review article peroxynitrite and peroxiredoxin in the pathogenesis...

Review ArticlePeroxynitrite and Peroxiredoxin in the Pathogenesis ofExperimental Amebic Liver Abscess

Judith Pacheco-Yepez1 Rosa Adriana Jarillo-Luna2 Manuel Gutierrez-Meza1

Edgar Abarca-Rojano1 Bruce Allan Larsen3 and Rafael Campos-Rodriguez3

1 Seccion de Estudios de Posgrado e Investigacion Escuela Superior de Medicina IPN Plan de San Luis y Dıaz Miron sn11340 Mexico DF Mexico

2Departamento de Morfologıa Escuela Superior de Medicina IPN Plan de San Luis y Dıaz Miron sn 11340 Mexico DF Mexico3 Departamento de Bioquimica Escuela Superior de Medicina IPN Plan de San Luis y Dıaz Miron sn 11340 Mexico DF Mexico

Correspondence should be addressed to Rafael Campos-Rodriguez citlicamposgmailcom

Received 5 November 2013 Accepted 12 March 2014 Published 15 April 2014

Academic Editor Marlene Benchimol

Copyright copy 2014 Judith Pacheco-Yepez 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

Themolecularmechanisms bywhichEntamoeba histolytica causes amebic liver abscess (ALA) are still not fully understood Amebicmechanisms of adherence and cytotoxic activity are pivotal for amebic survival but apparently do not directly cause liver abscessAbundant evidence indicates that chronic inflammation (resulting from an inadequate immune response) is probably the maincause of ALA Reports referring to inflammatory mechanisms of liver damage mention a repertoire of toxic molecules by theimmune response (especially nitric oxide and reactive oxygen intermediates) and cytotoxic substances released by neutrophils andmacrophages after being lysed by amoebas (eg defensins complement and proteases) Nevertheless recent evidence downplaysthese mechanisms in abscess formation and emphasizes the importance of peroxynitrite (ONOOminus) It seems that the defensemechanism of amoebas against ONOOminus namely the amebic thioredoxin system (including peroxiredoxin) is superior to thatof mammals The aim of the present text is to define the importance of ONOOminus as the main agent of liver abscess formationduring amebic invasion and to explain the superior capacity of amoebas to defend themselves against this toxic agent through theperoxiredoxin and thioredoxin system

1 Introduction

Amoebiasis is a result of infection with the enteropathogenprotozoan Entamoeba histolytica (E histolytica) Once theamoeba has established itself in the host its dissemination tothe liver and the formation of abscesses in this organ lead tohigh morbidity and mortality

Patients with amebic liver abscess (ALA) arrive to thehospital in the chronic phase of this pathogenesis whenthe abscess has already formed Therefore observations inpatients of the first phase of ALA involving the inflammatoryreaction of the immune response have been sporadic (whenpatients arrive for other reasons and inflammation is found)In order to understand the first phase of ALA in humansanimal models have been employed These models haveproven useful even though there are important differences

between hamsters and humans in the chronic phase of ALA[1 2]

Themolecular mechanisms by which E histolytica causesALA are still not fully understood Based on evidence fromthe hamster model of amoebiasis it seems that an inadequateimmune response in individuals susceptible to amoebiasisfails to impede an amebic invasion thus leading to uncon-trolled inflammation Chronic inflammation is accompaniedby the production of several toxic molecules including nitricoxide (NO) and reactive oxygen intermediates (ROIs) suchas the superoxide anion (O

2

minus) Recently it has becomeclear that these molecules are substrates for synthesis of ahighly oxidizing agent known as peroxynitrite (ONOOminus)Furthermore reports have pointed out that E histolyticahas very effective defense mechanisms against the formationof ONOOminus as well as for its interception and inactivation

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 324230 17 pageshttpdxdoiorg1011552014324230

2 BioMed Research International

The principal mechanism for ONOOminus interception seemsto be the thioredoxin system The aim of present text is todefine the importance of ONOOminus as the main agent of liverabscess formation during amebic invasion and to explainthe apparently superior capacity of the amoeba to defenditself against this toxic agent through the peroxiredoxin andthioredoxin system

2 Adhesion and Cytotoxic Activity of Amoebasduring Amoebiasis and ALA

There is still sharp controversy among scientists today regard-ing the molecular mechanisms of amoebiasis and amebicliver invasion caused by the pathogen E histolytica Someresearchers pose that the direct damage to liver tissue byamoebas (through mechanisms of adherence and toxicity)causes the formation of liver abscess during amoebiasisOther researchers assert that the importance of amebicmechanisms in the initial stage of amoebiasis is to allow thepathogens to survive and multiply in microenvironmentsthus provoking the chronic inflammatory response thatprovides the principal mechanisms of damage to liver tissueeventually resulting inALA To a great extent the controversyhinges on an ambiguous use of language on both sides of thequestion

Adherence of amoebas to the colonic epithelium andother host cells is unquestionably of fundamental importancein amoebiasis during the initial stages of the pathogene-sis which include the initial infection and extraintestinalinvasion That is adherence is essential for the amoebato invade the host and establishes itself in microenviron-ments in which it can survive The virulence factor ofadherence is to a great extent mediated by the N-acetyl-D-galactosamine inhibitable (GalGalNAc) lectin [3 4]the 220 kDa lectin [5] and other adhesins such as the112 kDa adhesin [6] There are reports of reduced ame-bic adherence to human erythrocytes neutrophils colonicmucins and epithelia when amebic lectins are inhibited[5ndash7] Recently it was shown that EhCPADH a complexformed by a cysteine proteinase and an adhesin mediatesadherence phagocytosis and cytolysis Immunization witha recombinant polypeptide EhADH243 induces protectionin hamsters against development of ALA [8] It has alsobeen demonstrated that the recombinant enzyme rEhCP112digested gelatin collagen type I fibronectin hemoglobinand Madin-Darby canine kidney (MDCK) cell monolay-ers the EhCP112 enzyme is secreted by the trophozoites[9]

The GalGalNAc lectin is a virulence factor required fortrophozoite adherence to target cells as shown by inhibitionof this lectin with galactose [10 11] cell killing occurs in thetypical GalGalNAc manner It has been shown that perox-iredoxin (Prx) interacts with GalGalNAc lectin and that thelectin-peroxiredoxin complex is located at the amoeba-hostcell contact sites Therefore the recruitment of Prx probablyprotects the trophozoites against the ROS generated by hostcells (phagocytic and epithelial) which would facilitate theinvasion [12 13]

Molecular mechanisms of pathogenicity that allow Ehistolytica trophozoites to survive and proliferate within awide variety of host environments are related not only toadherence but also to cytotoxic activity Trophozoites candamage host cells through direct contact or close proximityas well as through phagocytic activity towards dead anddying host cells in a receptor-mediated fashion [14] Ofthe molecules secreted by amoebas cysteine proteases areparticularly important [15ndash20] They are responsible for acytolytic effect on host cells [7] the modulation of the cell-mediated immune response and the proteolysis of the hostextracellular matrix [16 21ndash25] Previous works show thatwhen mutated cysteine proteinase 5 (CP5) has a reducedactivity and the trophozoite has a reduced ability to generateALA [26]

Extensive tissue damage has been attributed to the cys-teine proteinases of E histolytica because they are (i) secretedin large quantities and can cleave extracellular matrix pro-teins thus facilitating amebic invasion (ii) secreted in higherquantities by virulent than nonvirulent trophozoites and(iii) found to participate in the inflammation of the gut andALA [15ndash20 27] However cysteine proteases are dispensablefor phagocytosis and cytopathogenicity [28 29] Their mainphysiological role could be the digestion of host cells as theyare required for rosette formation hemolysis and digestionof erythrocytes

Another molecule that could participate in the damageof host cell is the cyclooxygenase- (COX-) like enzyme in Ehistolytica responsible for the biosynthesis of prostaglandinE2

(PGE2

)The production of PGE2

by the COX-like enzymein amebic liver granuloma can downregulate effector andaccessory cell functions of infiltrate immune cells [30]

Although the aforementioned processes are fundamentalin the initial stages of amoebiasis they seem to be quitesecondary as mechanisms of necrosis leading to hepaticabscesses in the latter stages of this disorder Adhesion isimportant for the survival of amoebas and the establishmentof prolonged contact of amoebas and toxic molecules withhost endothelial cells However neither adhesion nor thesetoxic molecules seem to be responsible for the host tissuedamage that directly results in amebic colitis and ALAThesedisorders occur during the latter stages of amoebiasis whenthe majority of host cells in contact with amoebas appear notto be damaged [31ndash33] It seems likely that damage to hostcells at this time is carried out in function of the inflammatoryprocess [31]

The proposal that host cell damage results mainly from achronic inflammatory response is corroborated by a numberof different studies Some recent reports [34 35] are revealingin this sense as they analyze the pathogenesis of ALA inhamsters inoculated with engineered HGL-2 trophozoitesdefective in GalGalNAc function The authors show thatHGL-2 amoebas infect hamster liver despite lacking thisimportant adhesion molecule although the pattern of infec-tion is different from that produced by wild-type E histolyt-ica HGL-2 amoebas cause a large number of inflammatoryfoci with a disorganized structure and these foci are locatedin the vicinity of blood vessels Despite their reduced capacityfor adhering to the host endothelium and penetrating liver

BioMed Research International 3

tissue [34] these defective trophozoites are able to provokeALA [35]

In histopathological terms the chronic phase of ALA inhumans corresponds to lytic or liquefactive necrosis whereasin rodent models there is granulomatous inflammation [12 36ndash38] Hence hepatic damage in hamsters is causedby apoptosis and necrosis rather than the lytic necrosisfound in humans [39ndash41] However hamster models haveprovided important insights into the possible mechanisms ofthe inflammatory response to amebic invasion in the acutephase [1 2]

In a hamster model and at thirty minutes followinginoculation E histolytica amoebas were found in the portalvein (resulting in slightly dilated sinusoids) in the lumenof small branches of the portal vein and in the centralveins [2] After one hour trophozoites were located in thesinusoids throughout the hepatic lobules [2] At three hoursafter inoculation polymorphonuclear leukocytes (PMNs)surrounded the amoebas with one or several layers of cellsand thus impeded them from making direct contact withhepatocytes Nonetheless lysis continued Under these con-ditions the leukocytes that managed to make direct contactwith amoebas were undamaged [2] Afterwards there wasa continual increase in the quantity of PMNs and the lysisof leukocytes in liver sections during the development ofALA The massive destruction of leukocytes favored greaternecrosis and hemorrhaging of parenchymal tissue and theformation of ischemic areas Whereas few amoebas werefound in necrotic areas at this stage there are many in theperiphery of the lesion where mononuclear cells (histiocyteswith an epithelioid appearance) started to formapalisade thatseparated parenchymatous cells from the necrotic area

Since studies with hamster models demonstrate thatALAs generally develop in the absence of direct contact withamoebas it is necessary to explore the possible mechanismsthat could account for their formation The mechanisms ofchronic inflammation seem to be a likely candidate [2 42 43]Of course it is the adhesion of amoebas and their capacity toevade the host immune defenses that provoke a continuousinflammatory response [31 44ndash46]

3 Chronic Inflammation as thePossible Cause of ALA

31 Nitric Oxide and the Pathogenesis of ALA Some researchgroups have long suspected that chronic inflammation is thecause of ALA Until recently the mechanisms of chronicinflammation considered as the cause of abscess formationwere mainly the amebicidal effects of NO ROIs cytokinesand cysteine proteinases [45 47ndash51]

In this sense it has been emphasized that greater quan-tities of NO are found in the serum of hamsters with liverabscesses than in healthy animals [52 53] Some reports haveshown that the amebicidal activity of activated macrophagesismainly associatedwithNOsynthesis [54ndash56]Neverthelessthe amoebas continue to survive and proliferate

Some studies have reported that reactive oxygen species(ROS) and NO produced by activated neutrophils or

macrophages lead to the lysing of E histolytica [56ndash60]In vitro studies used a high concentration of NO (1mM)[48 54 61 62] whereas in vivo the concentration of NOin inflamed tissues is approximately 1 120583M [60 63] Duringthe development of ALA there is evidence of trophozoiteresistance to high concentrations of NO in vitro [48] andin vivo [52 53] Despite the relatively high iNOS mRNAexpression and NO production E histolytica continues toshow an invasive capacity in vivo Hence the percentages ofamoebas that remain viable represent a sufficient number tosustain an amebic invasion

Two hypotheses can be formulated based on the apparentlack of effectiveness of NO as an amebicidal agent in vivo(i) that NO is not really toxic enough to carry out an effectiveamebicidal function or (ii) that virulent amoebas have anadequate defense mechanism against NO activity Regardingthe latter conclusion several studies indicate that the capacityof E histolytica to resist the destructive action of NO andROIs whether in vivo or in vitro probably owes itself tothe expression by this Entamoeba species of high levels ofantioxidant proteins such as Prx flavoprotein A superoxidedismutase (SOD) and rubrerythrin [64ndash67]

32 The Possible Role of Peroxynitrite in Inflammation andALA It is now known that there are molecules of theinflammatory response much more detrimental than NO Infact NO is a precursor of one of these molecules Duringthe inflammatory process NO and the O

2

minus are producedsimultaneously and they react at diffusion-controlled ratesto produce the ONOOminus anion [63 68] The reaction is asfollows

NO +O2

minus

997888rarr ONOOminus (1)

Unlike NO and O2

minus ONOOminus is not a free radical but it isa highly oxidizing agent The coupling of NO with O

2

minus toyield ONOOminus in biological systems is currently accepted asthe main biological source of ONOOminus which is extremelyreactive with biological molecules and highly toxic to cells

There is no direct in vivo evidence of ONOOminus probablydue to its high reactivity [69 70] However nitrate and nitritethe products of spontaneous decomposition of this moleculehave been detected in the serum of hamsters with ALA [5253]

The two substrates of ONOOminus NO and the O2

minus seemto be abundantly produced in inflammatory sites during thehost response to amebic invasion [31 52 53 71] For instanceO2

minus is produced by the reaction of NADPH oxidase (orxanthine oxidase) and dioxygen (O

2

) (Figure 1) via electronleakage in the mitochondrial respiratory chain and at theendoplasmic reticulum [70] The levels of O

2

minus can undergo a3- to 4-fold increase during the inflammatory response whenthe NADPH oxidase complex expressed in phagocytic cellsand endothelial cells is activated and reacts in the oxygenatedenvironment to generate O

2

minus (Figure 1) [68 72 73]To some extent in vitro and in vivo reports are con-

tradictory On the one hand in vitro studies report thatmacrophages isolated from ALA in gerbils are deficient intheir capacity to produce NO as a result of the modulation


E histolyticaNADPHoxidase

Endothelial cell

Oxidativeinjury

Oxidativeinjury

Hepatocytes

Peroxynitrite

Apoptosisand

necrosis

Neutrophil

Macrophage

Apoptoticneutrophil

Arg + O2

Arg + O2

NO

NO

NOS

NOS

NOS

O2

pH 65ndash68

NADPHoxidase

O2

O2minus

O2minus

NO + O2minus

∙NO2 +∙OH

∙NO2 +∙OH

Figure 1 Hypothetical model of the generation of ONOOminus in ALA Under inflammatory conditions the two substrates of ONOOminus nitricoxide (NO) and the superoxide anion (O

2

minus) are produced simultaneously This allows for the production of large quantities of ONOOminuswhich is a highly oxidizing agent Indeed the products of spontaneous decomposition of ONOOminus nitrate and nitrite are found at higherthan normal levels in inflamed tissueThe superoxide anion is produced byNADPHoxidase expressed in activated neutrophils macrophagesand endothelial cells NO is produced by NOS found in inflammatory cells or in the vascular endotheliumThe protonated form of ONOOminus isdisintegrated to form free nitrogen dioxide (∙NO

2

) and the hydroxyl radical (∙OH) Under conditions of hepatic hypoxia low concentrationsof L-arginine and oxygen enhance arginase activity that hydrolyzes L-arginine thus competing with the substrate for NOS enzymes Thisleads to the uncoupling of NOS simultaneously producing O

2

minus and NO and thus resulting in additional ONOOminus generation ONOOminus reactsdirectly with thiol groups of host cells High levels of ONOOminus lead to the development of ALA by inducing apoptosis and necrosis in hepaticcells

of iNOS mRNA [74] On the other hand there are studiesusing a hamster model that report the in vivo production ofNO in the zone of liver abscess mediated by iNOS mRNAexpression [53] The deficient capacity of macrophages iso-lated from ALA for developing a respiratory burst does notdenote the absence of the O

2

minus anion because the productionof this anion does not depend solely on this mechanism

The production of NO requires the presence of a nitricoxide synthase (NOS) a family of enzymes that includesiNOS eNOS andnNOS iNOS can be found in the inflamma-tory cells while eNOS is present in the vascular endothelium[63 75] In sites of inflammation the cytokine-induced activ-ity of iNOS increases The iNOS-mediated formation of O

2

via a novel pathway in L-Arg depletes murine macrophagesand the O

2

formed in this way interacts with NO to form

ONOOminus which then enhances macrophage immune func-tion However the overproduction of these oxidants couldalso trigger cell death (Figure 1) [76]

Both NADPH oxidase and NOS are found in phagocytesand activated NOS is found in endothelial cells (Figure 1)[69] Although NO is a relatively stable and highly diffusiblefree radical O

2

minus is very short lived and has restricted diffu-sion across biomembranes Thus the presence of O

2

minus wouldseem to be the limiting factor for the synthesis of ONOOminusNutrient deprivation ischemia and cytokines (eg TNF120572and IL-1) under the hypoxic conditions of inflammation allincrease the activity of endothelial NADPH oxidase and theconsequent production of O

2

minus [72]Under some inflammatory conditions the production

of NO and O2

minus is strongly activated which could lead to


a 1000-fold increase in the production of each This in turnwould cause a 1000000-fold increase in the formation ofONOOminus [63 69 70] High levels of ONOOminus lead to thedysfunction of critical cellular processes the disruption of cellsignaling pathways and the induction of cell death throughboth apoptosis and necrosis [63]Thus ONOOminus is thought tobe the potential cause of a number of inflammatory diseases[68] and could possibly lead to the development of ALA

The production of ONOOminus is increased by two mech-anisms under conditions of ischemia or hypoxia Firstlyhypoxia in the liver leads to low concentrations of L-arginine and oxygen (hypoxia and anoxia) which enhancemacrophage arginase activity [77ndash80] Arginase the principalenzyme of the urea cycle hydrolyzes L-arginine to urea andL-ornithine On the other hand L-arginine and a cofactor(tetrahydrobiopterin BH4) are substrates for NOS enzymesThus arginase competes with NOS enzymes for their com-mon substrate (L-arginine) leading to the uncoupling of allthree isoforms of NOS [80]

Secondly hypoxia induces production of the hypoxiainducible factor which also promotes the uncoupling ofNOS [81ndash83] as well as the activation of arginase-2 [84]This uncoupling simultaneously produces O

2

minus and NO [85]leading to additional ONOOminus generation [69 70 76 86]Indeed more ONOOminus is formed in hypoxic regions of theliver where the pH is low leading to the production ofhydroxyl-like free radical species in the absence of oxygen[87]

ONOOminus is capable of reacting directly or indirectlywith biological tissues It reacts directly with thiol groupsthe preferential targets for ONOOminus reactivity in vivo [70]causing oxidative damage to iron-sulfur centers and theactive site of -SH groups in tyrosine phosphatases lipidsand CO

2

On the other hand ONOOminus can act indirectlyby decomposing into highly reactive radicals The ONOOminusion is more stable than its protonated form (ONOOH pKa65 to 68) which decomposes rapidly via homolysis of theOndashO bond to form free nitrogen dioxide (∙NO

2

) and thehydroxyl (∙OH) radical (Figure 3) The latter radical hasa highly oxidizing effect on many biomolecules includingtyrosine residues thiols DNA and unsaturated fatty-acid-containing phospholipids [68 88]

Compared to the reactivity of the ∙NO2

and ∙OH radicalsthe reactions of ONOOminus are relatively slow Thus ONOOminusshould certainly bemore selective for its target molecules andbetter able to react relatively far from its site of formation[70] When generated from a cellular source ONOOminus couldinfluence surrounding target cells within one to two cellularlayers (sim5ndash20120583m) [69]

By reacting with lipids DNA and proteins via directoxidative reactions or via indirect radical-mediated mecha-nisms ONOOminus can trigger cellular responses ranging fromsubtle modulations of cell signaling to overwhelming oxida-tive injury that commits cells to necrosis or apoptosis [69]Whendamagemediated byONOOminus induces cell necrosis therelease of the intracellular content of cells (eg HMGB1 highmobility group protein B1) into the extracellular space cantrigger an additional inflammatory response representing apositive-feedback cycle of further ONOOminus generation [69]

DNA damage induced by ONOOminus leading to the acti-vation of poly (ADP-ribose) polymerase (PARP) can alsoincrease the inflammatory response It has been shown thatPARP coactivates many inflammatory cascades and increasestissue infiltration by activated phagocytes in experimentalmodels of inflammation circulatory shock and ischemia-reperfusion [63 89] Through these mechanisms ONOOminusamplifies neutrophil-dependent responses (adhesion migra-tion and activation of neutrophils) under inflammatoryconditions and thus contributes to the detrimental effectsof inflammation in arthritis colitis and other inflammatorydiseases [63 70]

4 Defense Mechanisms against ONOOminus

In mammalian tissue peroxides like H2

O2

and ONOOminus areproduced as a result of normal cellular processes includ-ing metabolism and inflammation As a result host tissueand amoebas must have defense mechanisms against thesemolecules To be able to provoke an uncontrolled inflam-matory response the trophozoite defense system againstONOOminus would have to be better than that of the hostBiological protection against ONOOminus is organized in twomain categories prevention and interception [90 91]

41 Prevention of ONOOminus Formation One defense mecha-nism by amoebas and mammalian tissue against ONOOminus isthe prevention of its formation through the inhibition of itsprecursors NO andO

2

minus [92]The steady state concentrationsof O2

minus are normally kept relatively low in the nanomolarto picomolar range The main route of O

2

minus consumption inbiological systems is its reaction with superoxide dismutase(SOD) to form hydrogen peroxide (H

2

O2

) and dioxygen Onthe other hand NO is scavenged by hemoglobin decreasingits levels in the blood [93] NO readily reacts with oxy-hemoglobin or oxymyoglobin to give nitrate (NO

3

minus) andoxidized hemoproteins (methemoglobin andmetmyoglobin)as follows

Hb (Fe minusO2

) +NO 997888rarr metHb (Fe III) +NO3

minus (2)

Due to the high concentrations of oxyhemoglobin in thebody this molecule may provide the primary metabolic aswell as detoxification mechanism for NO in vivo [93ndash95]Thus red blood cell-encapsulated hemoglobin can react veryquickly with NO and scavenge virtually all the moleculesproduced by endothelial and inflammatory cells [94 95]However the inactivation of NO by Hb and by self-oxidationcould be less efficient in a hypoxic environment as thisreaction requires oxygen

One generally recognizedmechanism for the inactivationof NO is its reaction with O

2

[93] Since NO andO2

are muchmore soluble in lipid layers than aqueous fractions biologicalmembranesmay act as a ldquolensrdquo that can focus andmagnify theself-oxidation of NO [96 97]

E histolytica has developed some mechanisms to avoidNO production For example soluble amoeba proteins sup-press INF120574 induced amebicidal activity thus affecting theexpression of the mRNA iNOS gene and consequently NO


production [74] Moreover there is evidence that the mono-cyte locomotion inhibitory factor of E histolytica inhibitsthe in vitro NO production normally induced by cytokinesin human leukocytes [98] It has also been reported that Ehistolytica arginase inhibits NO production by consuming L-arginine the substrate of iNOS in activated macrophages Allof these amebic mechanisms should certainly contribute tothe survival E histolytica [99]

42 Interception or Inactivation of ONOOminus Due to a directreaction with low-molecular weight compounds (eg carbondioxide thiols ascorbate selenocompounds and syntheticmetalloporphyrins) and other proteins (eg certain peroxi-dases hemoglobin albumin and selenoproteins) ONOOminus isdecomposed into nontoxic products Efficient ONOOminus scav-engers include selenium compounds particularly 2-phenyl-12 benzisoselenazol-3(2H)-one (ebselen) selenomethionineand selenocysteine as well as selenium-containing proteinssuch as glutathione peroxidase and thioredoxin-reductase[70 90] Additionally (minus)-epicatechin and other flavonoidscan contribute to the cellular defense against ONOOminus [91]Considering their approximate concentrations in vivo andtheir reaction rate constants in relation to ONOOminus the bestof these low-weightmolecules for the interception ofONOOminuswould seem to be CO

2

hemoglobin and glutathione [90]

5 The Thioredoxin System

Theprincipalmechanism for interception ofONOOminus is prob-ably the thioredoxin system which is comprised of thiore-doxin (Trx) and thioredoxin reductase (TrxR) This systemtogether with peroxiredoxin (Prx) mediates the NADPH-dependent reduction of H

2

O2

and tert-butyl hydroperoxidein Entamoeba species [100] For Trx to carry out most of itsfunctions the disulfide active site in this protein must bereduced in the following manner

Oxidized Trx + TrxR (with NADPH) 997888rarr Dithiol Trx (3)

Once Trx accepts electrons from TrxR it can reduce PrxAnother NADH enzyme flavin oxidoreductase also carriesout this function [100] The resulting form of Prx can protectagainst oxidative stress by decomposing H

2

O2

into water(H2

O) and probably ONOOminus into nitrite [101]

51 TrxR The different functions of Trx are entirely depen-dent upon the activity of TrxR [102] and the latter has twomajor forms in natureWhereas the larger form of TrxR has aselenoprotein (simMr = 55 kDa) and corresponds to mammalsthe relatively small nonselenoprotein form of TrxR is foundin bacteria plants archaea and most unicellular eukaryotes[102 103]

Homodimeric mammalian TrxR consists of two subunitsin a head-to-tail arrangement Both subunits are absolutelyrequired for normal catalysis during the catalytic cycle[102] The C-terminal -Gly-Cys-Sec-Gly-COOHmotif of thehuman selenoprotein (about 16 amino acids long) is found inall mammalian TrxR and the Cys-Sec dyad of this motif hasbeen identified as a reversible selenenylsulfideselenothiol

constituting the active site of the enzyme [102] The electronsfrom NADPH reduce a redox active disulfide and transferthem to the C-terminal active site of a selenothiol locatedin the sequence Gly-Cys-Sec-Gly which is conserved in allisoforms of TrxR From this site electrons are transferred toTrx allowing the latter to carry out its functions such as thereduction of protein disulfides or other substrates [104]

For example the twomajor forms of humanTrx cytosolicTrx1 (the TXN1 gene product) and mitochondrial Trx2 (theTXN2 gene product) are reduced by TrxR and NADPH [101102] As a result these two forms of Trx can provide electronsto proteins such as ribonucleotide reductase (formation ofdeoxyribonucleotides from ribonucleotides) In this way Trxreacts with ribonucleotide precursors to synthesize deoxyri-bonucleotides peroxiredoxins and methionine sulfoxidereductases [102]

As can be appreciated the Trx-reducing activity ofmammalian TrxR is totally selenium dependent [102] andaberrations in selenium metabolism must have a directimpact on the functions and levels of different selenoproteinsas well as on many cellular systems that are linked to Trxactivity

52 The Amebic Trx System Previous works reported themolecular cloning expression purification and functionalcharacterization of two genes from E histolytica that encodefor TrxR and Trx (EhTrxR and EhTrx) It turns out thatEhTrxR belongs to the low-molecular-weight family of TrxRswhich have a redox active site containing two key cysteineresidues without a selenoprotein [100]This is a homodimericcovalent protein that catalyzes the NAD(P)H-dependentreduction of thioredoxins (Figure 2) and S-nitrosothiols [92]

Unlike the TrxR of E histolytica that of mammalsis dependent on selenoproteins On the other hand bothmammalian TrxR and EhTrxR reduce the correspondingform of Trx by using NADPH [100] The resulting form ofTrx reduces Prx which in turn can reduce H

2

O2

to H2

Oand probably ONOOH to nitrite (Figure 2) thus acting as anantioxidant system [100]

EhTrxR EhTrx and Prx have been immunolocalizedunder the plasma membrane in E histolytica Contrarilymammalian TrxR Trx and Prx have not been found underthemembrane (see Section 534) [64 105]This cellular loca-tion in E histolytica favors the potential in vivo functionalityof the ROS detoxification system On the one hand oneamebic mechanism of resistance to lipid peroxidation is thecomposition of the amebic membrane which has a highconcentration of saturated lipids [106]On the other hand theEhTrx systemcould offer an alternative protectivemechanismagainst lipid peroxidation thus maintaining intracellularproteins and DNA safe from highly toxic reactive oxygen andnitrogen species In such a case this would represent a keymechanism in relation to the virulence of E histolytica whenexposed to highly toxic ROIs [105] and should be pivotal toamebic survival during extraintestinal infection [92]

53 Peroxiredoxin Peroxiredoxins (Prxs) are a familyof antioxidant enzymes that are principally reduced by


NADPH EhTrxR ox EhTrx red EhPrx ox

NADP+ EhTrxR red EhTrx ox EhPrx red

ONOOH

H2OH2O2

NO2 + H2O

Figure 2 E histolytica thioredoxin systemThe Entamoeba thioredoxin system is comprised of thioredoxin (Trx) and thioredoxin reductase(TrxR) coupled with peroxiredoxin (Prx) and NADPH Trx has to be reduced to fulfill its functions and this reduction is carried out throughcatalyzation by TrxR using NADPH as a cofactor Once Trx accepts electrons from TrxR it can reduce peroxiredoxin (Prx) The resultingform of Prx can protect against oxidative stress by decomposing H

2

O2

into H2

O Based on the figure by Arias et al [105]

Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2

Figure 3 Amebic peroxiredoxin in ALA During the developmentof ALA activated phagocytes produce increased levels of superoxide(O2

minus) and NO thus generating the ONOOminus anion which in turncauses oxidative damage to hepatocytes ONOOminus can decomposeinto nitrogen dioxide (∙NO

2

) and the highly oxidizing hydroxyl rad-ical (∙OH)The ONOOminus anion can be intercepted by direct reactionwith carbon dioxide decomposing it into nontoxic products ThePrx enzymes of parasites (eg E histolytica) and other bacteria areinvolved in protection of these organisms by the reduction of host-produced hydrogen peroxides and ONOOminus

Trx [107] Once reduced Prx can in turn reduce H2

O2

and perhaps ONOOminus to harmless forms These peroxide-eliminating enzymes are truly ubiquitous existing in yeastplant and animal cells including protozoa helminthsparasites and most (if not all) eubacteria and archaea [108]Prxs are preferentially expressed under conditions of stressinduced by elevated levels of reactive oxygen species (ROS)and reactive nitrogen species

531 Mammalian Prx There are at least five subfamilies ofmammalian Prx enzymes which are categorized on the basisof their protein sequence and cellular location Althoughlocated primarily in the cytosol Prx is also found withinmitochondria chloroplasts and peroxisomes associatedwith nuclei and membranes [108 109] The two categoriesof Prx consist of 1-Cys and 2-Cys Each form has a distinctnumber of cysteinyl residues involved in catalysis [108 109]

532 Amebic Prx Although parasites are in an anaerobicenvironment during colonization of the colonwhen invadingtissue they are exposed to aerobic conditions Several studieshave identified H

2

O2

as lethal to Entamoeba species [57 5864 110] Thus in the oxygen-rich microenvironment thatexists at the site of hepatic lesions the capacity of Prx toprevent the deleterious production of H

2

O2

in the host mustcertainly play an important role in the survival of parasites[65 111ndash113]

E histolytica Prx (EhPrx) can degrade H2

O2

[111 114] aswell as alkyl and aryl hydroperoxides [64] Itmay also degradeONOOminus to nitrite Like mammalian Prx the peroxidaseactivity of EhPrx depends on Trx and TrxR activity [115]EhPrx removes H

2

O2

in vitro in the presence of NADPHEhTrxR and EhTrx (Figure 2) This capacity of Prx has beenconfirmed in both native and recombinant proteins [111 114115]

Virulent E histolytica is reportedly more resistant toH2

O2

than E dispar and the nonvirulent strains of E histolyt-ica [64 116 117] There are reports of an increase in proteinand gene expression of Prx by E histolytica in an oxygen-richenvironment [65 112] By employing a fluorometric stopped-flow assay the H

2

O2

detoxification activity of EhPrx and Prxof E dispar was found to be similar [64] Thus it seemsthat the principal difference between EhPrx and the Prx ofE dispar is the location of this antioxidant Accordinglythe ability of E dispar to become virulent under certainconditions may be due in part to the possible change oflocation of Prx in distinct environments [114] Trophozoitesdeficient in Prx are more sensitive than the wild type to anoxygenated environment and to H

2

O2

in axenic culture [118]Logically the size of liver abscesses is lower in hamsters

inoculated with Prx-downregulated E histolytica tropho-zoites compared to normal HM1IMSS (6ndash9 versus 20ndash25mm) [118] Similarly overexpression of Prx in E histolyticaRahman rendered the transgenic trophozoites more resistantto killing by H

2

O2

(5mM) in vitro [116] and Rahman tropho-zoites expressing higher levels of Prx have been associatedbased on histological analysis of human colonic xenograftswith higher levels of intestinal inflammation andmore severedisease [116]

Trophozoites cultured axenically are less virulent bothin the hamster model of ALA and in the mouse modelof amebic colitis [119 120] RNA from such low virulence


trophozoites was compared with the transcripts of amoebaswhose virulence was maintained by passing them throughhamster liver abscesses Among the upregulated transcriptsfound in the virulent amoebas were those involved in bothoxidative and stress defense including three Prx transcripts[121] Hence Prx levels of E histolytica HM1IMSS tropho-zoites may increase in response to the presence of hostinflammatory cells [116]

Moreover immunization of gerbils and hamsters withrecombinant Prx results in partial protection against ALAformation andor a reduction in the size of the abscesses [122ndash124] Thus a strengthened immune response against amebicPrx may protect hamsters against the development of ALA[123]

The evidence regarding the activity and location of EhPrxtogether with the resistance provided by Prx to the toxiceffects of H

2

O2

clearly suggests the importance of thisantioxidant enzyme for the survival and virulence of Ehistolytica trophozoites [116 118]

533 Prx Structure and Action Mechanisms EhPrx hasa 52 identity with the human PrxII-2 Cys the maindifference being the extended cysteine-rich N-terminalregion in the amebic version probably contributing toits enzymatic activity [111] Several authors have demon-strated that peroxiredoxin forms a 60 kDa dimer throughdisulfide bonds [113 124] and forms an oligomer oroligomers with high-molecular mass on the cell surface(gt200 kDa) [112] Thus EhPrx is typically found as anobligate homodimer (2-Cys Prxs) with identical active sites[108]

Furthermore EhPrx can form a high-molecular massoligomer on the cell surface [111 112] because it contains twoconserved sequence motifs (region I sequence PLDWTFand region II sequence DSVYCHQAWCEA) that are nec-essary for decamer formation in 2-Cys Prxs [108 125] Thisdecameric structure (ring-like higher oligomers or toroids) iscomprised of five dimers (thatmay ormay not be functionallyactive) linked end-on through predominantly hydrophobicinteractions [109 126]

EhPrx retains arginine 163 and the two conserved cysteineresidues (VCP motifs Cys 87 and 208) that are required forits catalytic activity [108 109 115]The structure and sequenceof the peroxidatic active site is highly conserved among Prxclasses (1-Cys typical 2-Cys and atypical 2-Cys Prxs) Thefeatures of typical 2-Cys Prx are highly conserved acrossall kingdoms with 30 or greater sequence identity Themost conserved regions include (i) the peroxidative cysteine(generally near residue 50) within the DFTFVCPTEI motifin mammals and the DWTFVCPTEI motif in E histolyticaand (ii) the resolving cysteine (near residue 170) within theVCPmotif [108]Therefore the catalytic activity of the Prx ofEntamoeba spp must be similar to other typical Cys-2 Prx

Various studies have demonstrated that Prx whetherof mammalian bacterial or parasitic origin can reduceONOOminus to nitrite in vitro [69 127ndash130] One study confirmeda protective role of Prx against ONOOminus in vivo (Figure 3)[101]

534 Location of Prx Mammalian Prx is mainly located inthe cytosol membrane and mitochondria [108] ContrarilyEhPrx has been detected not only in the nucleus and cyto-plasm [131] but also on the peripheral membrane [12 132133] EhPrx probably forms a high-molecular-mass oligomer(gt200 kDa) on the cell surface [111 112] Indeed peroxire-doxin (29 kDa) is the major thiol-containing surface proteinof E histolytica and has both peroxidase and antioxidantactivities [64 132]

Prx interacts with galactose and the GalNAc lectin whichare both found at sites of amoeba-host cell contact Therecruitment of Prx at this site probably protects trophozoitesagainst the ROIs generated by host phagocytic and epithelialcells [12 13] The membrane location of EhPrx suggests animportant role for the Trx-dependent metabolic pathwayas a redox interchanger which could be critical for themaintenance and virulence of the parasite when exposed tohighly toxic ROIs [105] In this location the system protectsthemembrane against lipid peroxidation andmay participatein the protection of hemoglobin and other intracellularproteins against free radical damage by stimulating potassiumefflux

The enzymatic activity quantity and location of EhPrxprobably account for the greater resistance of this amebicstrain to oxidants like H

2

O2

Hence the Trx-Prx system of Ehistolytica seems to help it survive and proliferate in a highlyoxygenated environment leading to greater invasiveness[114] and virulence

6 Other Defense Mechanisms of E histolyticaduring Amebic Invasion

61 Flavodiiron Proteins Flavodiiron proteins are enzymesfound in strict and facultative anaerobic bacteria and archaeaas well as a limited number of eukaryotes such as thepathogenic protozoa Trichomonas vaginalis Entamoeba andGiardia intestinalis [66 134ndash138] Due to the role of flavodi-iron proteins in the catalysis or reduction of O

2

to H2

O andNO to N

2

O they have been proposed as a protective mecha-nism against nitrosative stress or oxygen toxicity in anaerobes[137 139 140] E histolytica has four copies of flavoproteinA [66] suggesting that this enzyme plays a significant rolein these twomechanisms However the substrate preferences(O2

or NO) of the E histolytica flavodiiron proteins and theirrole during infection have not been determined [141]

62 Erythrophagocytosis Phagocytosis is considered a viru-lence sign in E histolytica [14 142ndash145] Recent data suggestthat transmembrane kinases (TMK) have a role in phagocy-tosis of human erythrocytes [146] Erythrophagocytosis maybe important for the survival growth and proliferation of Ehistolytica in an aerobic or even microaerobic environment[147 148] E histolytica trophozoites preferentially interactwith red blood cells meaning that the phagocytic activity oferythrocytes by E histolytica trophozoites should certainlyhave a very important function in amoebiasis Indeed thephagocytic capacity of trophozoites has been taken as a


qualitative marker of pathogenicity [142 145 149] Ery-throphagocytosis may help the amoeba to scavenge NO andother reactive oxygen and nitrogen intermediates and thushelp the parasite to survive in the oxygen-rich environmentof oxidative stress [147]

Hemoglobin and Prx are two major proteins present inerythrocytes For example Prx2 is an abundant erythrocyteprotein [109 147] Therefore the process of erythrophago-cytosis may serve two important functions in the amebictrophozoites that would contribute to parasite virulenceFirstly erythrocytes rich in hemoglobin (Hb) may helptrophozoites to scavenge NO which readily reacts withoxyhemoglobin and oxymyoglobin to give nitrate (NO

3

minus)and the oxidized hemoproteins methemoglobin and met-myoglobin The reaction with hemoglobin is the primarydetoxificationmechanism forNO [93ndash95] Secondly erythro-cytes rich in Prx may help amoebas to scavenge ONOOminusHb and Prx-2 of the erythrocytes are extremely efficient atscavenging H

2

O2

NO and ONOOminusThese mechanisms are probably pivotal for parasite sur-

vival under the nitrogen and oxygen-rich environment ofinflammation (Figure 3) [148] Also phagocytosis of bacte-ria containing Prx could increase the pathogenicity of Ehistolytica in the host intestine and possibly also act as astimulus to induce the invasive behavior of trophozoites [150]E histolytica might have a mechanism of incorporating Prxsimilar to that of Plasmodium falciparum which imports Prx-2 from human erythrocytes into its cytosol This Prx-2 existsin a functional form and a significant concentration [151]

7 An Overview of the PrxModel of Amoebiasis

Abundant evidence in the literature suggests that inflam-mation and inflammatory mediators are the cause of tissuedamage in ALA (see Sections 31 and 32) as few trophozoitesare present in the extensive areas of apoptosis and necrosis[152] Furthermore in the absence of inflammatory cellsvirulent E histolytica trophozoites lead to little or no abscessformation in hamsters [44 153]

N-Acetylcysteine (NAC) also inhibits tissue damage inthe latter stages of ALA in hamster In the liver of ham-sters treated with NAC there are many clusters of well-preserved amoebas in close contact with hepatocytes sparseinflammatory infiltrate and minimal or no tissue damage[42] The inhibitory effect of NAC on tissue damage duringlate stages of EALAH was explained [42] as the inhibitionof leukocyte-endothelium adhesion molecules and reducedmigration of leukocytes leading to an inhibitory effect ofNAC on NO production and ROS activity and consequentlya reduction of the toxic effect on cells According to Olivos-Garcıa et al [42] ldquoROS and NOS (NO and 119874119873119874119874minus) may bethe principalmolecules responsible for tissue destruction duringthe late stages of ALArdquo [42] However NAC also reducesONOOminus mediated toxicity in various pathophysiologicalconditions [69 154 155] Hence the point of view in theaforementioned quote is perhaps better explained by the Prxmodel which holds that the inhibition of the synthesis of

ONOOminus would not only maintain amoebas alive but alsoreduce both inflammatory infiltrate and tissue damage

71 Initial Phase Acute Inflammation It is well known thatsome individuals are susceptible to E histolytica trophozoiteswhile others are resistant [31 156] In susceptible individualstrophozoites invade the intestine survive the host immuneresponse and arrive to the liver through the blood flowTheirpresence in the sinusoids is associated with an influx of hostimmune cells We propose that the key to susceptibility is theincapacity of the immune response to eliminate the amoebasbefore the latter are able to establish colonies and provoke achronic inflammatory response

During this initial stage of amoebiasis in hamstersthe two major components of the inflammatory responseare PMNs and mononuclear cells (MO) both involved incellular infiltration [1 2] PMNs cause the formation ofnumerous inflammatory foci that produce large amountsof NO and O

2

minus which in turn lead to the productionof ONOOminus (Figure 4) This highly reactive oxidizing agentprobably lyses neutrophils endothelial cells hepatocytes andother parenchymal cells Lysed neutrophils release proteolyticenzymes adding to the assortment of toxic compounds thatcause damage to endothelial parenchymal and inflamma-tory cells (Figure 4) [1 2] It has been proposed that withinflammation there is massive trophozoite death [157] whichdoes indeed seem to be the case for individuals resistant toE histolytica However for susceptible individuals it seemsmore likely that amoebas take advantage of the inflammatoryenvironment in the first stage of the pathogenesis by ingestingnutrients from dead inflammatory cells

72 Chronic Phase Uncontrolled Inflammation Amoebiclesions in hamsters are formed by necrosis and apoptosis ofhepatocytes [1 2 37 39 40 152 157] The evidence outlinedin the present review seems to indicate that these abscessesare not formed principally through direct contact betweenamoebas and host cells but instead by the uncontrolledinflammatory response produced when susceptible individ-uals are unable to eliminate amoebas

Under conditions of uncontrolled inflammation thereis an ever greater quantity of amoebas amebic molecules(cysteine proteinases LPFG GalGalNAc-specific lectin andprostaglandin E2) and cytokines (IL-1a IL-6 IL-8 and TNF-a) that induce the production of adhesion molecules (egICAM-1 and E-selectin) which in turn attract macrophagesand lymphocytes [31] These cells together with Kupffer cellsproduce large amounts of cytokines which further amplifythe inflammatory response [31 158ndash160]The abundant num-ber of monocytes macrophages and Kupffer cells as well asactivated endothelial cells and host mitochondria produceever greater quantities of NO and O

2

minus which probably leadto the formation of large amounts of the ONOOminus anion(Figure 4)

The Prx model proposes that diffusible molecules suchas ONOOminus and TNF-120572 are able to commit cells to necrosisor apoptosis in the liver (Figure 4) [68 69] Corroboratingthis idea one study reported that inflammation is induced


E histolyticaE histolytica

Hepatocytes

Peroxynitrite

Neutrophil lysisKupffer cellNeutrophils

Endothelialcell

Ito cellNecrosis and apoptosis

of hepatocytesEntamoeba defenselowast Peroxiredoxinlowast Flavoproteinslowast Hb caption

Cytokines(TNF-120572)

NO + O2minus

Figure 4 Role of ONOOminus and E histolytica Prx in the pathogenesis of ALA The early stages of ALA are characterized by an acute andchronic host inflammatory response where an abundant number of monocytes macrophages Kupffer cells and activated endothelial cellsproduce large amounts of nitric oxide and the superoxide anion Moreover these molecules may be released after immune cells are lysedTherefore there is probably a high concentration of ONOOminus at inflammatory sites which in turn would promote necrosis andor apoptosisof hepatocytes Amebic flavoproteins and Prx play a protective role against the oxidative and nitrosative attack of phagocytic cells and againstthe H

2

O2

produced by the amebic metabolism

when parasites or immune cells secrete or release diffusibleproducts after cell death [51] ONOOminus interacts with lipidsDNA and proteins via direct oxidative reactions or indirectradical-mediated mechanisms Among the latter mecha-nisms nitrogen dioxide (∙NO

2

) and hydroxyl radicals (∙OH)are of particular importance (Figure 3)

The continuous prolonged and uncontrolled inflamma-tory response seems to be responsible for the productionof large granulomas or ldquoabscessesrdquo in hamsters which arecharacteristic of the chronic phase of ALA in experimentalanimal models [31] At 3-4 days after inoculation of axenicamoebas the chronic granulomatous reaction in the liver isformed by typical multiple granulomas which have a centralnecrosis limited peripherally by a palisade of epithelioidcells and more externally by connective fibers Amoebas arerecognizable between the necrosis and epithelioid cells [1 2]

With chronic inflammation and necrosis comes ischemiaof the lesions [31 153 161] Consequently there is reducedavailability of the host molecules that could counteractONOOminus (eg carbon dioxide hemoglobin and albumin)[69 70] Indeed it has been shown that in ischemic tissuesthere is a greater ONOOminus concentration [87]

73 Defense against ONOOminus Amoebas versus Host Cells Tobe able to provoke an uncontrolled inflammatory responsethe E histolytica defense system against ONOOminus would haveto be better than that of the host According to evidence inthe literature there are four important factors in this sense

Firstly since Prx-2 is an abundant erythrocyte pro-tein [109 147] E histolytica trophozoites may ingest high

quantities of Prx through erythrocyte phagocytosis as occurswith agents of other parasitic diseases [151] This would notbe the case for host cells Hence the quantity of this enzymein amoebas is probably greater than in mammals Moreovererythrocytes which are rich in hemoglobin (Hb) may helpthe amoeba to scavenge NO

Secondly in E histolytica Prx has a prominent surfaceand subcortical distribution [12 131ndash133] which probablymakes these trophozoitesmore capable of surviving the attackof ONOOminus produced externally Contrarily Prx is mainlylocated in the cytosol and mitochondria of hamsters

Thirdly the structure of mammalian cells leaves themmore susceptible to an ONOOminus attackThat is mitochondriaare found in mammalian cells but not in trophozoites andmitochondria play an essential role in the mechanisms of celldeath (by apoptosis and necrosis) triggered by ONOOminus [63]

Fourthly the effectiveness of Prx in mammals is com-pletely selenium dependent and plasma selenium concentra-tion and the synthesis of selenoproteins (probably includingTrxR) seem to be markedly reduced during inflammationevidenced by a severe drop in the concentration of theseproteins in the liver during sepsis or sepsis-like illness [162163] In addition if indeed selenium inhibits the activation ofNF-kappaB as has been reported [164] the deficit of seleniumwould tend to feed uncontrolled inflammation which wouldfurther reduce the levels of selenium in liver and serum

Most ingested selenium enters specific seleniummetabolism pathways in the liver thus providing theraw material for the synthesis of selenoproteins Onesuch protein produced in the liver is selenoprotein P


(SelP) whose main function is the transport of seleniumto remote tissues Additionally locally expressed SelPmay have other functions as demonstrated in the brain[163 165] One of these functions is the chelation of heavymetals presumably by forming nontoxic selenium-metalcomplexes thus preventing cell toxicity and protectingagainst ONOOminus mediated oxidation and nitration [165]A recent report indicated that SelP protects mice againsttrypanosomiasis [166] Therefore the reduction of seleniummetabolism and selenoprotein synthesis by hepatic damageshould certainly diminish the synthesis and activityof mammalian TrxR in hepatocytes and inflammatorycells

The apparent importance of the scarcity of selenium in theliver during the pathogenesis of ALA is further supported bythe effect of supplementation with selenium during chronicinflammationThe consequence is the restoration of depletedhepatic and serum selenium levels caused by an increasein selenoprotein biosynthesis leading to the suppression ofCRP production and an attenuation of the inflammatoryprocess [164] However selenium supplementation can alsoproduce undesirable effects such as increasing susceptibilityto infection by reducing inflammation or inducing sexu-ally dimorphic effects [167] The relationship between sele-nium selenoprotein synthesis and host-pathogen interac-tions is an important issue and warrants further investigation[168]

Hence EhPrx is considered to be an important protectivemechanism against two elements (i) the oxidative attack byhost phagocytic cells activated during the amebic invasionand (ii) the H

2

O2

produced by amebic metabolism [114]Ample evidence indicates that the protection afforded by Prxagainst ONOOminus is essential not only for the survival but alsofor the pathogenicity of trophozoites

8 Drug Design

The Trx system provides interesting drug design targetsThe structural and mechanistic differences between humanand P falciparum TrxR and more generally between themammalian and the bacterial TrxR shouldmake this proteina new drug design target for antibacterial agents [103 165169] Additionally the stimulation of host or the inhibitionof amebic Prx might be a useful strategy for the treatment ofamoebic liver abscess

This area of research is only beginning to be exploredRecently it has been reported that auranofin a gold-containing drug that inhibits TrxR has a higher amebi-cidal activity than metronidazole in vitro In a hamstermodel of amebic liver abscess orally administered aura-nofin markedly decreased the number of parasites thedetrimental host inflammatory response and hepatic dam-age However many trophozoites are resistant to auranofin[170]

Selenocysteine 496 of human TrxR is a major target ofthe antirheumatic gold-containing drug auranofin Althoughauranofin is a potent inhibitor of mammalian TrxR in vitro itis practically ineffective in vivo [171ndash173]

9 Perspective

Amoebiasis caused by E histolytica is an important publichealth problem in emerging countries It is generally thepoorest who have the greatest exposure to this amebic speciesand at the same time the greatest susceptibility Therefore itis important to understand the pathogenic mechanisms ofthis disease in order to define molecular targets and designeconomical drugs to act against such targets

The hypothesis of the ldquouncontrolled inflammatoryresponserdquo as the main cause of amoebiasis has been evolvingfor approximately three decades The understanding of themechanisms of chronic inflammation and their possiblerelation to amebic colitis and ALA may be reaching theproper depth of understanding in order to be able to finallyproduce novel therapies Intervention in the Trx systemof the host or parasite could possibly provide the long-sought solution to this public health problem In relationto E histolytica trophozoites the surface and subcorticaldistribution of Prx which provides this amebic species withmany advantages in terms of survival and virulence mightalso provide an easily accessible drug target

Conflict of Interests

Authors have no affiliations or financial involvement withany organization or entity that has a financial interest inor conflict of interests with the subject matter or materialsdiscussed in the paper

Acknowledgment

This work was supported in part by grants from SEPI-IPN Judith Pacheco-Yepez RosaAdriana Jarillo-Luna EdgarAbarca-Rojano and Rafael Campos-Rodriguez are fellows ofCOFAA-IPN and EDI-IPN

References

[1] V Tsutsumi and A Martinez-Palomo ldquoInflammatory reactionin experimental hepatic amebiasis An ultrastructural studyrdquoAmerican Journal of Pathology vol 130 no 1 pp 112ndash119 1988

[2] V Tsutsumi R Mena-Lopez F Anaya-Velazquez and AMartinez-Palomo ldquoCellular bases of experimental amebic liverabscess formationrdquo American Journal of Pathology vol 117 no1 pp 81ndash91 1984

[3] K Chadee W A Petri Jr D J Innes and J I Ravdin ldquoRat andhuman colonic mucins bind to and inhibit adherence lectin ofEntamoeba histolyticardquoThe Journal of Clinical Investigation vol80 no 5 pp 1245ndash1254 1987

[4] J I Ravdin C F Murphy R A Salata R L Guerrant and EL Hewlett ldquoN-acetyl-d-galactosamine-inhibitable adherencelectin of Entamoeba histolytica I Partial purification andrelation to amoebic virulence in vitrordquo Journal of InfectiousDiseases vol 151 no 5 pp 804ndash815 1985

[5] I Meza F Cazares J L Rosales-Encina P Talamas-Rohanaand M Rojkind ldquoUse of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolyticardquo Journalof Infectious Diseases vol 156 no 5 pp 798ndash805 1987


[6] GGarcıa-RiveraMA Rodrıguez ROcadiz et al ldquoEntamoebahistolytica a novel cysteine protease and an adhesin form the 112kDa surface proteinrdquoMolecular Microbiology vol 33 no 3 pp556ndash568 1999

[7] WA Petri Jr and J I Ravdin ldquoCytopathogenicity of Entamoebahistolytica the role of amebic adherence and contact-dependentcytolysis in pathogenesisrdquo European Journal of Epidemiologyvol 3 no 2 pp 123ndash136 1987

[8] C Martınez-Lopez E Orozco T Sanchez R M Garcıa-Perez F Hernandez-Hernandez and M A Rodrıguez ldquoTheEhADH112 recombinant polypeptide inhibits cell destructionand liver abscess formation by Entamoeba histolytica tropho-zoitesrdquo Cellular Microbiology vol 6 no 4 pp 367ndash376 2004

[9] R Ocadiz E Orozco E Carrillo et al ldquoEhCP112 is anEntamoeba histolytica secreted cysteine protease that may beinvolved in the parasite-virulencerdquo Cellular Microbiology vol7 no 2 pp 221ndash232 2005

[10] W A Petri Jr ldquoPathogenesis of amebiasisrdquo Current Opinion inMicrobiology vol 5 no 4 pp 443ndash447 2002

[11] M Espinosa-Cantellano and A Martınez-Palomo ldquoPathogen-esis of intestinal amebiasis from molecules to diseaserdquo ClinicalMicrobiology Reviews vol 13 no 2 pp 318ndash331 2000

[12] M A Hughes C W Lee C F Holm et al ldquoIdentification ofEntamoeba histolytica thiol-specific antioxidant as a GalNAclectin-associated proteinrdquoMolecular and Biochemical Parasitol-ogy vol 127 no 2 pp 113ndash120 2003

[13] C A Gilchrist and W A Petri Jr ldquoUsing differential geneexpression to study Entamoeba histolytica pathogenesisrdquo Trendsin Parasitology vol 25 no 3 pp 124ndash131 2009

[14] N C V Christy andW A Petri Jr ldquoMechanisms of adherencecytotoxicity and phagocytosis modulate the pathogenesis ofEntamoeba histolyticardquo Future Microbiology vol 6 no 12 pp1501ndash1519 2011

[15] W E Keene M E Hidalgo E Orozco and J H McKerrowldquoEntamoeba histolytica correlation of the cytopathic effect ofvirulent trophozoites with secretion of a cysteine proteinaserdquoExperimental Parasitology vol 71 no 2 pp 199ndash206 1990

[16] J Matthiesen A K Bar A K Bartels et al ldquoOverexpressionof specific cysteine peptidases confers pathogenicity to a non-pathogenic Entamoeba histolytica clonerdquo mBio vol 4 no 22013

[17] S L Stanley Jr T Zhang D Rubin and E Li ldquoRole ofthe Entamoeba histolytica cysteine proteinase in amebic liverabscess formation in severe combined immunodeficient micerdquoInfection and Immunity vol 63 no 4 pp 1587ndash1590 1995

[18] S Ankri T Stolarsky R Bracha F Padilla-Vaca and DMirelman ldquoAntisense inhibition of expression of cysteine pro-teinases affects Entamoeba histolytica-induced formation ofliver abscess in hamstersrdquo Infection and Immunity vol 67 no1 pp 421ndash422 1999

[19] M Tillack N Nowak H Lotter et al ldquoIncreased expression ofthe major cysteine proteinases by stable episomal transfectionunderlines the important role of EhCP5 for the pathogenicity ofEntamoeba histolyticardquoMolecular and Biochemical Parasitologyvol 149 no 1 pp 58ndash64 2006

[20] D Bansal P Ave S Kerneis et al ldquoAn ex-vivo human intestinalmodel to study Entamoeba histolytica pathogenesisrdquo PLoSNeglected Tropical Diseases vol 3 no 11 article e551 2009

[21] R D Horstmann M Leippe and E Tannich ldquoHost tissuedestruction by Entamoeba histolytica molecules mediatingadhesion cytolysis and proteolysisrdquo Memorias do InstitutoOswaldo Cruz vol 87 supplement 5 pp 57ndash60 1992

[22] X Que and S L Reed ldquoCysteine proteinases and the pathogen-esis of amebiasisrdquo Clinical Microbiology Reviews vol 13 no 2pp 196ndash206 2000

[23] S Pertuz Belloso P Ostoa Saloma I Benitez G Soldevila AOlivos and E Garcıa-Zepeda ldquoEntamoeba histolytica cysteineprotease 2 (EhCP2) modulates leucocyte migration by prote-olytic cleavage of chemokinesrdquoParasite Immunology vol 26 no5 pp 237ndash241 2004

[24] X Que S-H Kim M Sajid et al ldquoA surface amebic cysteineproteinase inactivates interleukin-18rdquo Infection and Immunityvol 71 no 3 pp 1274ndash1280 2003

[25] Z Zhang L Wang K B Seydel et al ldquoEntamoeba histolyticacysteine proteinases with interleukin-1 beta converting enzyme(ICE) activity cause intestinal inflammation and tissue damagein amoebiasisrdquo Molecular Microbiology vol 37 no 3 pp 542ndash548 2000

[26] C A Gilchrist and W A Petri Jr ldquoVirulence factors ofEntamoeba histolyticardquo Current Opinion in Microbiology vol 2no 4 pp 433ndash437 1999

[27] Y Hou L Mortimer and K Chadee ldquoEntamoeba histolyticacysteine proteinase 5 binds integrin on colonic cells andstimulates NF120581B-mediated pro-inflammatory responsesrdquo TheJournal of Biological Chemistry vol 285 no 46 pp 35497ndash35504 2010

[28] H Irmer M Tillack L Biller et al ldquoMajor cysteine peptidasesof Entamoeba histolytica are required for aggregation anddigestion of erythrocytes but are dispensable for phagocytosisand cytopathogenicityrdquo Molecular Microbiology vol 72 no 3pp 658ndash667 2009

[29] AOlivos-Garcıa E TelloMNequiz-Avendano et al ldquoCysteineproteinase activity is required for survival of the parasitein experimental acute amoebic liver abscesses in hamstersrdquoParasitology vol 129 no 1 pp 19ndash25 2004

[30] I Dey K Keller A Belley and K Chadee ldquoIdentificationand characterization of a cyclooxygenase-like enzyme fromEntamoeba histolyticardquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 23 pp13561ndash13566 2003

[31] R Campos-Rodrıguez R A Jarillo-Luna B A Larsen VRivera-Aguilar and J Ventura-Juarez ldquoInvasive amebiasis amicrocirculatory disorderrdquo Medical Hypotheses vol 73 no 5pp 687ndash697 2009

[32] J L Griffin ldquoHuman amebic dysentery Electron microscopyof Entamoeba histolytica contacting ingesting and digestinginflammatory cellsrdquo American Journal of Tropical Medicine andHygiene vol 21 no 6 pp 895ndash906 1972

[33] F Pittman J C Pittman and W K el-Hashimi ldquoHumanamebiasis Light and electron microscopy findings in colonicmucosal biopsies from patients with acute amebic colitisrdquo inProceedings of the International Conference on Amebiasis BSepulveda and L S Diamond Eds pp 398ndash417 Mexico CityMexico 1976

[34] S Blazquez M-C Rigothier M Huerre and N GuillenldquoInitiation of inflammation and cell death during liver abscessformation by Entamoeba histolytica depends on activity ofthe galactoseN-acetyl-d-galactosamine lectinrdquo InternationalJournal for Parasitology vol 37 no 3-4 pp 425ndash433 2007

[35] P Tavares M-C Rigothier H Khun P Roux M Huerre andN Guillen ldquoRoles of cell adhesion and cytoskeleton activityin Entamoeba histolytica pathogenesis a delicate balancerdquoInfection and Immunity vol 73 no 3 pp 1771ndash1778 2005


[36] J Aguirre-Garcıa ldquoHistopathological peculiarities of the ame-bic lesionrdquo Archivos de Investigacion Medica vol 1 supplementpp 147ndash156 1970

[37] K Chadee and E Meerovitch ldquoThe pathogenesis of experi-mentally induced amebic liver abscess in the gerbil (Merionesunguiculatus)rdquoAmerican Journal of Pathology vol 117 no 1 pp71ndash80 1984

[38] W B Lushbaugh A B Kairalla A F Hofbauer and F EPittman ldquoSequential histopathology of cavitary liver abscessformation induced by axenically grown Entamoeba histolyticardquoArchivos de InvestigacionMedica vol 11 no 1 pp 163ndash168 1980

[39] O Berninghausen and M Leippe ldquoNecrosis versus apoptosisas the mechanism of target cell death induced by Entamoebahistolyticardquo Infection and Immunity vol 65 no 9 pp 3615ndash36211997

[40] K B Seydel and S L Stanley Jr ldquoEntamoeba histolytica induceshost cell death in amebic liver abscess by a non-fas-dependentnon-tumor necrosis factor alpha-dependent pathway of apopto-sisrdquo Infection and Immunity vol 66 no 6 pp 2980ndash2983 1998

[41] C Velazquez M Shibayama-Salas J Aguirre-Garcia V Tsut-sumi and J Calderon ldquoRole of neutrophils in innate resis-tance to Entamoeba histolytica liver infection in micerdquo ParasiteImmunology vol 20 no 6 pp 255ndash262 1998

[42] A Olivos-Garcıa J C Carrero E Ramos et al ldquoLateexperimental amebic liver abscess in hamster is inhibited bycyclosporine and N-acetylcysteinerdquo Experimental and Molecu-lar Pathology vol 82 no 3 pp 310ndash315 2007

[43] R Perez-Tamayo R DMartinez IMontfort I Becker E Telloand R Perez-Montfort ldquoPathogenesis of acute experimentalamebic liver abscess in hamstersrdquo Journal of Parasitology vol77 no 6 pp 982ndash988 1991

[44] R Perez-Tamayo I Montfort A O Garcıa E Ramos and CB Ostria ldquoPathogenesis of acute experimental liver amebiasisrdquoArchives of Medical Research vol 37 no 2 pp 203ndash209 2006

[45] R B I Perez-Tamayo I Montfort and R Perez-MontfortldquoPathobiology of amebiasisrdquo inAmebiasis Infection and Diseaseby Entamoeba Histolytica R R Krestchmer Ed pp 123ndash157CRC Press Boca Raton Fla USA 1990

[46] S L Stanley Jr ldquoPathophysiology of amoebiasisrdquo Trends inParasitology vol 17 no 6 pp 280ndash285 2001

[47] A Olivos-Garcıa E Saavedra E Ramos-Martınez M Nequizand R Perez-Tamayo ldquoMolecular nature of virulence in Enta-moeba histolyticardquo Infection Genetics and Evolution vol 9 no6 pp 1033ndash1037 2009

[48] E Ramos A Olivos-Garcıa M Nequiz et al ldquoEntamoebahistolytica apoptosis induced in vitro by nitric oxide speciesrdquoExperimental Parasitology vol 116 no 3 pp 257ndash265 2007

[49] L Mortimer and K Chadee ldquoThe immunopathogenesis ofEntamoeba histolyticardquo Experimental Parasitology vol 126 no3 pp 366ndash380 2010

[50] K S Ralston and W A Petri Jr ldquoTissue destruction andinvasion by Entamoeba histolyticardquo Trends in Parasitology vol27 no 6 pp 254ndash263 2011

[51] J Santi-Rocca M-C Rigothier and N Guillen ldquoHost-microbeinteractions and defense mechanisms in the development ofamoebic liver abscessesrdquo Clinical Microbiology Reviews vol 22no 1 pp 65ndash75 2009

[52] J Pacheco-Yepez R Campos-Rodrıguez M Shibayama JVentura-Juarez J Serrano-Luna and V Tsutsumi ldquoEntamoebahistolytica production of nitric oxide and in situ activityof NADPH diaphorase in amebic liver abscess of hamstersrdquoParasitology Research vol 87 no 1 pp 49ndash56 2001

[53] J Ramırez-Emiliano A Gonzalez-Hernandez and S Arias-Negrete ldquoExpression of inducible nitric oxide synthase mRNAand nitric oxide production during the development of liverabscess in hamster inoculated with Entamoeba histolyticardquoCurrent Microbiology vol 50 no 6 pp 299ndash308 2005

[54] J Y Lin and K Chadee ldquoMacrophage cytotoxicity againstEntamoeba histolytica trophozoites is mediated by nitric oxidefrom L-argininerdquo Journal of Immunology vol 148 no 12 pp3999ndash4005 1992

[55] J-Y Lin R Seguin K Keller and K Chadee ldquoTumor necrosisfactor alpha augments nitric oxide-dependent macrophagecytotoxicity against Entamoeba histolytica by enhanced expres-sion of the nitric oxide synthase generdquo Infection and Immunityvol 62 no 5 pp 1534ndash1541 1994

[56] R A Jarillo-Luna R Campos-Rodrıguez and V TsutsumildquoEntamoeba histolytica immunohistochemical study of hepaticamoebiasis in mouse Neutrophils and nitric oxide as possiblefactors of resistancerdquo Experimental Parasitology vol 101 no 1pp 40ndash56 2002

[57] E Ghadirian S D Somerfield and P A L KongshavnldquoSusceptibility of Entamoeba histolytica to oxidantsrdquo Infectionand Immunity vol 51 no 1 pp 263ndash267 1986

[58] H W Murray S B Aley and W A Scott ldquoSusceptibility ofEntamoeba histolytica to oxygen intermediatesrdquo Molecular andBiochemical Parasitology vol 3 no 6 pp 381ndash391 1981

[59] R A Salata R D Pearson and J I Ravdin ldquoInteraction ofhuman leukocytes and Entamoeba histolytica killing of virulentamebae by the activated macrophagerdquo The Journal of ClinicalInvestigation vol 76 no 2 pp 491ndash499 1985

[60] K B Seydel S J Smith and S L Stanley Jr ldquoInnate immunityto amebic liver abscess is dependent on gamma interferonand nitric oxide in a murine model of diseaserdquo Infection andImmunity vol 68 no 1 pp 400ndash402 2000

[61] R Siman-Tov and S Ankri ldquoNitric oxide inhibits cysteine pro-teinases and alcohol dehydrogenase 2 of Entamoeba histolyticardquoParasitology Research vol 89 no 2 pp 146ndash149 2003

[62] J Santi-Rocca S Smith C Weber et al ldquoEndoplasmic retic-ulum stress-sensing mechanism is activated in Entamoebahistolytica upon treatment with nitric oxiderdquo PLoS ONE vol 7no 2 Article ID e31777 2012

[63] P Pacher J S Beckman and L Liaudet ldquoNitric oxide andperoxynitrite in health and diseaserdquo Physiological Reviews vol87 no 1 pp 315ndash424 2007

[64] M-H Choi D Sajed L Poole et al ldquoAn unusual surfaceperoxiredoxin protects invasive Entamoeba histolytica fromoxidant attackrdquo Molecular and Biochemical Parasitology vol143 no 1 pp 80ndash89 2005

[65] M A Akbar N S Chatterjee P Sen et al ldquoGenes induced by ahigh-oxygen environment in Entamoeba histolyticardquoMolecularand Biochemical Parasitology vol 133 no 2 pp 187ndash196 2004

[66] B Loftus I Anderson R Davies et al ldquoThe genome of theprotist parasite Entamoeba histolyticardquo Nature vol 433 no7028 pp 865ndash868 2005

[67] R C MacFarlane and U Singh ldquoIdentification of differentiallyexpressed genes in virulent and nonvirulent Entamoeba speciespotential implications for amebic pathogenesisrdquo Infection andImmunity vol 74 no 1 pp 340ndash351 2006

[68] S Goldstein and G Merenyi ldquoThe chemistry of peroxynitriteimplications for biological activityrdquoMethods in Enzymology vol436 pp 49ndash61 2008


[69] C Szabo H Ischiropoulos and R Radi ldquoPeroxynitrite bio-chemistry pathophysiology and development of therapeuticsrdquoNature Reviews Drug Discovery vol 6 no 8 pp 662ndash680 2007

[70] R Radi A Denicola B Alvarez G Ferrer-Sueta andH RubboldquoThe biological chemistry of peroxynitriterdquo in Nitric OxideBiology and Pathobiology L J Ignarro Ed pp 57ndash82 AcademicPress San Diego Calif USA 2000

[71] M E Quintanar-Quintanar A Jarillo-Luna V Rivera-Aguilaret al ldquoImmunosuppressive treatment inhibits the developmentof amebic liver abscesses in hamstersrdquoMedical Science Monitorvol 10 no 9 pp BR317ndashBR324 2004

[72] J-M Li and A M Shah ldquoEndothelial cell superoxide genera-tion regulation and relevance for cardiovascular pathophysi-ologyrdquo American Journal of PhysiologymdashRegulatory Integrativeand Comparative Physiology vol 287 no 5 pp R1014ndashR10302004

[73] W Droge ldquoFree radicals in the physiological control of cellfunctionrdquo Physiological Reviews vol 82 no 1 pp 47ndash95 2002

[74] W Wang K Keller and K Chadee ldquoEntamoeba histolyticamodulates the nitric oxide synthase gene and nitric oxideproduction by macrophages for cytotoxicity against amoebaeand tumour cellsrdquo Immunology vol 83 no 4 pp 601ndash610 1994

[75] S Moncada ldquoNitric oxide discovery and impact on clinicalmedicinerdquo Journal of the Royal Society of Medicine vol 92 no4 pp 164ndash169 1999

[76] Y Xia and J L Zweier ldquoSuperoxide and peroxynitrite gener-ation from inducible nitric oxide synthase in macrophagesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 13 pp 6954ndash6958 1997

[77] J E Albina M D Caldwell W L Henry Jr and C D MillsldquoRegulation of macrophage functions by L-argininerdquo Journal ofExperimental Medicine vol 169 no 3 pp 1021ndash1029 1989

[78] J E Albina W L Henry Jr B Mastrofrancesco B-A Martinand J S Reichner ldquoMacrophage activation by culture in ananoxic environmentrdquo Journal of Immunology vol 155 no 9 pp4391ndash4396 1995

[79] J E Albina C D Mills W L Henry Jr and M D CaldwellldquoRegulation of macrophage physiology by L-arginine roleof the oxidative L-arginine deiminase pathwayrdquo Journal ofImmunology vol 143 no 11 pp 3641ndash3646 1989

[80] J E Albina C D Mills W L Henry Jr and M D Cald-well ldquoTemporal expression of different pathways of l-argininemetabolism in healing woundsrdquo Journal of Immunology vol144 no 10 pp 3877ndash3880 1990

[81] M A Robinson J E Baumgardner V P Good and C M OttoldquoPhysiological and hypoxic O

2

tensions rapidly regulate NOproduction by stimulated macrophagesrdquo American Journal ofPhysiologymdashCell Physiology vol 294 no 4 pp C1079ndashC10872008

[82] C Peyssonnaux V Datta T Cramer et al ldquoHIF-1120572 expressionregulates the bactericidal capacity of phagocytesrdquoThe Journal ofClinical Investigation vol 115 no 7 pp 1806ndash1815 2005

[83] V Nizet and R S Johnson ldquoInterdependence of hypoxic andinnate immune responsesrdquo Nature Reviews Immunology vol 9no 9 pp 609ndash617 2009

[84] K Krotova J M Patel E R Block and S Zharikov ldquoHypoxicupregulation of arginase II in human lung endothelial cellsrdquoAmerican Journal of PhysiologymdashCell Physiology vol 299 no6 pp C1541ndashC1548 2010

[85] W Durante F K Johnson and R A Johnson ldquoArginase a crit-ical regulator of nitric oxide synthesis and vascular functionrdquo

Clinical and Experimental Pharmacology and Physiology vol 34no 9 pp 906ndash911 2007

[86] Y Xia V L Dawson T M Dawson S H Snyder and JL Zweier ldquoNitric oxide synthase generates superoxide andnitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injuryrdquo Proceedings of the National Academyof Sciences of the United States of America vol 93 no 13 pp6770ndash6774 1996

[87] G E Arteel M B Kadiiska I Rusyn et al ldquoOxidativestress occurs in perfused rat liver at low oxygen tension bymechanisms involving peroxynitriterdquoMolecular Pharmacologyvol 55 no 4 pp 708ndash715 1999

[88] C Szabo ldquoPathopysiological roles of nitric oxide in inflamma-tionrdquo in Nitric Oxide Biology and Pathobiology L J IgnarroEd pp 841ndash872 Academic Press San Diego Calif USA 2000

[89] YGao ldquoThemultiple actions ofNOrdquoPflugers ArchivmdashEuropeanJournal of Physiology vol 459 no 6 pp 829ndash839 2010

[90] G E Arteel K Briviba and H Sies ldquoProtection againstperoxynitriterdquo FEBS Letters vol 445 no 2-3 pp 226ndash230 1999

[91] L-O Klotz andH Sies ldquoDefenses against peroxynitrite seleno-compounds and flavonoidsrdquo Toxicology Letters vol 140-141 pp125ndash132 2003

[92] D G Arias E L Regner A A Iglesias and S A GuerreroldquoEntamoeba histolytica thioredoxin reductase molecular andfunctional characterization of its atypical propertiesrdquo Biochim-ica et Biophysica Acta vol 1820 pp 1859ndash1866 2012

[93] L Ignarro ldquoIntroduction and overviewrdquo inNitric Oxide Biologyand Pathobiology L Ignarro Ed pp 3ndash19 Academic Press SanDiego Calif USA 2000

[94] J R Lancaster Jr ldquoSimulation of the diffusion and reactionof endogenously produced nitric oxiderdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 91 no 17 pp 8137ndash8141 1994

[95] X Liu Q Yan K L Baskerville and J L Zweier ldquoEstimationof nitric oxide concentration in blood for different rates ofgeneration evidence that intravascular nitric oxide levels aretoo low to exert physiological effectsrdquo The Journal of BiologicalChemistry vol 282 no 12 pp 8831ndash8836 2007

[96] X Liu M J S Miller M S Joshi D D Thomas and JR Lancaster Jr ldquoAccelerated reaction of nitric oxide withO2

within the hydrophobic interior of biological membranesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 5 pp 2175ndash2179 1998

[97] K L Davis E Martin I V Turko and F Murad ldquoNovel effectsof nitric oxiderdquoAnnual Review of Pharmacology and Toxicologyvol 41 pp 203ndash236 2001

[98] G Rico E Leandro S Rojas J A Gimenez and R RKretschmer ldquoThe monocyte locomotion inhibitory factor pro-duced by Entamoeba histolytica inhibits induced nitric oxideproduction in human leukocytesrdquoParasitology Research vol 90no 4 pp 264ndash267 2003

[99] K Elnekave R Siman-Tov and S Ankri ldquoConsumption of L-argininemediated by Entamoeba histolytica L-arginase (EhArg)inhibits amoebicidal activity and nitric oxide production byactivated macrophagesrdquo Parasite Immunology vol 25 no 11-12pp 597ndash608 2003

[100] D G Arias C E Gutierrez A A Iglesias and S A GuerreroldquoThioredoxin-linked metabolism in Entamoeba histolyticardquoFree Radical Biology andMedicine vol 42 no 10 pp 1496ndash15052007


[101] S G Rhee H Z Chae andK Kim ldquoPeroxiredoxins a historicaloverview and speculative preview of novel mechanisms andemerging concepts in cell signalingrdquo Free Radical Biology andMedicine vol 38 no 12 pp 1543ndash1552 2005

[102] E S J Arner ldquoFocus on mammalian thioredoxin reductasesmdashimportant selenoproteins with versatile functionsrdquo Biochimicaet Biophysica Acta vol 1790 no 6 pp 495ndash526 2009

[103] C H Williams Jr L D Arscott S Muller et al ldquoThioredoxinreductase two modes of catalysis have evolvedrdquo EuropeanJournal of Biochemistry vol 267 no 20 pp 6110ndash6117 2000

[104] A Holmgren and J Lu ldquoThioredoxin and thioredoxin reduc-tase current research with special reference to human diseaserdquoBiochemical andBiophysical ResearchCommunications vol 396no 1 pp 120ndash124 2010

[105] D G Arias P G Carranza H D Lujan A A Iglesias and S AGuerrero ldquoImmunolocalization and enzymatic functional char-acterization of the thioredoxin system inEntamoeba histolyticardquoFree Radical Biology andMedicine vol 45 no 1 pp 32ndash39 2008

[106] M K Sawyer J M Bischoff M A Guidry and R E ReevesldquoLipids from Entamoeba histolyticardquo Experimental Parasitologyvol 20 no 3 pp 295ndash302 1967

[107] H Z Chae S J Chung and S G Rhee ldquoThioredoxin-dependent peroxide reductase from yeastrdquo The Journal ofBiological Chemistry vol 269 no 44 pp 27670ndash27678 1994

[108] ZAWood E Schroder J RobinHarris and L B Poole ldquoStruc-ture mechanism and regulation of peroxiredoxinsrdquo Trends inBiochemical Sciences vol 28 no 1 pp 32ndash40 2003

[109] E Schroder J A Littlechild A A Lebedev N Errington AA Vagin and M N Isupov ldquoCrystal structure of decameric 2-cys peroxiredoxin from human erythrocytes at 17 a resolutionrdquoStructure vol 8 pp 605ndash615 2000

[110] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[111] I Bruchhaus S Richter and E Tannich ldquoRemoval of hydrogenperoxide by the 29 kDa protein of Entamoeba histolyticardquoBiochemical Journal vol 326 part 3 pp 785ndash789 1997

[112] B M Flores M A Batzer M A Stein C Petersen D LDiedrich and B E Torian ldquoStructural analysis and demonstra-tion of the 29 kDa antigen of pathogenic Entamoeba histolyticaas the major accessible free thiol-containing surface proteinrdquoMolecular Microbiology vol 7 no 5 pp 755ndash763 1993

[113] B M Flores S L Reed J I Ravdin and B E Torian ldquoSerologicreactivity to purified recombinant and native 29-kilodaltonperipheral membrane protein of pathogenic Entamoeba his-tolyticardquo Journal of ClinicalMicrobiology vol 31 no 6 pp 1403ndash1407 1993

[114] X-J Cheng E Yoshihara T Takeuchi and H TachibanaldquoMolecular characterization of peroxiredoxin from Entamoebamoshkovskii and a comparison with Entamoeba histolyticardquoMolecular and Biochemical Parasitology vol 138 no 2 pp 195ndash203 2004

[115] L B Poole H Z Chae B M Flores S L Reed S G Rheeand B E Torian ldquoPeroxidase activity of a TSA-like antioxidantprotein from a pathogenic amoebardquo Free Radical Biology andMedicine vol 23 no 6 pp 955ndash959 1997

[116] P H Davis X Zhang J Guo R R Townsend and S LStanley Jr ldquoComparative proteomic analysis of two Entamoebahistolytica strains with different virulence phenotypes identifiesperoxiredoxin as an important component of amoebic viru-lencerdquo Molecular Microbiology vol 61 no 6 pp 1523ndash15322006

[117] E Ramos-Martınez A Olivos-Garcıa E Saavedra et al ldquoEnta-moeba histolytica oxygen resistance and virulencerdquo Interna-tional Journal for Parasitology vol 39 no 6 pp 693ndash702 2009

[118] A Sen N S Chatterjee M A Akbar N Nandi and P DasldquoThe 29-kilodalton thiol-dependent peroxidase of Entamoebahistolytica is a factor involved in pathogenesis and survival ofthe parasite during oxidative stressrdquo Eukaryotic Cell vol 6 no4 pp 664ndash673 2007

[119] I Bruchhaus T Roeder H Lotter M Schwerdtfeger and ETannich ldquoDifferential gene expression in Entamoeba histolyticaisolated from amoebic liver abscessrdquo Molecular Microbiologyvol 44 no 4 pp 1063ndash1072 2002

[120] E R Houpt D J Glembocki T G Obrig et al ldquoThe mousemodel of amebic colitis reveals mouse strain susceptibility toinfection and exacerbation of disease by CD4+ T cellsrdquo Journalof Immunology vol 169 no 8 pp 4496ndash4503 2002

[121] J Santi-Rocca C Weber G Guigon O Sismeiro J-Y Coppeeand N Guillen ldquoThe lysine- and glutamic acid-rich proteinKERP1 plays a role in Entamoeba histolytica liver abscesspathogenesisrdquo Cellular Microbiology vol 10 no 1 pp 202ndash2172008

[122] B Jimenez-Delgadillo P P Chaudhuri L Baylon-Pacheco ALopez-Monteon P Talamas-Rohana and J L Rosales-EncinaldquoEntamoeba histolytica cDNAs cloned as 30 kDa collagen-binding proteins (CBP) belong to an antioxidant moleculefamily Protection of hamsters from amoebic liver abscess byimmunization with recombinant CBPrdquo Experimental Parasitol-ogy vol 108 no 1-2 pp 7ndash17 2004

[123] M C Gonzalez-Vazquez A Carabarin-Lima L Baylon-Pacheco P Talamas-Rohana and J L Rosales-Encina ldquoObtain-ing of three recombinant antigens of Entamoeba histolytica andevaluation of their immunogenic ability without adjuvant in ahamster model of immunoprotectionrdquo Acta Tropica vol 122no 2 pp 169ndash176 2012

[124] C-J G Soong B E Torian M D Abd-Alla T F H G JacksonVGatharim and J I Ravdin ldquoProtection of gerbils from amebicliver abscess by immunization with recombinant Entamoebahistolytica 29-kilodalton antigenrdquo Infection and Immunity vol63 no 2 pp 472ndash477 1995

[125] Z A Wood L B Poole R R Hantgan and P A KarplusldquoDimers to doughnuts redox-sensitive oligomerization of 2-cysteine peroxiredoxinsrdquo Biochemistry vol 41 no 17 pp 5493ndash5504 2002

[126] J R Harris E Schroder M N Isupov et al ldquoComparison ofthe decameric structure of peroxiredoxin-II by transmissionelectron microscopy and X-ray crystallographyrdquo Biochimica etBiophysica Acta vol 1547 no 2 pp 221ndash234 2001

[127] R Bryk P Griffin and C Nathan ldquoPeroxynitrite reductaseactivity of bacterial peroxiredoxinsrdquo Nature vol 407 no 6801pp 211ndash215 2000

[128] M Trujillo H Budde M D Pineyro et al ldquoTrypanosomabrucei and Trypanosoma cruzi tryparedoxin peroxidases cat-alytically detoxify peroxynitrite via oxidation of fast reactingthiolsrdquoThe Journal of Biological Chemistry vol 279 no 33 pp34175ndash34182 2004

[129] M Trujillo G Ferrer-Sueta and R Radi ldquoKinetic studieson peroxynitrite reduction by peroxiredoxinsrdquo Methods inEnzymology vol 441 pp 173ndash196 2008

[130] M Dubuisson D Vander Stricht A Clippe et al ldquoHumanperoxiredoxin 5 is a peroxynitrite reductaserdquo FEBS Letters vol571 no 1ndash3 pp 161ndash165 2004


[131] H Tachibana S Kobayashi Y Kato K Nagakura Y Kanedaand T Takeuchi ldquoIdentification of a pathogenic isolate-specific30000-M(r) antigen of Entamoeba histolytica by using a mono-clonal antibodyrdquo Infection and Immunity vol 58 no 4 pp 955ndash960 1990

[132] B E Torian B M Flores V L Stroeher F S Hagen and WE Stamm ldquocDNA sequence analysis of a 29-kDa cysteine-richsurface antigen of pathogenic Entamoeba histolyticardquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 87 no 16 pp 6358ndash6362 1990

[133] S L Reed B M Flores M A Batzer et al ldquoMolecularand cellular characterization of the 29-kilodalton peripheralmembrane protein of Entamoeba histolytica differentiationbetween pathogenic and nonpathogenic isolatesrdquo Infection andImmunity vol 60 no 2 pp 542ndash549 1992

[134] J O Andersson R P Hirt P G Foster and A J RogerldquoEvolution of four gene families with patchy phylogeneticdistributions influx of genes into protist genomesrdquo BMCEvolutionary Biology vol 6 article 27 2006

[135] A diMatteo FM Scandurra F Testa et al ldquoTheO2

-scavengingflavodiiron protein in the human parasite Giardia intestinalisrdquoThe Journal of Biological Chemistry vol 283 no 7 pp 4061ndash4068 2008

[136] P Sarti P L Fiori E Forte et al ldquoTrichomonas vaginalisdegrades nitric oxide and expresses a flavorubredoxin-likeprotein a new pathogenic mechanismrdquo Cellular andMolecularLife Sciences vol 61 no 5 pp 618ndash623 2004

[137] J B Vicente F Testa D Mastronicola et al ldquoRedox propertiesof the oxygen-detoxifying flavodiiron protein from the humanparasite Giardia intestinalisrdquo Archives of Biochemistry andBiophysics vol 488 no 1 pp 9ndash13 2009

[138] T Smutna V L Goncalves L M Saraiva J Tachezy MTeixeira and I Hrdy ldquoFlavodiiron protein from Trichomonasvaginalis hydrogenosomes the terminal oxygen reductaserdquoEukaryotic Cell vol 8 no 1 pp 47ndash55 2009

[139] J B Vicente M A Carrondo M Teixeira and C FrazaoldquoStructural studies on flavodiiron proteinsrdquo Methods in Enzy-mology vol 437 pp 3ndash19 2008

[140] C M Gomes A Giuffre E Forte et al ldquoA novel type of nitric-oxide reductase Escherichia coli flavorubredoxinrdquoThe Journalof Biological Chemistry vol 277 no 28 pp 25273ndash25276 2002

[141] J B Vicente V Tran L Pinto M Teixeira and U SinghldquoA detoxifying oxygen reductase in the anaerobic protozoanEntamoeba histolyticardquo Eukaryotic Cell vol 11 pp 1112ndash11182012

[142] V Tsutsumi A Ramirez-Rosales H Lanz-Mendoza et alldquoEntamoeba histolytica erythrophagocytosis collagenolysisand liver abscess production as virulencemarkersrdquoTransactionsof the Royal Society of TropicalMedicine andHygiene vol 86 no2 pp 170ndash172 1992

[143] K K Hirata X Que S G Melendez-Lopez et al ldquoA phago-cytosis mutant of Entamoeba histolytica is less virulent dueto deficient proteinase expression and releaserdquo ExperimentalParasitology vol 115 no 2 pp 192ndash199 2007

[144] R M Mukherjee K C Bhol S Mehra T K Maitra andK N Jalan ldquoStudy of Entamoeba histolytica isolates fromCalcutta with special reference to their zymodeme animalpathogenicity and erythrophagocytosisrdquo Journal of DiarrhoealDiseases Research vol 9 no 1 pp 11ndash15 1991

[145] E Orozco G Guarneros A Martinez-Palomo and T SanchezldquoEntamoeba histolytica Phagocytosis as a virulence factorrdquo

Journal of Experimental Medicine vol 158 no 5 pp 1511ndash15211983

[146] L A Baxt and U Singh ldquoNew insights into Entamoeba histolyt-ica pathogenesisrdquoCurrent Opinion in Infectious Diseases vol 21no 5 pp 489ndash494 2008

[147] B L Tekwani and R K Mehlotra ldquoMolecular basis of defenceagainst oxidative stress in Entamoeba histolytica and GiardialambliardquoMicrobes and Infection vol 1 no 5 pp 385ndash394 1999

[148] R Bracha andDMirelman ldquoVirulence ofEntamoeba histolyticatrophozoites Effects of bacteria microaerobic conditions andmetronidazolerdquo Journal of Experimental Medicine vol 160 no2 pp 353ndash368 1984

[149] D Trissl A Martinez-Palomo and M de la Torre ldquoSurfaceproperties of Entamoeba increased rates of human erythrocytephagocytosis in pathogenic strainsrdquo Journal of ExperimentalMedicine vol 148 no 5 pp 1137ndash1145 1978

[150] J M Galvan-Moroyoqui M del Carmen Domınguez-RoblesE Franco and I Meza ldquoThe interplay between Entamoeba andenteropathogenic bacteria modulates epithelial cell damagerdquoPLoS Neglected Tropical Diseases vol 2 no 7 article e266 2008

[151] S Koncarevic P Rohrbach M Deponte et al ldquoThe malarialparasite Plasmodium falciparum imports the human proteinperoxiredoxin 2 for peroxide detoxificationrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 32 pp 13323ndash13328 2009

[152] L C Pelosof P H Davis Z Zhang X Zhang and S L StanleyJr ldquoCo-ordinate but disproportionate activation of apoptoticregenerative and inflammatory pathways characterizes the liverresponse to acute amebic infectionrdquo Cellular Microbiology vol8 no 3 pp 508ndash522 2006

[153] A Olivos-Garcıa M Nequiz-Avendano E Tello et al ldquoInflam-mation complement ischemia and amoebic survival in acuteexperimental amoebic liver abscesses in hamstersrdquo Experimen-tal and Molecular Pathology vol 77 no 1 pp 66ndash71 2004

[154] A Cabassi E C Dumont H Girouard et al ldquoEffects of chronicN-acetylcysteine treatment on the actions of peroxynitriteon aortic vascular reactivity in hypertensive ratsrdquo Journal ofHypertension vol 19 no 7 pp 1233ndash1244 2001

[155] M ZafarullahW Q Li J Sylvester andM Ahmad ldquoMolecularmechanisms of N-acetylcysteine actionsrdquo Cellular and Molecu-lar Life Sciences vol 60 no 1 pp 6ndash20 2003

[156] R Campos-Rodrıguezp andA Jarillo-Luna ldquoThepathogenicityof Entamoeba histolytica is related to the capacity of evadinginnate immunityrdquo Parasite Immunology vol 27 no 1-2 pp 1ndash8 2005

[157] M-C Rigothier H Khun P Tavares A Cardona M Huerreand N Guillen ldquoFate of Entamoeba histolytica during estab-lishment of amoebic liver abscess analyzed by quantitativeradioimaging and histologyrdquo Infection and Immunity vol 70no 6 pp 3208ndash3215 2002

[158] E Helk H Bernin T Ernst et al ldquoTnfalpha-mediated liverdestruction by kupffer cells and ly6chi monocytes duringEntamoeba histolytica infectionrdquo PLoS Pathogens vol 9 ArticleID e1003096 2013

[159] C Maldonado-Bernal C J Kirschning Y Rosenstein et alldquoThe innate immune response toEntamoeba histolytica lipopep-tidophosphoglycan is mediated by toll-like receptors 2 and 4rdquoParasite Immunology vol 27 no 4 pp 127ndash137 2005

[160] M Sharma H Vohra and D Bhasin ldquoEnhanced pro-inflammatory chemokinecytokine response triggered bypathogenic Entamoeba histolytica basis of invasive diseaserdquoParasitology vol 131 no 6 pp 783ndash796 2005


[161] R Perez-Tamayo I Montfort E Tello and A Olivos ldquoIschemiain experimental acute amebic liver abscess in hamstersrdquo Inter-national Journal for Parasitology vol 22 no 1 pp 125ndash129 1992

[162] X Forceville D Vitoux R Gauzit A Combes P Lahilaire andP Chappuis ldquoSelenium systemic immune response syndromesepsis and outcome in critically ill patientsrdquo Critical CareMedicine vol 26 no 9 pp 1536ndash1544 1998

[163] R F Burk and K E Hill ldquoSelenoprotein P an extracellularprotein with unique physical characteristics and a role inselenium homeostasisrdquo Annual Review of Nutrition vol 25 pp215ndash235 2005

[164] L H Duntas ldquoSelenium and inflammation underlying anti-inflammatory mechanismsrdquo Hormone and Metabolic Researchvol 41 no 6 pp 443ndash447 2009

[165] L V Papp J Lu A Holmgren and K K Khanna ldquoFromselenium to selenoproteins synthesis identity and their role inhuman healthrdquo Antioxidants and Redox Signaling vol 9 no 7pp 775ndash806 2007

[166] T Bosschaerts M Guilliams W Noel et al ldquoAlternativelyactivated myeloid cells limit pathogenicity associated withAfrican trypanosomiasis through the IL-10 inducible geneselenoprotein Prdquo Journal of Immunology vol 180 no 9 pp6168ndash6175 2008

[167] ZHuangAH Rose andP RHoffmann ldquoThe role of seleniumin inflammation and immunity from molecular mechanismsto therapeutic opportunitiesrdquoAntioxidants andRedox Signalingvol 16 no 7 pp 705ndash743 2012

[168] K Renko P J Hofmann M Stoedter et al ldquoDown-regulationof the hepatic selenoprotein biosynthesis machinery impairsseleniummetabolism during the acute phase response in micerdquoThe FASEB Journal vol 23 no 6 pp 1758ndash1765 2009

[169] K Becker S Gromer R H Schirmer and S Muller ldquoThiore-doxin reductase as a pathophysiological factor and drug targetrdquoEuropean Journal of Biochemistry vol 267 no 20 pp 6118ndash61252000

[170] A Debnath D Parsonage R M Andrade et al ldquoA high-throughput drug screen for Entamoeba histolytica identifies anew lead and targetrdquo Nature Medicine vol 18 no 6 pp 956ndash960 2012

[171] O Rackham A-M J Shearwood RThyer et al ldquoSubstrate andinhibitor specificities differ between human cytosolic andmito-chondrial thioredoxin reductases implications for developmentof specific inhibitorsrdquo Free Radical Biology andMedicine vol 50no 6 pp 689ndash699 2011

[172] A Bindoli M P Rigobello G Scutari C Gabbiani A Casiniand L Messori ldquoThioredoxin reductase a target for goldcompounds acting as potential anticancer drugsrdquo CoordinationChemistry Reviews vol 253 no 11-12 pp 1692ndash1707 2009

[173] C F Shaw III ldquoGold-based therapeutic agentsrdquo ChemicalReviews vol 99 no 9 pp 2589ndash2600 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research



Microbiology


The principal mechanism for ONOOminus interception seemsto be the thioredoxin system The aim of present text is todefine the importance of ONOOminus as the main agent of liverabscess formation during amebic invasion and to explainthe apparently superior capacity of the amoeba to defenditself against this toxic agent through the peroxiredoxin andthioredoxin system

2 Adhesion and Cytotoxic Activity of Amoebasduring Amoebiasis and ALA

There is still sharp controversy among scientists today regard-ing the molecular mechanisms of amoebiasis and amebicliver invasion caused by the pathogen E histolytica Someresearchers pose that the direct damage to liver tissue byamoebas (through mechanisms of adherence and toxicity)causes the formation of liver abscess during amoebiasisOther researchers assert that the importance of amebicmechanisms in the initial stage of amoebiasis is to allow thepathogens to survive and multiply in microenvironmentsthus provoking the chronic inflammatory response thatprovides the principal mechanisms of damage to liver tissueeventually resulting inALA To a great extent the controversyhinges on an ambiguous use of language on both sides of thequestion

Adherence of amoebas to the colonic epithelium andother host cells is unquestionably of fundamental importancein amoebiasis during the initial stages of the pathogene-sis which include the initial infection and extraintestinalinvasion That is adherence is essential for the amoebato invade the host and establishes itself in microenviron-ments in which it can survive The virulence factor ofadherence is to a great extent mediated by the N-acetyl-D-galactosamine inhibitable (GalGalNAc) lectin [3 4]the 220 kDa lectin [5] and other adhesins such as the112 kDa adhesin [6] There are reports of reduced ame-bic adherence to human erythrocytes neutrophils colonicmucins and epithelia when amebic lectins are inhibited[5ndash7] Recently it was shown that EhCPADH a complexformed by a cysteine proteinase and an adhesin mediatesadherence phagocytosis and cytolysis Immunization witha recombinant polypeptide EhADH243 induces protectionin hamsters against development of ALA [8] It has alsobeen demonstrated that the recombinant enzyme rEhCP112digested gelatin collagen type I fibronectin hemoglobinand Madin-Darby canine kidney (MDCK) cell monolay-ers the EhCP112 enzyme is secreted by the trophozoites[9]

The GalGalNAc lectin is a virulence factor required fortrophozoite adherence to target cells as shown by inhibitionof this lectin with galactose [10 11] cell killing occurs in thetypical GalGalNAc manner It has been shown that perox-iredoxin (Prx) interacts with GalGalNAc lectin and that thelectin-peroxiredoxin complex is located at the amoeba-hostcell contact sites Therefore the recruitment of Prx probablyprotects the trophozoites against the ROS generated by hostcells (phagocytic and epithelial) which would facilitate theinvasion [12 13]

Molecular mechanisms of pathogenicity that allow Ehistolytica trophozoites to survive and proliferate within awide variety of host environments are related not only toadherence but also to cytotoxic activity Trophozoites candamage host cells through direct contact or close proximityas well as through phagocytic activity towards dead anddying host cells in a receptor-mediated fashion [14] Ofthe molecules secreted by amoebas cysteine proteases areparticularly important [15ndash20] They are responsible for acytolytic effect on host cells [7] the modulation of the cell-mediated immune response and the proteolysis of the hostextracellular matrix [16 21ndash25] Previous works show thatwhen mutated cysteine proteinase 5 (CP5) has a reducedactivity and the trophozoite has a reduced ability to generateALA [26]

Extensive tissue damage has been attributed to the cys-teine proteinases of E histolytica because they are (i) secretedin large quantities and can cleave extracellular matrix pro-teins thus facilitating amebic invasion (ii) secreted in higherquantities by virulent than nonvirulent trophozoites and(iii) found to participate in the inflammation of the gut andALA [15ndash20 27] However cysteine proteases are dispensablefor phagocytosis and cytopathogenicity [28 29] Their mainphysiological role could be the digestion of host cells as theyare required for rosette formation hemolysis and digestionof erythrocytes

Another molecule that could participate in the damageof host cell is the cyclooxygenase- (COX-) like enzyme in Ehistolytica responsible for the biosynthesis of prostaglandinE2

(PGE2

)The production of PGE2

by the COX-like enzymein amebic liver granuloma can downregulate effector andaccessory cell functions of infiltrate immune cells [30]

Although the aforementioned processes are fundamentalin the initial stages of amoebiasis they seem to be quitesecondary as mechanisms of necrosis leading to hepaticabscesses in the latter stages of this disorder Adhesion isimportant for the survival of amoebas and the establishmentof prolonged contact of amoebas and toxic molecules withhost endothelial cells However neither adhesion nor thesetoxic molecules seem to be responsible for the host tissuedamage that directly results in amebic colitis and ALAThesedisorders occur during the latter stages of amoebiasis whenthe majority of host cells in contact with amoebas appear notto be damaged [31ndash33] It seems likely that damage to hostcells at this time is carried out in function of the inflammatoryprocess [31]

The proposal that host cell damage results mainly from achronic inflammatory response is corroborated by a numberof different studies Some recent reports [34 35] are revealingin this sense as they analyze the pathogenesis of ALA inhamsters inoculated with engineered HGL-2 trophozoitesdefective in GalGalNAc function The authors show thatHGL-2 amoebas infect hamster liver despite lacking thisimportant adhesion molecule although the pattern of infec-tion is different from that produced by wild-type E histolyt-ica HGL-2 amoebas cause a large number of inflammatoryfoci with a disorganized structure and these foci are locatedin the vicinity of blood vessels Despite their reduced capacityfor adhering to the host endothelium and penetrating liver













2


NO +O2

minus


Unlike NO and O2


2






2


2


2





Endothelial cell

Oxidativeinjury

Oxidativeinjury

Hepatocytes

Peroxynitrite

Apoptosisand

necrosis

Neutrophil

Macrophage

Apoptoticneutrophil

Arg + O2

Arg + O2

NO

NO

NOS

NOS

NOS

O2

pH 65ndash68

NADPHoxidase

O2

O2minus

O2minus

NO + O2minus

∙NO2 +∙OH

∙NO2 +∙OH


2


2


2



2



2


2




2


2


2


of NO and O2






2



2


2








O2



2



2


2

O2


3


Hb (Fe minusO2


minus (2)



2

[93] Since NO andO2






2




2

O2




2

O2

into water(H2









2

O2

to H2







ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













2


NO +O2

minus


Unlike NO and O2


2






2


2


2





Endothelial cell

Oxidativeinjury

Oxidativeinjury

Hepatocytes

Peroxynitrite

Apoptosisand

necrosis

Neutrophil

Macrophage

Apoptoticneutrophil

Arg + O2

Arg + O2

NO

NO

NOS

NOS

NOS

O2

pH 65ndash68

NADPHoxidase

O2

O2minus

O2minus

NO + O2minus

∙NO2 +∙OH

∙NO2 +∙OH


2


2


2



2



2


2




2


2


2


of NO and O2






2



2


2








O2



2



2


2

O2


3


Hb (Fe minusO2


minus (2)



2

[93] Since NO andO2






2




2

O2




2

O2

into water(H2









2

O2

to H2







ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Endothelial cell

Oxidativeinjury

Oxidativeinjury

Hepatocytes

Peroxynitrite

Apoptosisand

necrosis

Neutrophil

Macrophage

Apoptoticneutrophil

Arg + O2

Arg + O2

NO

NO

NOS

NOS

NOS

O2

pH 65ndash68

NADPHoxidase

O2

O2minus

O2minus

NO + O2minus

∙NO2 +∙OH

∙NO2 +∙OH


2


2


2



2



2


2




2


2


2


of NO and O2






2



2


2








O2



2



2


2

O2


3


Hb (Fe minusO2


minus (2)



2

[93] Since NO andO2






2




2

O2




2

O2

into water(H2









2

O2

to H2







ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2



2


2








O2



2



2


2

O2


3


Hb (Fe minusO2


minus (2)



2

[93] Since NO andO2






2




2

O2




2

O2

into water(H2









2

O2

to H2







ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2




2

O2




2

O2

into water(H2









2

O2

to H2







ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




ONOOH

H2OH2O2

NO2 + H2O


2

O2

into H2


Peroxynitrite

Oxidativeinjury

Macrophage

Neutrophil

Prx

E histolytica

Hepatocytes

CO2

CO3

Nitrite

Nitrate

NO3minus

NO2minus

NOminus+ O2

minus

∙OH + NO2



2



O2




2

O2


2

O2



O2


2

O2



O2


2

O2


2

O2



2

O2







2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2

O2









2

O2




2

to H2

O andNO to N

2







3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




3


2

O2









2




2





Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Hepatocytes

Peroxynitrite


Endothelialcell




NO + O2minus


2

O2



2















2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2

O2


8 Drug Design




9 Perspective





Acknowledgment


References





















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































































2












































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article peroxynitrite and peroxiredoxin in the pathogenesis...

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