 of either) were exposed to O2-saturatedHanks medium as mentioned above for 15 min (sub-lethal dose)at 36 �C in the presence of the following fluorescent probes:5 lM dihydroethidum (DHE) for O��2 (Zhao et al., 2003) or10 lM dihydrorhodamine (DHR) for H2O2 (Henderson and Chap-pell, 1993). Controls of amoebae in Hanks medium or TYI-S-33media plus DHE or DHR were prepared at the same time. Thesamples were further centrifuged at 2000g and the cellular

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pellets fixed by adding 1 ml of 1% paraformaldehyde (w/v) for30 min. The pellets were washed twice with PBS and resus-pended in 0.1 ml of the same buffer. Finally, 30 ll of the sampleswere dropped on a slide and confocal images were obtained onan inverted Zeiss LSM 510 META confocal microscope (Carl Zeiss,Jena, Germany) using a 20� (NA 0.5 Plan Neofluar) objective.Excitation: 543 nm line of a HeNe for rhodamine and 514 nmof Ar laser for oxyethidium. Both fluorescent compounds weredetected with the LP560 emission filter. Because DHR does notshow fluorescence until it is oxidized to rhodamine by peroxi-dases in the presence of H2O2 as a substrate (Masaki et al.,1995), the test reveals both peroxidase activity and H2O2 pro-duction (peroxidase-H2O2).

2.8. O��2 in amoebic lysates

Pellets of 1 � 106 of either vEh or nvEh were frozen at �70 �C;the cellular lysates were resuspended in 1.2 ml of high O2-satu-rated Hanks medium for 1 h at 36 �C as described above, in thepresence of 5 lM DHE, 100 lM NADPH and 10 lM tiron (an FeSODinhibitor to allow superoxide accumulation). At the end of theincubation, fluorescence was determined in a fluorometer (Fluo-roskan ascent) at 480 nm excitation and 567 nm emission. Thefluorescence of control samples without parasites (DHE orDHE + O2) was always subtracted from the experimental results.

2.9. Thiol peroxidase activity and H2O2 production in amoebic lysates

To determine amoebic thiol peroxidase activity, pellets of1 � 106 of either vEh or nvEh were frozen at �70 �C and the cel-lular lysate resuspended with 1.2 ml of normal or O2-saturatedHanks medium in the presence of 10 lM DHR and 100 lMNADPH and further incubated for 1 h at 36 �C as described above.Duplicate tubes were pre-incubated with 1 mM iodoacetamide(an alkylating inhibitor of thiol-containing enzymes). In order todetermine H2O2 generation, in a parallel experiment 1 U/ml HRPwas added. Fluorescence was monitored at 488 nm excitationand 525 nm emission. The background fluorescence (DHR orDHR + O2) without parasites was always subtracted from theexperimental results.

2.10. ROS scavengers in culture

vEh trophozoites (1 � 106) were incubated in 5 ml of TYI-S-33medium containing 1, 10, 50 and 100 lM of edaravone (an.OHscavenger) (Kawai et al., 1997), EUK-8 (an O��2 and H2O2 scavenger)(Brandier et al., 1997) and tempol (an O��2 scavenger) (Gariboldiet al., 2000). Viability was determined by Trypan blue exclusionafter 6 and 24 h incubation. Four independent experiments wereperformed. Results were expressed as means ± SD.

2.11. Metabolites

Twenty-five to 45 Eppendorf tubes containing 1 � 106 vEh ornvEh per tube were exposed to O2-saturated media for 15 min asdescribed above, but using PBS with 5 mM glucose instead ofHanks medium. Parallel control tubes (amoebae + PBS-glucosewithout O2) were simultaneously incubated. The samples fromeach condition were pooled and the cells collected by centrifuga-tion at 2000g. Supernatants (1 ml) were used for ethanol (EtOH)determination and the cellular pellets were resuspended in 1 mlof PBS. The samples were treated on ice with 3% (v/v) ice-cold per-chloric acid in the presence of 1 mM EDTA and vortexed until awhite precipitate was visualized. Each sample was centrifuged at12,000g for 5 min and the supernatant was separated and neutral-ized with different volumes of a 100 mM KOH �3 M Tris solution

and stored at �70 �C. Quantitative determinations of glycolyticintermediaries glucose-6-phosphate (G6P), fructose-6-phosphate(F6P), pyruvate, ATP and EtOH were performed by enzymatic as-says as previously described (Saavedra et al., 2007) with couplingenzymes and the neutralized samples as substrates. Reactionswere started with the specific enzyme for each metabolite andNADH oxidation or NAD(P)+ reduction were monitored at 340 nmin a spectrophotometer.

2.12. PFOR activity assays

nvEh or vEh amoebae (8 � 106) were treated for 15 min in O2-saturated PBS as described above, the cells were harvested andthe pellets resuspended in 0.5 ml of 100 mM KH2PO4 buffer pH7.5, 25 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluo-ride (PMSF), 5 mM EDTA and 1% Triton X-100 (previously purgedwith N2). The cells were lysed three times by freezing and thawingusing liquid nitrogen, centrifuged at 21,000g and the supernatantseparated and stored under anaerobic conditions at �20 �C. PFORactivity was determined under anaerobic conditions (N2 environ-ment) in a 1 ml cuvette containing a mixture of 100 mM Na2HPO4

pH 7.5 buffer (previously purged with N2), 0.25 mM nitroblue tet-razolium (NBT), 0.1 mM coenzyme A, 10 mM pyruvate and 2–6 lgof amoebic extracts. The reaction was started by addition of coen-zyme A or pyruvate and the basal value in their absence was sub-tracted. NBT reduction was monitored at 560 nm in aspectrofluorometer (Shimadzu, Kyoto, Japan). For PFOR inactiva-tion, aliquots of the cellular extracts from amoebae incubated inPBS were treated at room environment for 1 h on ice and theremaining PFOR activity was determined. For PFOR reactivation,aliquots of extracts inactivated by air from amoebae incubated inPBS or cellular extracts from amoebae incubated in O2-saturatedPBS were treated for 1 h with 1 mM Fe++ (in the form of ferrousammonium sulfate) and 5 mM DTT under an N2 environment,and the PFOR activity was determined.

2.13. Lipid peroxidation assay

vEh or nvEh (1 � 106) were exposed to high O2 concentration inHanks medium as mentioned above for 2 h at 36 �C. Parallel con-trols (Hanks or TYI-S-33 medium) were incubated without O2 un-der the same conditions. The tubes were further stored at �70 �C inthe presence of 2% EDTA plus 0.05% butylhydroxytoluene (BHT).One hundred microliters of each sample were used to determine li-pid peroxidation, measuring thiobarbituric acid reactive sub-stances (TBARS) using a fluorescence method (Fraga et al., 1987).Briefly, 0.05 ml of 4% (w/v) BHT and 1 ml PBS were added to0.1 ml of each sample. After incubating for 30 min at 36 �C,1.5 ml of 20% (v/v) acetic acid and 1.5 ml 0.8% (w/v) 2-thiobarbitu-ric acid were added. The mixture was heated for 45 min in boilingwater and TBARS were extracted into 5 ml of n-butanol. After briefcentrifugation, the fluorescence of the butanol layer was measuredat 515 nm excitation and 553 nm emission in a Perkin-Elmer LS50Bspectrofluorometer. The value is expressed as pmol TBARS (mal-ondialdehyde (MDA) equivalents) per mg protein. A MDA standardwas prepared from 1,1,3,3-tetraethoxypropane (El Hafidi andBaños, 1997).

2.14. Amoebic H2O2 resistance

vEh or nvEh (1 � 106) were incubated for 1 h at 36 �C in 1 ml ofTYI-S-33 medium containing 2 mM H2O2. Viability was determinedby Trypan blue exclusion and compared with parasites incubatedin the same medium without H2O2. Five independent experimentswere performed in duplicate and results were expressed asmeans ± SD.

Fig. 1. Comparison of amoebic functions in virulent Entamoeba histolytica (vEh) andnon-virulent E. Histolytica (nvEh). The functions were evaluated as described in theMaterials and methods section. For the first five functions we determined themeans ± SD. of the data from nvEh assays and compared those with the control vEhthat was considered as 100%; for the rest, the absolute values of viability percentageare displayed. All assays (except survival in mouse intestine) were performed induplicate in five independent experiments.

Fig. 2. Amoebic survival in normal and O2-saturated Hanks medium. VirulentEntamoeba histolytica (vEh) or non-virulent E. histolytica (nvEh) were incubated innormal or oxygen-saturated (+O2) Hanks medium. Viability was determined at theindicated times by Trypan blue exclusion. Five independent experiments wereperformed in duplicate. Results are expressed as means ± SD.

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2.15. Protein oxidation

After 15 min exposure of 1 � 106 of vEh or nvEh to O2, the para-sites were centrifuged at 500g for 3 min, the pellets resuspended in0.5 ml PBS-glucose buffer plus 1 mM DTT and frozen at �70 �C. Inorder to determine the carbonyl protein content (Levine et al.,1990), 1 mg of protein from each sample in 1.5-ml Eppendorf tubeswas precipitated by addition of TCA (10% w/v final) and centrifugedat 11,000g for 3 min. The pellets were resuspended in 0.5 ml of10 mM 2,4-dinitrophenylhydrazine (dissolved in 2 N HCl) and al-lowed to stand at room temperature for 1 h with vortexing every10–15 min, 0.5 ml of 20% TCA was added and the tubes centrifugedat 11,000g for 3 min. The pellets were washed three times with 1 mlethanol–ethyl acetate (1:1) and then solubilized in 0.6 ml of 6 Mguanidine, 20 mM potassium phosphate (adjusted to pH 2.3 withtrifluoroacetic acid). The insoluble material was removed by centri-fugation at 11,000g for 3 min and the carbonyl content determinedfrom the absorbance at 370 nm, using a molar absorption coefficientof 22,000 M�1 cm�1. Three independent experiments were per-formed in duplicate and results were expressed as means ± SD.

2.16. Hyperbaric O2 therapy and menadione on ALAH development

vEh or nvEh (5 � 105) were incubated for 24 h in TYI-S-33 med-ium with 1 mM menadione (Md-vEh). The cells were recovered,washed twice with PBS and injected into the portal vein of differ-ent groups of hamsters mentioned below (four per group). Thirtymin after parasite injection, some groups received hyperbaric oxy-gen therapy (HBO) in a small experimental REMISA hyperbaricchamber, at 2.0 atmosphere absolute (ATA) for 60 min. The cham-ber was pressurized and depressurized at the rate of 0.2 ATA/min.Other animal groups received menadione (30 mg/kg) i.p. (Md-treatment), 4 h before parasite injection and then every 12 h. Thetreatment groups were as follows: (1) vEh, (2) vEh + HBO, (3)vEh + Md-treatment (4) vEh + HBO+Md-treatment, (5) Md-vEhand (6) Md-vEh + HBO. Groups were sacrificed 6, 24, 72 and144 h after parasite injection by pentobarbital overdose and theirlivers removed, weighed and processed for histology, as mentionedabove. Liver weights were expressed as means ± SD.

3. Results

3.1. Erythrophagocytosis, haemolytic and proteolytic activities,complement resistance, cytotoxicity on PMNs and survival in mousecaecum

Several in vitro tests have been developed to evaluate the viru-lence of E. histolytica strains, which include erythrophagocytic andhaemolytic capabilities, proteolytic activity content, complementresistance, cytotoxicity on PMNs and amoebic survival in mousecaecum. The activities of diverse ‘‘virulence factors” such as amoe-bapores, phospholipases, adhesins and cysteine proteases havebeen reported to be implicated in such virulent functions (Ravdinet al., 1985; Keller et al., 1988; Reed et al., 1989; Ankri et al.,1998; González-Garza et al., 2000; Bracha et al., 2003; Hustonet al., 2003). In order to evaluate whether phenotypic differencesexist between vEh and nvEh, these amoebic functions were ana-lyzed. Despite the fact that all of these amoebic functions havebeen previously correlated with an amoebic virulent phenotype,unexpectedly no differences were observed between vEh and nvEh,as shown in Fig. 1.

3.2. O2 resistance

During early in vivo infection of hamster liver by E. histolytica,vEh are visible before 24 h p.i. whereas nvEh are cleared before this

time. During this time period it is supposed that amoebae are chal-lenged with complement and high oxygen concentration. Becauseit is known that amoebic complement resistance does not partici-pate in nvEh clearance (Olivos et al., 2005), we then comparedamoebic oxygen resistance in both amoebae. When vEh or nvEhwere incubated in Hanks medium saturated with O2 (0.66 mM),nvEh viability decreased significantly after 30 min and was com-pletely absent after 2 h. In contrast, vEh showed higher resistanceto O2 and their viability only decreased to 65% after 2 h (Fig. 2).Control Hanks medium was slightly toxic for both kinds of amoe-bae, probably because of the low redox environment and the airdissolved in solution (0.18 mM O2) (Fig. 2).

3.3. O��2 and peroxidase-H2O2 in live parasites

As a good correlation was found between amoebic oxygen resis-tance and virulence, the amoebic oxygen detoxification pathwayswere analyzed in both amoebae. The intracellular levels of O��2(monitored through the conversion of dihydroethidium to oxyethi-dium) (Fig. 3A) in live vEh and nvEh grown in TYI-S-33 mediumwere barely detectable; however, in Hanks medium the positivesignal increased slightly in both groups of amoebae. Moreover, in

Fig. 3. Confocal images of amoebic O��2 production and H2O2 generation-peroxidase activity after 15 min in high O2 concentration. (A) O��2 production detected bydihydroethidium oxidation and (B) peroxidase activity and H2O2 production detected by dihydrorhodamine oxidation. (a) virulent Entamoeba histolytica (vEh) or (b) non-virulent E. histolytica (nvEh) in TYI-S-33 culture medium (control); (c) vEh and (d) nvEh in Hanks medium; (e) vEh and (f) nvEh in O2-saturated Hanks medium. The imagesshown are confocal equatorial sections of 4 lm thickness, with their corresponding phase contrast images on the left.

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O2-saturated Hanks medium, vEh showed an increased O2�� signal

compared with nvEh (Fig. 3A). Similar behavior was observed inthe peroxidase activity-H2O2 production detection assay (dihydro-rhodamine to rhodamine conversion) (Fig. 3B), except that inO2-saturated Hanks medium, vEh showed a remarkably higherrhodamine-positive signal than nvEh.

3.4. Effect of ROS scavengers in culture

High ROS production is observed when amoebae are challengedwith a high oxygen concentration for short periods of time as de-scribed above; however, it was interesting to test a putative phys-iological requirement of ROS for in vitro amoebic growth as it hasbeen described for mammalian cells. Thus, ROS requirement forin vitro growth was analyzed. The ROS scavengers edaravone (forOH), EUK-8 (for O��2 and H2O2) and tempol (for O��2 ) at 10, 50 or100 lM were lethal for vEh after 24 h culture. However, at low con-centrations (1 lM) the amoebic viability was 90 ± 1.05%, 71 ± 5.4%,and 88 ± 2.0%, respectively.

3.5. O��2 and H2O2 production, and thiol peroxidase activity in amoebiclysates

As demonstrated above, there were increased O��2 and peroxi-dase-H2O2 signals of the fluorescent probes observed in live vEhafter high O2 exposure (Fig. 3); however it was necessary to quan-tify the differences in these parameters between nvEh and vEh andthis was done by using amoebic lysates. To avoid the interferenceof FeSOD activity that would prevent O��2 accumulation and detec-tion, this radical was quantified in amoebic lysates in the presenceof tiron (a Fe scavenger; essential for FeSOD activity). In Hanksmedium vEh showed higher O��2 production than nvEh, whereas

in O2-saturated Hanks medium, only vEh increased O��2 production(2.7 relative fluorescence units) (Fig. 4B).

In normal Hanks medium thiol peroxidase activity was similarin both vEh and nvEh lysates, whereas in O2-saturated Hanks med-ium it increased significantly (7.3-fold) only in vEh lysates (Fig. 4C).H2O2 production in normal Hanks medium and in the presence ofHRP (Fig. 4A) was slightly higher in vEh than nvEh (0.79-fold);however, in O2-saturated Hanks medium, only the lysate fromvEh increased H2O2 production (41%). In addition, the thiol-depen-dent enzyme inhibitor iodoacetamide abolished activity in all sam-ples. These results are in agreement with our observations in liveparasites by confocal microscopy, as mentioned above.

3.6. Thiobarbituric acid reactivity (MDA)

Despite the fact that nvEh died after 2 h exposure to high O2

concentration (Fig. 1), TBARS determination (an index of lipid per-oxidation) in both vEh and nvEh did not increase after treatment(Fig. 4D). Furthermore, the levels were very similar to those pro-duced in TYI-S-33 medium or Hanks medium without O2.

3.7. Metabolites

In order to determine metabolic differences between vEh andnvEh incubated in saturated O2 conditions, glycolytic intermediar-ies were determined under the four experimental conditions. vEhor nvEh incubated in PBS-glucose did not show significant differ-ences in either the intracellular content of G6P, F6P, pyruvateand ATP or ethanol production. The values (nmol/106 cells) forG6P, F6P, ethanol and ATP in these control amoebae were: vEh,2.35 ± 0.54, 0.77 ± 0.28, 3054 ± 2073, 1.9 ± 0.9; and nvEh,2.2 ± 0.68, 0.67 ± 0.24, 3835 ± 2784, 2.2 ± 0.8, respectively. The

Fig. 4. Oxygen reduction and malondialdehyde levels in virulent Entamoeba histolytica (vEh) and non-virulent E. histolytica (nvEh) after high O2 exposure in amoebic lysates.Hydrogen peroxide (A) and O2

�� (B) production and thiol peroxidase activity (C) in vEh and nvEh lysates incubated 1 h in normal or O2-saturated Hanks medium. (D) MDAlevels determined in vEh and nvEh after 2 h incubation under the same conditions. The plots represent the means ± S.D. of five independent experiments performed induplicate. RFU: relative fluorescence units.

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pyruvate concentration was below the limit of detection of theenzymatic assay (<0.18 nmol/106 cells) for both types of controlamoebae. Strikingly, the content of G6P, F6P and pyruvate was2.4- 6.6-fold significantly increased in both amoebae incubatedin O2 saturated PBS whereas a 29–43% reduction in intracellularATP content as well as in EtOH production was observed (Fig. 5)compared with the control PBS + glucose amoebae. These resultssuggest that glycolytic flux is being affected due to (i) oxidative

Fig. 5. Glycolytic intermediaries in virulent Entamoeba histolytica (vEh) and non-virulent E. histolytica (nvEh) exposed to O2 saturated conditions. Amoebae wereincubated in O2-saturated PBS + glucose for 15 min and glycolytic intermediarieswere determined in perchloric acid extracts as described in the Materials andmethods section. The plots represent the means ± S.D. of the variation foldcompared with control amoebae without O2 treatment run in parallel (see maintext for absolute values for controls). Three independent experiments wereperformed with different amoebic culture batches.

stress damaging glycolytic enzymes, or (ii) direct O2 inhibition ofamoebic glycolytic proteins.

3.8. PFOR activity

PFOR is an enzyme which is highly susceptible to inhibition bymolecular oxygen through the oxidation of its iron–sulfur (Fe–S)centers, which are structural components in the protein necessaryfor electron transfer during the enzymatic reaction. PFOR activitywas examined to explain failure in the glycolytic flux and thechanging pattern in the glycolytic intermediaries (particularly thehigh pyruvate accumulation) of amoebae incubated with O2. Asshown in Fig. 6, in both vEh and nvEh amoebae, PFOR is 80–90%inactivated either in vivo, when the cells were incubated with O2

or when amoebic extracts without O2 treatment were exposed toroom environment. However, an important difference betweenboth types of PFORs is that vEhPFOR inactivation was reversibleat a high rate (1.6- and 6.1-fold from their in vivo or in vitro inhib-ited state) when the extracts were incubated in the presence of1 mM Fe++ under reducing and anaerobic conditions; on the otherhand, nvEhPFOR inactivation was essentially irreversible. Further-more, O2 treatment in extracts did not significantly affect otherglycolytic enzymes such as hexokinase or 3-phosphoglycerate ki-nase (data not shown) suggesting oxygen-specific inhibition atthe PFOR level. These results strongly suggest that oxidative stresscaused by O2 treatment might damage nvEh PFOR more than vEh.

3.9. H2O2 resistance and protein oxidation

The experiments described above indicate that vEh displayhigher ROS production, however, their damaging effects in theseamoeba are less drastic than the effects observed in nvEh. Thus,the anti-oxidant capacity was indirectly tested by challenging theamoebae with external addition of a high H2O2 concentration(2 mM). vEh showed 27.8 ± 6.0% viability after incubation with

Fig. 6. Oxygen pyruvate: ferredoxin oxidoreductase inactivation in virulentEntamoeba histolytica (vEh) and non-virulent E. histolytica (nvEh). Live amoebaewere incubated in O2-saturated PBS (amoebae + O2) for 15 min and PFOR remainingactivity was determined in cellular extracts. Aliquots from these extracts weretreated under reactivation conditions (amoebae + O2 + DTT + Fe++) and activitydetermined. Clarified extracts from amoebae incubated in PBS for 30 min (amoebaewithout O2) were incubated at room environment for 1 h on ice (extract + O2);further, aliquots were treated under reactivation conditions (extract + O2 +DTT + Fe++) and remaining pyruvate: ferredoxin oxidoreductase activity wasdetermined. The plots represent the average ± SD of four experiments performedwith independent amoebic culture batches.

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2 mM H2O2, whereas nvEh viability was 8.5 ± 4.9%. Furthermore,vEh showed higher basal levels of carbonylated proteins than nvEh(6.9 ± 0.5 and 4.6 ± 1 nmol/mg protein, respectively). In addition,these levels increased in both amoebae after high oxygen exposure,with nvEh showing higher levels (vEh 9.5 ± 1.1 and nvEh12.1 ± 1.2).

3.10. Hyperbaric O2 therapy and menadione on ALAH development

The increased liver weight during ALAH correlates with tissuedamage. However, HBO did not modify the outcome of ALAH asthe average liver weight of control animals was 10.4 ± 0.39 g ver-sus 9.91 ± 1.12 g after HBO treatment. Furthermore, microscopicanalysis showed similar features (numbers of amoebae, inflamma-tion and tissue damage). On the other hand, hamsters infected withamoebae and treated with menadione showed smaller increases intheir average liver weight (7.35 ± 0.45 g). The microscopic aspectwas similar to that found with the anti-inflammatory treatment(Olivos-García et al., 2007); many amoebae in clusters surroundedby few inflammatory cells and with minimal tissue damage. Theaddition of HBO also resulted in less increased liver weight(5.61 ± 1.31 g) and the microscopic aspect was similar to the grouptreated with menadione. On the other hand, menadione was nottoxic to amoebae when incubated in TYI-S-33 medium for 24 h.Nevertheless, when these amoebae were injected into the portalvein of hamsters, the parasites disappeared in a short time (3 h)in the absence of inflammation, and liver weights were the sameas normal animals (3.96 ± 0.56 g).

4. Discussion

Amoebic liver abscess in hamster is an experimental modelwidely used for evaluating amoebic virulence: once the liver is in-fected by the parasite, tissue damage progresses, ultimately caus-ing host death (Tsutsumi and Shibayama, 2006). Humanamoebiasis is characterized by extensive necrotizing lesions, forwhich the parasite was named (Brandt and Pérez-Tamayo, 1970).Tissue destruction is usually attributed to different parasite com-ponents, such as amoebapores, cysteine proteases, adhesins, phos-pholipases and collagenase, which are considered virulence factors,

although the data supporting such views are indirect and contro-versial (Stanley and Reed, 2001; Ackers and Mirelman, 2006;Pérez-Tamayo et al., 2006; Santi-Rocca et al., 2008). Although inhi-bition or absence of these presumed virulence factors interferewith the ability of axenic E. histolytica trophozoites to cause ALAH,their in vivo histolytic activity have not been demonstrated. Analternative explanation would be that the parasite is unable to sur-vive the in vivo conditions encountered in the liver without theiractivity. Although it has been claimed that the presumed virulencefactors are required for some in vitro amoebic functions, such asreplication, complement resistance, cytotoxicity, erythrophagocy-tosis, haemolytic and proteolytic capacity, in our experiments wedid not observe differences in such functions between vEh andnvEh.

It has been suggested that redox amoebic capacity could beimplicated in E. histolytica virulence (Bracha and Mirelman, 1984;Mirelman, 1987; Kumar et al., 1992). In addition, this parasite isa microaerotolerant microorganism as it grows in micro-anaerobicenvironments, such as TYI-S-33 medium or human large intestine,and tolerates up to 5% O2 (Band and Cirrito, 1979; Reeves, 1984).When E. histolytica invades human tissues, it is challenged by anhigh O2 environment �0.24 mM (Nöth et al., 1999) and by theROS produced by inflammation; only if the parasite survives suchconditions will it be able to cause tissue damage. This suggests thatO2 and ROS resistance may also be important features of the viru-lent phenotype of E. histolytica.

In this work we found that nvEh and vEh showed a marked dif-ference in viability when challenged with high O2 concentration(0.66 mM), nvEh being far more susceptible (Fig. 1). After exposureto high O2 concentration, increased levels of O��2 and H2O2 weremainly observed in vEh, whereas there was no increase in MDAlevels in either type of amoebae, suggesting a lipid oxidation-inde-pendent cytotoxicity mechanism (Fig. 4). Dihydroethidium used inour experiments is oxidized specifically by O��2 to oxyethidium torender a fluorescent compound (Zhao et al., 2003). Although theparasite contains an FeSOD responsible for O��2 reduction (Bruch-haus and Tannich, 1994), the specific O��2 production in E. histolyticahas not been determined; moreover, the amoebic O��2 producer isunder characterization. Amoebic O2 detoxification through O2

monovalent reduction and O��2 reduction by FeSOD is not an effi-cient pathway because O2 is recycled. However, as in aerobic cells,amoebic O��2 and the other ROS may also play an important func-tion in several signaling pathways, gene expression or cell prolifer-ation (Sauer et al., 2001). In our experiments, O��2 , H2O2 and .OHscavengers interfered with amoebic viability during axenic culture.On the other hand, it has been demonstrated that amoebicNADPH:flavin oxidoreductase (Ehp34) and thioredoxin reductaseare O2 scavengers since they are able to perform divalent O2 reduc-tion to produce H2O2 and they also reduce peroxiredoxin (Eh29)which subsequently detoxifies H2O2 (Bruchhaus et al., 1997; Ariaset al., 2008). The higher H2O2 production in live (Fig. 3B) and lysatevEh amoebae (Fig. 4), after O2 exposure, point to the NADPH:flavinoxidoreductase and/or thioredoxin reductase as the principal en-zyme(s) responsible for intracellular O2 reduction in vEh. Researchis being conducted in the translational or post-translational regula-tion of such enzymes, over-expression in nvEh, and its specificinhibition in live vEh.

ROS are necessary for living cells and are well tolerated in mod-erate concentrations by aerobic cells, but when they are excessiveand cannot be neutralized, they become lethal (Kimura et al., 2005;Halliwell, 2007). In contrast, the same molecules (including O2) areextremely toxic for anaerobic microorganisms (Imlay, 2003). Toavoid their toxic effects, ROS are transformed into water througha divalent or tetravalent O2 reduction, and by the over-expressionof anti-oxidant enzymes and molecules. Frequently, anaerobicmicroorganisms express superoxide reductase and rubrerythrin

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enzymes, instead of superoxide dismutase and catalase, to avoid O2

cycling (Jenney et al., 1999; Fournier et al., 2003). On the otherhand, the main cellular targets of molecular O2 in anaerobic organ-isms are proteins containing Fe–S centers which function as elec-tron-carriers in enzymes such as fumarate reductase, aconitaseand PFOR (Pan and Imlay, 2001). O2 is capable of inactivating theseenzymes through O��2 or H2O2 (the first two enzymes) or by directlyreducing the ferrous atom to a ferric state in the case of PFOR (Im-lay, 2006). Interestingly, once PFOR is inhibited in vivo by O2 in theobligate anaerobe bacterium Bacteroides thetaiotaomicron, activitycan be regained in vivo in the absence of protein synthesis andin vitro by incubation with DTT and Fe++ (Kennedy et al, 1983;Pan and Imlay; Djaman et al., 2004). It has been previously re-ported that PFOR activity in amoebic lysates is lost when the ex-tracts are prepared under aerobic conditions (Reeves et al., 1977).In our experiments, clarified extracts of nvEh and vEh amoebaeincubated with O2 and prepared under anaerobic conditions dis-played a significant decrease (>80%) in PFOR activity comparedwith activity displayed by amoebae without O2 treatment(Fig. 6). These results are in agreement with the high accumulationof glycolytic intermediaries before the PFOR reaction (G6P, F6P andpyruvate) and the decrease in EtOH and ATP content (Fig. 5). How-ever, a remarkable difference between the enzymes from bothamoebae is that nvEhPFOR is irreversibly inactivated, bothin vivo and in vitro. Due to the higher carbonylated protein levelobserved in nvEh after high O2 challenge, the irreversible nvEhPFOR inactivation could be caused by this protein oxidation path-way which resulted from H2O2 accumulation plus free Fe++ lostfrom the Fe-S centers.

Taken together, we suggest that nvEh O2 in vitro susceptibilitycould be attributed to high O2 and H2O2 accumulation caused bylow O2 reduction and low H2O2 detoxifying capacity (Fig. 4) whichgreatly affects PFOR activity (Fig. 6) and glycolytic flux (Fig. 5) lead-ing to metabolic failure and death.

In the light of our findings, what is the possible relationship ofin vitro amoebic O2 resistance and anti-oxidative capacity, withvirulence?

Frequently, E. histolytica virulence is evaluated by the parasite’scapacity to cause ALAH. A few minutes after vEh is injected into thehamster portal vein, it can be found in the liver sinusoids withoutinflammatory cells and in contact with blood. The sinusoidal liverenvironment contains a constant O2 concentration of 23.5 Torr(Jiang et al., 1996) that is toxic for the amoeba. After 4–6 h, inflam-matory cells arrive (Tsutsumi et al., 1984) and create an ischaemicinflammatory focus that remains until the animal dies (Pérez-Ta-mayo et al., 1992). O2 concentration in this microenvironmentmay be reduced by both parasite and inflammatory infiltrate. Onthe other hand, it has been observed that a high proportion ofvEh are dead before 12 h of ALAH and the remaining viable para-sites are capable of proliferating to continue with ALAH develop-ment (Rigothier et al., 2002). PMN cells have been suggested asthe principal cells that kill the parasites during early ALAH, sinceparasite debris can be observed near and inside those. However,the possibility that parasites might be killed by O2 (without disin-tegration) and cleared by PMNs cannot be excluded. The selectionof an O2-resistant parasite population during this exposure is unli-kely since: (i) as soon as the surviving parasites were recoveredfrom ALAH (1 week) and injected into the portal vein again, a sim-ilar percentage of parasites disappeared (data not shown) and (ii)when less than 2 � 105 virulent parasites were injected into thehamster portal vein, all died within 6 h with minimal or no inflam-matory infiltrate and without tissue damage. In addition, it hasbeen observed that vEh disappear from the hamster liver before24 h in the absence of inflammatory cells, and complement deple-tion does not modify this phenomenon (Olivos-García et al., 2004).The above previous findings together with the results presented in

this paper, suggest that O2 is the principal element responsible forextensive vEh elimination during the initial steps of ALAH (0–6 h).After this period, ischaemia generated by inflammation becomes anecessary condition for vEh survival. Another finding that supportsthis conclusion is that during ALAH development, the relevance ofthe ischaemic focus to avoid high level O2 concentration was evi-dent since hyperbaric O2 treatment of the animals (which increasestissue O2 concentration (Peloso, 1982) did not modify inflamma-tion or ischaemia and failed to kill vEh. On the other hand, mena-dione (a cycling O��2 producer) was non-toxic for vEh duringin vitro microanaerobiosis but when O2 was increased to a sub-lethal concentration, it became toxic. The same result was ob-served when vEh was pre-incubated with menadione and injectedinto the hamster portal vein, where the parasites disappeared be-fore 4 h in the absence of inflammatory reaction. Also, microanaer-obiosis was present in ischaemic areas during ALAH developmentas a high dose of menadione failed to kill vEh. Moreover, in contrastto vEh, nvEh disappeared from the hamster liver in a short time(<24 h), even in hypocomplementemic animals (Olivos et al.,2005).

On the other hand, unlike vEh, the low viability observed innvEh after H2O2 exposure suggests low anti-oxidative capacity, asobserved by Ghadirian et al. (1986). Also, increased transcriptionin ROS and anti-oxidant coding genes of E. histolytica (FeSOD andEh29) was observed after in vitro sub-lethal O2 challenge (Akbaret al., 2004). The Eh29 enzyme might play an important role inamoebic anti-oxidant capacity since it could detoxify the increasedH2O2 observed in vEh after high O2 exposure. Furthermore, Eh29higher basal up-regulation has been observed in vEh comparedwith nvEh Rahman strain and the non-pathogenic Entamoeba dis-par (Choi et al., 2005; Davis et al., 2006; MacFarlane and Singh,2006) as well as increased transcription in vEh during ALAH (San-ti-Rocca et al., 2008). Recently, the importance of Eh29 for amoebicsurvival through H2O2 reduction during ALAH was demonstratedby anti-sense inhibition (Sen et al., 2007). However, the participa-tion of the rest of the amoebic anti-oxidant enzymatic system(SOD, flavoprotein, rubrerythrin) or non-enzymatic anti-oxidantsystem (trypanothione, cysteine, vitamins) in virulence remainsto be analyzed.

Acknowledgements

This work was supported by CONACyT Grants 59175 and 83084,and DGAPA Grant IN-205407. We received excellent technical helpfrom Marco E. Gudiño, Dr. Jorge Rojas-Serrano, Pedro Balderas, aswell as the professional animal care of Daniel Sánchez Almarazand Ricardo Vargas. This work includes part of the doctoral disser-tation of E.R.

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