Entamoeba histolytica Interaction with Enteropathogenic … · Entamoeba histolytica Interaction...

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Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis Luz A. Fernández-López, a Karla Gil-Becerril, a Silvia Galindo-Gómez, a Teresa Estrada-García, b Cecilia Ximénez, c Aralia Leon-Coria, d France Moreau, d Kris Chadee, d Víctor Tsutsumi a a Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, Mexico b Departamento Biomedicina Molecular, CINVESTAV-IPN, Mexico City, Mexico c Medicina Experimental, Facultad de Medicina, UNAM, Mexico City, Mexico d Cumming School of Medicine, Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada ABSTRACT Epidemiological studies suggest frequent association of enteropatho- genic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5) could modify the virulence of E. histo- lytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5 for 2.5 h. This was associated with increased E. histo- lytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 sub- strate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP- A1,-A2,-A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked up- regulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inocu- lated with either E. histolytica-EPEC or E. histolytica-E. coli DH5. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reac- tions, ischemia, and necrosis with high expression of il-1, ifn-, and tnf- proinflam- matory cytokine genes compared with that in livers of E. histolytica-E. coli DH5- inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cy- tokine genes il-6, il-12, and mcp-1. These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease. KEYWORDS Entamoeba histolytica, Escherichia coli, enteropathogenic, virulence, liver abscess, intestinal amebiasis, amebic liver abscess, virulence factors I n countries of endemicity, the human protozoan parasite Entamoeba histolytica has become a common resident of the large intestine, where it survives by feeding on dead cells and bacteria without causing harm to the host (1, 2). However, for reasons not quite understood, E. histolytica transition to an invasive form and following invasion gives rise to amebic colitis with symptoms ranging from diarrhea, ameboma, and life-threatening extraintestinal invasion to the liver (3, 4). This suggests that changes in the gut environment may contribute to the pathogenesis of E. histolytica, leading to invasive amebiasis (5–7). Citation Fernández-López LA, Gil-Becerril K, Galindo-Gómez S, Estrada-García T, Ximénez C, Leon-Coria A, Moreau F, Chadee K, Tsutsumi V. 2019. Entamoeba histolytica interaction with enteropathogenic Escherichia coli increases parasite virulence and inflammation in amebiasis. Infect Immun 87:e00279-19. https:// doi.org/10.1128/IAI.00279-19. Editor De’Broski R. Herbert, University of Pennsylvania Copyright © 2019 American Society for Microbiology. All Rights Reserved. Address correspondence to Kris Chadee, [email protected], or Víctor Tsutsumi, [email protected]. Received 11 April 2019 Returned for modification 27 May 2019 Accepted 26 August 2019 Accepted manuscript posted online 16 September 2019 Published FUNGAL AND PARASITIC INFECTIONS crossm December 2019 Volume 87 Issue 12 e00279-19 iai.asm.org 1 Infection and Immunity 18 November 2019 on December 12, 2020 by guest http://iai.asm.org/ Downloaded from

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Entamoeba histolytica Interaction with EnteropathogenicEscherichia coli Increases Parasite Virulence and Inflammationin Amebiasis

Luz A. Fernández-López,a Karla Gil-Becerril,a Silvia Galindo-Gómez,a Teresa Estrada-García,b Cecilia Ximénez,c

Aralia Leon-Coria,d France Moreau,d Kris Chadee,d Víctor Tsutsumia

aDepartamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicobDepartamento Biomedicina Molecular, CINVESTAV-IPN, Mexico City, MexicocMedicina Experimental, Facultad de Medicina, UNAM, Mexico City, MexicodCumming School of Medicine, Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada

ABSTRACT Epidemiological studies suggest frequent association of enteropatho-genic bacteria with Entamoeba histolytica during symptomatic infection. In thisstudy, we sought to determine if the interaction with enteropathogenic (EPEC) ornonpathogenic Escherichia coli (strain DH5�) could modify the virulence of E. histo-lytica to cause disease in animal models of amebiasis. In vitro studies showed a2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted withEPEC but not with E. coli DH5� for 2.5 h. This was associated with increased E. histo-lytica proteolytic activity as revealed by zymogram analysis and degradation of theE. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 sub-strate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked up-regulation of E. histolytica virulence factors, nonsignificant macroscopic differences inamebic liver abscess development were observed at early stages in hamsters inocu-lated with either E. histolytica-EPEC or E. histolytica-E. coli DH5�. Histopathology oflivers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3h postinoculation that progressively increased, producing large inflammatory reac-tions, ischemia, and necrosis with high expression of il-1�, ifn-�, and tnf-� proinflam-matory cytokine genes compared with that in livers of E. histolytica-E. coli DH5�-inoculated animals. In closed colonic loops from mice, intense inflammation wasobserved with E. histolytica-EPEC manifested by downregulation of Math1 mRNA witha corresponding increase in the expression of Muc2 mucin and proinflammatory cy-tokine genes il-6, il-12, and mcp-1. These results demonstrate that E. histolytica/EPECinteraction enhanced the expression and production of key molecules associatedwith E. histolytica virulence, critical in pathogenesis and progression of disease.

KEYWORDS Entamoeba histolytica, Escherichia coli, enteropathogenic, virulence, liverabscess, intestinal amebiasis, amebic liver abscess, virulence factors

In countries of endemicity, the human protozoan parasite Entamoeba histolytica hasbecome a common resident of the large intestine, where it survives by feeding on

dead cells and bacteria without causing harm to the host (1, 2). However, for reasonsnot quite understood, E. histolytica transition to an invasive form and following invasiongives rise to amebic colitis with symptoms ranging from diarrhea, ameboma, andlife-threatening extraintestinal invasion to the liver (3, 4). This suggests that changes inthe gut environment may contribute to the pathogenesis of E. histolytica, leading toinvasive amebiasis (5–7).

Citation Fernández-López LA, Gil-Becerril K,Galindo-Gómez S, Estrada-García T, Ximénez C,Leon-Coria A, Moreau F, Chadee K, Tsutsumi V.2019. Entamoeba histolytica interaction withenteropathogenic Escherichia coli increasesparasite virulence and inflammation inamebiasis. Infect Immun 87:e00279-19. https://doi.org/10.1128/IAI.00279-19.

Editor De’Broski R. Herbert, University ofPennsylvania

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Kris Chadee,[email protected], or Víctor Tsutsumi,[email protected].

Received 11 April 2019Returned for modification 27 May 2019Accepted 26 August 2019

Accepted manuscript posted online 16September 2019Published

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Coinfection with pathogenic organisms, especially enteropathogenic bacteria, maybe an important factor that contributes to alteration of normal enteric microbiota andimmune regulation, enhancing the virulence of E. histolytica in disease pathogenesis (5,7, 8). Epidemiological studies have reported frequent presence of enteropathogenicbacteria in coinfection with symptomatic intestinal amebic infection (9–11). Under invitro culture conditions, E. histolytica interaction with enteropathogenic bacteria for aslittle as 1 h enhanced parasite adherence and cysteine protease activity, with increasedcytopathic activity (12–14). Another study showed that short-term coculture (12 h) of apathogenic Escherichia coli serotype with an E. histolytica strain that had lost its capacityto produce amebic liver abscess (ALA) in hamsters restored parasite virulence byproducing ALA (15). Likewise, Entamoeba dispar strain ADO cultured under axenicconditions did not produce ALA, but when it was maintained in culture with microbiotafrom patients, it produced liver damage in hamsters similar to that produced by axenicE. histolytica (16). The influence of bacteria on intestinal amebiasis has been observedin gnotobiotic athymic mice (17), in which the percentage of cecum colonization withE. histolytica strains HK-9 and NIH:200 increased when they interacted with E. coli orClostridium perfringens. Similarly, in C3H/Hej mice, cecum damage produced by E.histolytica increased from 17% to 39% when parasites were cocultured with bacterialorganisms (18). Furthermore, innate host immune responses also play a key role insusceptibility to E. histolytica infection. In HT-29 cells, exposure to E. histolytica in thepresence of E. coli DH5� resulted in a synergistic increase in the expression ofinterleukin 8 (IL-8), IL-1�, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (19). Bacterial components in the healthy gut maintain protective innate immuneresponses against E. histolytica, as seen in antibiotic-treated animals, that rapidlyincrease susceptibility of mice to E. histolytica invasion due to decreased CXCR2 inneutrophils (6). An increased percentage of pathogenic bacteria can lead to dysbiosisand contribute to chronic intestinal inflammation through the induction of proinflam-matory cytokines gamma interferon (IFN-�), tumor necrosis factor alpha (TNF-�), IL-1�,and IL-6 (8).

The aim of this study was to determine if E. histolytica interactions with entero-pathogenic E. coli (EPEC) modulated parasite virulence factors and host innate immuneresponses associated with disease pathogenesis using several novel pathological ap-proaches that are quantifiable to differentiate acute disease that affects gene transcrip-tion, proinflammatory cytokine release, and disease progression. Here we show thatshort-term interaction between E. histolytica and EPEC markedly upregulated cysteineprotease, amebapore A, and cyclooxygenase (Cox)-like gene expression and increasedparasite adherence and killing of host cells. In animal models of disease, E. histolytica-EPEC interaction enhanced cellular inflammatory reaction, granuloma formation, andnecrosis in ALA in hamsters and increased secretory and proinflammatory cytokineresponses in closed colonic loops in mice.

RESULTSE. histolytica interaction with EPEC increases parasite virulence and cysteine

protease activity. To determine if E. coli serotypes can directly alter the virulence of E.histolytica, parasites were exposed to either EPEC or E. coli DH5� (which is nonpatho-genic). Phagocytosis of green fluorescent protein (GFP)-labeled bacteria was deter-mined by confocal microscopy and flow cytometry from 1 to 6 h. Maximal phagocytosisby E. histolytica towards EPEC and E. coli DH5� occurred between 2 and 3 h (see Fig. S1Aand SB in the supplemental material), and based on this observation, we used 2.5 h asthe optimal time for E. histolytica interactions with bacteria for all subsequent studies.Although the optimal times for E. histolytica to phagocytose bacteria were similar, moreE. coli DH5� organsims were phagocytized than EPEC (Fig. S1A and B). E. histolyticainteraction with EPEC but not with E. coli DH5� significantly increased CaCo2 cellmonolayer destruction (Fig. 1A). The increase in cytopathic effect produced by E.histolytica-EPEC interaction correlated with increased E. histolytica adherence to cell

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monolayers compared to that obtained with E. histolytica-E. coli DH5� and untreated E.histolytica (Fig. 1B).

Even though increased cysteine protease activity has been reported for E. histolyticafollowing exposure to different serotypes of pathogenic bacteria (13, 14, 20), thespecific cysteine proteases have not been defined. To quantify the specific cysteineprotease, we determined if E. histolytica interaction with EPEC and E. coli DH5�

differentially degraded porcine gelatin by zymogram analysis and synthetic substrates.E. histolytica exposed to both bacteria showed increased proteolytic activity towardgelatin; however, E. histolytica-EPEC interaction showed significantly enhanced cleav-ages at 35 and 27/29 kDa (Fig. 1C, arrows) compared to the case with E. histolytica-E. coliDH5� or only E. histolytica. To determine if the major cysteine proteinases involved inparasite virulence were upregulated following E. histolytica-EPEC and E. histolytica-E. coliDH5� interaction, we quantified the degradation of E. histolytica CP-A1/5 (EhCP-A1/5)-specific synthetic substrate Z-RR and EhCP-A4-specific substrate Z-VVR (21). Surpris-

FIG 1 Phenotypic characterizations of E. histolytica (Eh) following interaction with E. coli DH5� and EPECfor 2.5 h. (A) E. histolytica destruction of CaCo2 monolayers after 30 min of exposure. (B) Adhesion indexof CFSE-labeled E. histolytica on CaCo2 cell monolayer after 15 min of exposure. (C) Zymogram analysisof proteolytic activity of E. histolytica (control), E. histolytica-E. coli DH5�, and E. histolytica-EPEC. Thearrows point to cleavage products that were enhanced following E. histolytica-EPEC interaction. Thehistogram on the right represents the band density produced by individual EhCP activity. (D) Kineticanalysis of E. histolytica CP enzymatic degradation on the substrates Z-Arg-Arg-pNA (left), specific forEhCP-A1/5, and Z-Val-Val-Arg-AMC, specific for EhCP-A4 (right). Enzymatic unity for each substrate wascalculated as the micromolar concentration per minute per milligram. For statistical analysis, theabsorbance of E. histolytica (reference value, control) was compared with that of either E. histolytica-E. coliDH5� or E. histolytica-EPEC. P values were determined using Student’s t test. *, P � 0.05; **, P � 0.01; ***,P � 0.001.

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ingly, both E. histolytica-EPEC and E. histolytica-E. coli DH5� interactions significantlydegraded the substrates compared to untreated E. histolytica controls (Fig. 1D). Basedon these results, we next determined if bacteria could module the virulence genes inE. histolytica. Interaction with EPEC significantly upregulated several E. histolytica viru-lence genes in a time-dependent manner (Table 1). Maximal gene expression occurredafter 2 h of E. histolytica-EPEC interaction, significantly increasing the expression ofEhCP-A1, EhCP-A2, EhCP-A4, EhCP-A5, Gal/GalNAc lectin (Hgl), amebapore A (Apa) andcyclooxygenase (Cox-1)-like mRNA. In contrast, E. histolytica-E. coli DH5� interactioninduced only a slight overexpression of Hgl and Apa. Taken together, these resultssuggest that E. histolytica interaction with EPEC has a stronger effect in modulating E.histolytica virulence genes with increased enzymatic and cytopathic activity.

E. histolytica-EPEC interaction does not alter macroscopic liver damage butaffects the evolution of lesions compared to that with E. histolytica-E. coli DH5.Hamsters inoculated with E. histolytica following 2.5 h of interaction with either EPEC orE. coli DH5� showed �25 to 40% ALA at 3 and 7 days postinoculation (p.i.) (Fig. 2A). Thelesions produced by E. histolytica-E. coli DH5� and E. histolytica-EPEC showed similarwhitish and irregular anatomic changes on the liver surfaces (Fig. 2B, arrows). Lesionmorphology was consistent with the normal development of ALA produced by E.histolytica (22, 23). The percentages of liver damage produced by axenic E. histolytica,E. histolytica-E. coli DH5�, and E. histolytica-EPEC were comparable at 3 days p.i.;however, at 7 days p.i. the percent ALA in response to E. histolytica-EPEC was signifi-cantly higher (Fig. 2A). Animals inoculated with E. histolytica-EPEC showed noticeabletotal body weight loss at 3 days p.i. that became worse at day 7 p.i., whereas the bodyweight of animals inoculated with E. histolytica-E. coli DH5� decreased slightly at day3 p.i. but thereafter was similar to that of E. histolytica controls. Animals inoculated withEPEC showed an increase in body weight similar to those of animals inoculated with E.histolytica and E. histolytica-E. coli DH5� at 6 days p.i. (Fig. 2C).

Despite the clear absence of gross anatomic differences in ALA development,histological analysis (Fig. 3A to C) showed marked cytopathological differences amongthe groups at early stages. At 3 h p.i., animals inoculated with E. histolytica-EPECpresented inflammatory foci with more dense, greater, and compact infiltration ofpolymorphonuclear leukocytes (PMNs) associated with E. histolytica (Fig. 3A). Compar-ative two-dimensional (2D) morphometric analysis of the inflammatory foci between E.histolytica-EPEC and E. histolytica-E. coli DH5� at 3 h p.i. showed significant differencesin sizes of the foci, whereas no differences were observed between E. histolytica-E. coliDH5� and E. histolytica (Fig. 3D). Besides showing larger foci in E. histolytica-EPEC, eachcontained more PMNs, whereas livers inoculated with E. histolytica/E. coli DH5� oraxenic E. histolytica showed smaller foci and less PMN infiltration (Fig. 3A, black arrows).Also, the inflammatory foci at the early stages of E. histolytica-EPEC showed the

TABLE 1 Expression of E. histolytica virulence genes following interaction with E. coliDH5� and EPECa

Gene

E. histolytica-E. coli DH5� E. histolytica-EPEC

30 min 1 h 2 h 30 min 1 h 2 h

EhCP-A1 1.2 � 0.05 1.3 � 0.21 1.31 � 0.23 1.0 � 0.24 1.4 � 0.17 2.2 � 0.25**EhCP-A2 0.9 � 0.23 0.7 � 0.18 1.1 � 0.28 1.0 � 0.25 1.2 � 0.14 3.2 � 0.39***EhCP-A4 1.0 � 0.37 1.0 � 0.43 1.67 � 0.41 2.1 � 0.3 2.14 � 0.06 6.6 � 0.59***EhCP-A5 0.9 � 0.1 0.9 � 0.19 1.1 � 0.59 1.4 � 0.22 1.11 � 0.18 4.6 � 0.46***Hgl 0.8 � 0.05 1.5 � 0.24 2.6 � 0.19*** 1.15 � 0.15 1.4 � 0.04 5.1 � 0.56***Apa 1.0 � 0.06 0.9 � 0.12 1.9 � 0.13 2.3 � 0.33** 1.9 � 0.29* 6.8 � 0.56***Cox-1 0.7 � 0.13 0.7 � 0.09 1.1 � 0.19 1.09 � 0.17 1.3 � 0.17 2.6 � 0.39***aQuantitative kinetic gene expression at 30 min, 1 h, and 2 h was evaluated by qPCR, taking as basalexpression wild-type virulent E. histolytica. The E. histolytica DNA gene was used as an endogenous control.EhCP-A1 to -5 encode cysteine proteinases, Hgl encodes Gal/GalNAc lectin heavy chain, Apa encodesamebapore, and Cox-1 encodes cyclooxygenase. Values are means � SDs from three independentexperiments repeated twice. P value was calculated by Student’s t test. *, P � 0.05; **, P � 0.01; ***,P � 0.001.

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presence of E. histolytica organisms scattered inside each focus, sometimes with two ormore E. histolytica organisms, in contrast to E. histolytica-E. coli DH5� or controlsinoculated with axenic E. histolytica, which usually showed one E. histolytica organismat the center of inflammatory focus. Structural evolution of hepatic lesions at day 3 afterinfection with E. histolytica or E. histolytica-E. coli DH5� showed typical amebic granu-lomas constituted by epithelioid cells at the outer limit with E. histolytica locatedbetween the palisade cells and the necrotic center; no clear difference in the extents ofdamage was observed between these two groups (Fig. 3B). In contrast, hamstersinoculated with E. histolytica-EPEC showed fewer, compact granulomas but with a liverparenchyma substituted by an extensive necrotic and ischemic areas peripherallysurrounded by acute and chronic inflammatory reactions with irregular outer limits (Fig.3B). Lesions produced at 3 h and day 3 after infection with E. histolytica-EPEC weresimilar to the lesions reported at 6 h and 7 days after infection, respectively, with onlyaxenic E. histolytica (22, 23). In hamsters inoculated only with E. coli DH5� or EPEC(without E. histolytica), a relatively small perivascular inflammatory infiltrate that pro-gressed to compact small areas of ischemia was observed at 24 h p.i., and by day 3 p.i.the inflammatory response was completely cleared, presenting a normal liver paren-chymal appearance (Fig. 3C). Although bacteria alone did not produce liver lesions,

FIG 2 Macroscopic changes in livers of hamsters inoculated with E. histolytica (1 � 106 in 200 �l ofTYI-S-33) following interaction with nonpathogenic E. coli (strain DH5�) or EPEC for 2.5 h. (A) ALA inducedby E. histolytica following interaction with E. coli DH5� and EPEC at 3 and 7 days p.i. No significantdamage was observed in the livers of animals inoculated with only EPEC or E. coli DH5�. **, P � 0.01. (B)Gross anatomy of liver lesions produced by E. histolytica following interaction with E. coli DH5� or EPECafter 3 and 7 days p.i. Note the whitish granular amebic lesions on the liver surfaces (arrows). At least sixhamsters were evaluated for each time and condition. Animals inoculated with TYI-S-33 were used ascontrols. (C) Changes in hamster body weights at 3 and 7 days after infection with axenic E. histolyticaand following interaction with E. coli DH5� and EPEC. Animals inoculated with EPEC only presentedchanges in body weight similar to those observed in the E. histolytica and E. histolytica-DH5� groups. Pvalue was calculated by Student’s t test. ***, P � 0.001.

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they induced early proinflammatory responses that could promote the survival of E.histolytica and progression of ALA.

Proinflammatory cytokine responses were more pronounced in the liver ofhamsters inoculated with E. histolytica-EPEC. E. histolytica-EPEC inoculations not onlyaffected granuloma development but also stimulated robust proinflammatory cytokineexpression during the induction of hepatic amebiasis. At 3 h p.i., E. histolytica-EPEC wasfound to have elicited higher expression of tnf-�, ifn-�, and il-1� than did E. histolytica-E.coli DH5� and E. histolytica (Fig. 4A). Surprisingly, kc expression was significant higher

FIG 3 Liver histopathology of hamsters inoculated with axenic E. histolytica, E. histolytica-E. coli DH5�, E. histolytica-EPEC, and E. coli DH5� or EPEC. (A) Inflammatory foci produced at 3 h after intrahepatic inoculation demonstratingE. histolytica (arrows) surrounded by PMNs. Inflammatory foci with E. histolytica-EPEC show more abundantinflammatory cells than with E. histolytica-DH5� or E. histolytica. (B) Three days after intrahepatic inoculation.Numerous inflammatory granulomas can be observed clearly in liver from E. histolytica-inoculated hamsters. E.histolytica-DH5�-inoculated animals show more irregular granulomas, whereas livers from animals inoculated withE. histolytica-EPEC present larger necrotic areas with small dense granulomas and intense inflammatory reaction.(C) Perivascular inflammatory infiltration around small ischemic zones produced by EPEC at 24 h p.i. and normalhepatic tissue 3 days after EPEC intrahepatic inoculation. Hematoxylin-eosin stain was used. (D) Histogram of 2Dmorphometry of inflammatory foci at 3 h post-intrahepatic inoculation. E. histolytica-EPEC yielded significantlylarger foci than E. histolytica and E. histolytica-DH5�. P value was calculated by Tukey’s multiple-comparison test.***, P � 0.05.

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towards EPEC, E. histolytica, and E. histolytica-DH5�. Proinflammatory cytokine re-sponses were low towards EPEC and E. coli DH5�. The anti-inflammatory cytokine geneil-10 was overexpressed only at 3 h p.i. in all groups (Fig. 4A). By 12 h p.i., only tnf-� andil-1� were significantly increased, but at lower levels than at 3 h p.i., and they remainedhigher in the E. histolytica-EPEC group (Fig. 4B). At 12 and 24 h p.i., kc and il-10 geneexpressions returned to basal levels in all groups (Fig. 4B and C). At 24 h p.i., except forifn-�, which was still upregulated in the E. histolytica-EPEC group, all the cytokinesreturned to basal level in all groups (Fig. 4C). These findings indicate that the proin-

FIG 4 RT-qPCR for cytokine expression in the liver of hamsters inoculated with E. histolytica, E.histolytica-E. coli DH5�, E. histolytica-EPEC, and EPEC. (A) At 3 h p.i., genes for all proinflammatorycytokines were overexpressed in animals inoculated with E. histolytica, E. histolytica-E. coli DH5�, and E.histolytica-EPEC. EPEC inoculation induced il-8 and il-10 overexpression, and E. coli DH5� only inducedil-10 overexpression. (B) At 12 h p.i., expression of tnf-� decreased and ifn-� increased in animalsinoculated with E. histolytica, E. histolytica-DH5�, and E. histolytica-EPEC. (C) At 24 h p.i., ifn-� was stilloverexpressed in E. histolytica-EPEC-inoculated hamsters. Hamsters inoculated with only E. histolyticaculture medium were used as a basal expression control (Ctl); the GAPDH gene was used as ahousekeeping gene to normalize mRNA levels. Data correspond to means � SDs from three independentexperiments repeated twice. Animals inoculated with TYI-S-33 were used as a control. P value wascalculated by two-way ANOVA. *, P � 0.05; **, P � 0.01; ***, P � 0.001 (compared with values for E.histolytica). Differences over Ctl (*), E. histolytica (�), and E. histolytica-E. coli DH5� (●) are shown.

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flammatory milieu evoked by E. histolytica-EPEC might be critical in shaping the earlydevelopment and progression of ALA.

E. histolytica-EPEC inhibited Muc2 secretory lineage with a correspondingincrease in activity in the distal colon driven by proinflammatory cytokines. Todetermine if E. histolytica-E. coli DH5� and E. histolytica-EPEC interaction altered thevirulence of E. histolytica, we tested this using a short-term colonic loop model withMath1GFP mice. Math1 protein is a transcription factor that promotes the differentiationof multipotential colonic cells to secretory goblet cells whose lineage is lost by theablation of this factor. We have recently shown that a decrease in Math1GFP activity(signal) correlates with E. histolytica-induced proinflammatory responses and microbialtranslocation in the colon (24). Interestingly, EPEC and E. histolytica-EPEC inoculation inthe proximal colon inhibited Math1GFP activity in both the proximal and distal colon(Fig. 5A and B). However, reduction in Math1 mRNA expression was higher in E.histolytica-EPEC- than in EPEC-inoculated loops. This contrasts with colonic loopsinoculated with E. histolytica, E. coli DH5�, or E. histolytica-E. coli DH5�, which showeda significant decrease in Math1GFP activity in the proximal colon with a correspondingincrease in activity in the distal colon (Fig. 5B and C) compared to that in phosphate-buffered saline (PBS)-inoculated controls. Predictably, Muc2 mRNA expression was thehighest towards E. histolytica-EPEC in both the proximal and distal colon (Fig. 5D). E.histolytica, E. coli DH5�, and E. histolytica-E. coli DH5� stimulated modest Muc2 mRNAexpression in the proximal colon, with a similar increase in the distal colon.

As host protection from E. histolytica colonic invasion has been attributed mainly toneutrophil infiltration via oxygen-free radicals induced by the presence of TNF-� andIFN-� (25, 26), we quantified changes in neutrophil recruitment using myeloperoxidase(MPO) activity as a readout and proinflammatory responses after 3 h in colonic loopsinoculated with E. histolytica, E. histolytica-EPEC, or E. histolytica-E. coli DH5�. However,no significant differences were observed in MPO activity induced by inoculating E.histolytica, E. histolytica-E. coli DH5�, or E. histolytica/EPEC (Fig. 6A). Moreover, colonictissue cytokine gene expression was upregulated in loops inoculated with E. histolytica-EPEC; the highest expression was found for il-6, il-12, mcp-1, tnf-�, and il-1� (Fig. 6B).EPEC inoculation alone significantly increased the expression of il-6, il-1�, and mcp-1(Fig. 6B), suggesting that the overexpression of these cytokines was a direct or partialresponse to bacterial components in the E. histolytica-EPEC inoculum. There were nodiscernible differences in loops inoculated with E. histolytica or E. histolytica-E. coliDH5�. ifn-� expression slightly increased in loops inoculated with E. histolytica and E.histolytica-E. coli DH5� and remained at basal levels in response to E. histolytica-EPEC orEPEC (Fig. 6B). il-10 levels were unaltered regardless of the inoculum. Taken together,these results demonstrate that E. histolytica-EPEC interaction enhanced parasite viru-lence in the colon to dampen Math1 and upregulate Muc2 and proinflammatorycytokine expression.

DISCUSSION

Here we report that E. histolytica interaction with EPEC significantly increasedparasite virulence, which led to enhanced destruction of CaCo2 cell monolayers andaggressive immune responses in hamster liver and mouse colon. E. histolytica-EPECinteractions markedly upregulated E. histolytica virulence genes EhCP-A1, EhCP-A2,EhCP-A4, EhCP-A5, Hgl, Apa, and Cox-1 in a time-dependent manner. In contrast, E.histolytica interaction with nonpathogenic E. coli DH5� modestly induced the expres-sion of Hgl and Apa without modifying parasite virulence. Correlated with geneexpression, high cysteine protease activity and adhesion to host cells mediated by theGal/GalNAc lectin were induced by E. histolytica-EPEC interaction. These virulencefactors have been directly associated with E. histolytica killing cells in vitro (27–30) andin vivo to alter secretory responses and tight junction proteins in the gut (31, 32).

In animal models of disease, increased expression of the Gal/GalNAc lectin and EhCPgenes has been observed during ALA development (30, 33–39). However, despite aconsiderable increase in proteolytic activity and adherence to mammalian cells induced

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by E. histolytica-EPEC interaction, no significant differences were observed in thepercentages of ALA produced in hamsters inoculated with E. histolytica-EPEC and withE. histolytica alone. The lack of correlation between the ability to induce ALA with E.histolytica proteolytic activity was previously observed (40) using two clones of HM1:IMSS_B that were not able to produce ALA in gerbils but had high CP activity. Thesefindings are partially in contrast to those of a previous study (15) that restored virulenceof E. histolytica strain HMI:IMMS to produce ALA in hamsters when interacting with apathogenic serotype of E. coli. This indicates that E. histolytica interaction with patho-genic E. coli may not increase E. histolytica capacity to produced larger lesions, but itcould enhance its adaptation and survival in the host by reinforcing E. histolytica

FIG 5 Math1 and Muc2 secretion and gene expression in colons of mice inoculated with E. histolytica, EPEC, E. coliDH5�, E. histolytica-EPEC, and E. histolytica-E. coli DH5�. (A) Math1GFP activity in closed proximal colonic loops fromcontrol animals and animals inoculated with E. histolytica, E. coli DH5�, EPEC, E. histolytica-EPEC, and E. histolytica-E.coli DH5�. The dotted line indicates the colonic loop ligations. The images show an overlay of the black-and-whitecolon tissue and the GFP signal (fluorescence images are shown in color, superimposed over a black-and-whiteimage of the dissected colon ex vivo). The fluorescence emission signal in the green channel was quantified andcorrected for background. (B) Histogram representation of Math1GFP signal in the proximal and distal colon. (C)Relative q-PCR for Math1 mRNA in the proximal and distal colonic tissues from panel A. (D) Differences in relativeMuc2 expression evaluated by RT-qPCR in the proximal and distal colon. Gene expression levels were normalizedusing �-actin. Animals inoculated with PBS were used as the control. P value was calculated by Student’s t est. AU,arbitrary units. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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pathogenesis. In accordance with this, preincubation of E. histolytica with live E. coli O55increased parasite resistance to oxidative stress by reversing changes in the expressionof genes involved in protein synthesis, intracellular trafficking, nutrition, homeostasis,and peroxidases altered by oxidative stress (41). It is well known that EhCPs playimportant roles in disease pathogenesis, as demonstrated by the inability of E. histo-lytica to induce lesions in gerbil liver when transfected with an antisense gene forEhCP-A5, despite conserving the cytopathic capacity over mammalian cell monolayers(42). Additionally, E. histolytica invasion in human intestinal xenografts was reduced�95% when EhCP-A1 was blocked with the specific inhibitor WRR483 or with EhCP-A4-specific inhibitor WRR605 in C3H/HeJ mice (43, 44). Therefore, it is possible that E.histolytica interaction with EPEC improved survival of E. histolytica under host condi-tions by increasing EhCP production and adhesion to mammalian cells.

Histological analysis of hepatic lesions suggested a faster progression of the lesionin response to E. histolytica-EPEC. This could be related to a more aggressive immuneresponse expressed by greater numbers of PMNs surrounding E. histolytica, observedexclusively at a very early stage of E. histolytica inoculation, and higher expression ofthe proinflammatory cytokines IL-1�, IFN-�, and TNF-� than with E. histolytica-E. coliDH5� or E. histolytica alone. No significant upregulation of inflammatory cytokines inresponse to EPEC occurred, suggesting that the sole presence of EPEC was not thecause for the more aggressive immune response during ALA. More likely, the combi-nation of E. histolytica virulence factors and host immune response to pathogenicbacterial components was responsible for the robust acute inflammatory response.Overexpression of the proinflammatory cytokines IL-1�, IL-8, IFN-�, and TNF-� waspreviously observed during the first 3 to 12 h after infection with E. histolytica in thelivers of hamsters and in a model of human liver tissue explants (33, 45). These resultssuggest that the expression of proinflammatory cytokines participated in tissue dam-age during ALA by inducing a more aggressive response of PMNs and other immunecells recruited in the liver.

Although the liver is a good organ to study E. histolytica pathogenesis, the naturalenvironment of E. histolytica is the colon, where it colonizes and preferentially feeds onbacterial components of the normal microbiota by phagocytosis (46). In our study, weevaluated whether the interaction of E. histolytica with E. coli DH5� or EPEC inducedchanges in stimulating mucus secretion, Muc2 mucin gene expression, and repressionof the transcription factor Math1, events critical in the pathogenesis of intestinalamebiasis (24). For these studies we used Math1GFP mice, which express the greenfluorescent protein (GFP) in goblet cells, to determine differences in intestinal homeo-stasis alteration. Somewhat surprisingly, inoculating E. histolytica or E. histolytica-E. coli

FIG 6 Proinflammatory responses in mouse proximal colon inoculated with E. histolytica, E. histolytica-EPEC, E. histolytica-E. coli DH5�, or EPEC. (A) Acute inflammation was evaluated by MPO activity;PBS-inoculated colons were used as controls. (B) Cytokine mRNA in the proximal colon (site of E.histolytica inoculation) was quantified by RT-qPCR. Gene expression levels were normalized using �-actin.For both panels, 5 mice were analyzed for each condition in two independent experiments. Animalsinoculated with PBS were used as a control. P value was calculated by Student’s t test. *, P � 0.05; **,P � 0.01; ***, P � 0.001. Differences over Ctl (*), E. histolytica (�), and E. histolytica-E. coli DH5� (●) areshown.

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DH5� caused a shift in Math1GFP signal from the proximal colon, where E. histolyticawas inoculated, towards the distal colon. In contrast, with E. histolytica-EPEC inocula-tion, Math1GFP signal was repressed in both the proximal and distal colon, indicating agreater imbalance in intestinal cellular homeostasis, related to the repression of thesecretory cell linage and robust proinflammatory responses. Muc2 mucin mRNA ex-pression was high in both the proximal and distal colon, with a corresponding increasein mucus secretion when E. histolytica-EPEC was inoculated. Muc2 is the main compo-nent of the intestinal mucus that forms the first line of innate host defense againstpathogenic organisms in the large intestine; deficiency of this glycoprotein inducessensitivity and greater damage to the epithelium by E. histolytica (47–49). We haverecently shown (50) that high production of Muc2 mucin in response to dextran sodiumsulfate (DSS) caused oxidative stress and apoptosis of goblet cells that led to the lossof the protective mucus barrier.

During the establishment of intestinal amebiasis, regulation of the immune re-sponse by cytokines is widely associated with tissue damage and/or protection againstE. histolytica invasion. In our study, we observed that E. histolytica-EPEC induced greaterexpression of tissue il-12, il-6, il-1�, tnf-�, and mcp-1 and inhibited the expression ofifn-� that remained at basal levels. Elimination of E. histolytica infection is associatedwith the presence of IFN-�, which plays a critical role in the destruction of E. histolyticaand inhibition of EhCP activity (51–53). In contrast, tnf-� and il-4 are correlated with theprogression of disease in murine models of amebic colitis, where it was found that theTh2 adaptive response determines the establishment of infection in the host. In ourstudy, mice inoculated with E. histolytica-EPEC showed high expression of Mcp-1protein, which is produced by epithelial cells and functions as a potent chemoattractantfor monocytes, naive dendritic cells, and basophils and is important for the recruitmentof antigen-presenting cells (APCs) to the site of damage, where IL-6 and transforminggrowth factor � (TGF-�) are secreted and induce the differentiation of naive CD4� cellsto Th17 type cells (54–56). Th17 cells secrete IL-23, contributing to expansion of theTh17 response, and the combination of IL-17 and IL-23 is apparently important for therecruitment of neutrophils, induction of mucus secretion, and inhibition of the Th1response (57). Interestingly, in this study, the cyclooxygenase enzyme that producedprostaglandin 2 (PGE2) was overexpressed when E. histolytica interacted with EPEC.PGE2 can condition dendritic cells to induce IL-23 and the migration of neutrophilsthrough the Th17 immune response, simultaneously inhibiting Th1 responses, which isnecessary for host defense against E. histolytica (57–59). However, despite high up-regulation of the cyclooxygenase enzyme in E. histolytica-EPEC and overexpression ofMcp-1 and IL-1�, no differences were observed in neutrophil recruitment in miceinoculated with axenic E. histolytica, E. histolytica-E. coli DH5�, E. histolytica-EPEC, andEPEC.

Coinfection with pathogenic bacteria normally occurs in intestinal amebiasis, andALAs harbor bacteria from the gut (10, 11, 60); however, it is unclear if pathogenicbacteria affect E. histolytica virulence. Unfortunately, an animal model that allows us tostudy in detail how the prevalence of pathogenic bacteria affects the establishment andprogression of intestinal amebiasis has not yet been established. In summary, theresults of this work show that E. histolytica interaction with EPEC or nonpathogenic E.coli markedly influences parasite virulence by upregulating key virulence genes criticalin disease pathogenesis. In particular, E. histolytica-EPEC interaction increased EhCP-A1,EhCP-A2, EhCP-A4, EhCP-A5, Hgl, Apa, and Cox-1 mRNA expression associated withincreased parasite adherence to and killing of CaCo2 cell monolayers. E. histolytica-EPECinteractions increased cellular necrosis in hamster liver and evoked intense mucussecretagogue activity and downregulation of Math1 activity associated with highexpression of proinflammatory cytokines in mouse colon. These results demonstratethat E. histolytica interaction with EPEC during parasite colonization in asymptomatichosts can potentially influence the expression and production of key molecules asso-ciated with E. histolytica virulence, critical in disease pathogenesis and progression ofthe disease.

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MATERIALS AND METHODSE. histolytica culture. E. histolytica strain HM1:IMSS, which produces large ALA in hamsters, was used

in this study. E. histolytica were axenically cultured at 37°C in TYI-S-33 medium supplemented with 20%adult bovine serum (ABS). For all experiments, cultures of E. histolytica were harvested and used inlogarithmic growth phase (72-h culture).

Bacterial cultures. The nonpathogenic E. coli strain DH5� and the enteropathogenic E. coli (EPEC)strain B171-0111:NM, donated by Teresa Estrada (CINVESTAV, Mexico), were grown in Luria-Bertani (LB)agar plates at 37°C for 15 to 18 h. For bacterium-E. histolytica interaction, a colony was taken and grownin LB broth at 37°C with shaking for 15 to 18 h. A total of 500 �l of the culture was seeded in 5 ml ofTYI-S-33 medium without ABS and incubated at 37°C with shaking for 4 to 6 h, until reaching an opticaldensity (OD) at 600 nm of 0.6 to 0.8.

E. histolytica-bacterium interaction and phagocytosis kinetics. E. histolytica organisms (1 � 106)in logarithmic phase of growth were transferred to TYI-S-33 medium without ABS and 1 � 108 CFU ofbacteria from one of the strains of E. coli were added for an E. histolytica-bacterium ratio of 1:100. TheE. histolytica-bacterium combinations were incubated at 37°C for the desired time, the supernatant wasdiscarded, and PBS was added and chilled on ice for 10 min. E. histolytica was recovered by centrifugationat 200 � g and 4°C for 5 min, washed 3 times with cold PBS containing 200 �g/ml of gentamicin, andcentrifuged at 200 � g for 5 min. E. histolytica was washed twice with PBS to remove antibiotic residue.To quantify bacteria phagocytosed by E. histolytica, bacteria were transfected to express the greenfluorescent protein (GFP) using the plasmid pEGFP and incubated with 5 � 105 E. histolytica organismsfor 1, 2, 3, 4, 5, and 6 h at 37°C. After incubation, E. histolytica organisms were recovered by centrifugationand washed with PBS-gentamicin. E. histolytica organisms were fixed with 3.7% formaldehyde for 20 min,washed 2 twice with PBS by centrifuging at 200 � g, and suspended in 500 �l of PBS. Intracellularfluorescence was measured by confocal microscopy and flow cytometry.

CaCo2 cell culture, cytopathic effect, and E. histolytica adhesion. CaCo2 cells were cultured inDulbecco modified Eagle minimal essential medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and 100 U/ml of penicillin-streptomycin and incubated at 37°C with 5% CO2. The cells were passagedwith 0.25% trypsin-EDTA once they reached 90% confluence. For cytopathic effect, cells were seeded in24-well plates in triplicate at a density of 5 � 105 and cultured until 80% confluence. Cells were thenincubated with 2.5 � 105 E. histolytica organisms for each condition for 30 min and 1 h at 37°C, then placedon ice for 15 min to detach E. histolytica, washed with cold PBS, and stained with 0.1% methylene blue. Cellswere extracted with 0.1 M HCl, and the samples were read at 650 nm with a spectrophotometer. Thequantified absorbance was compared with that given by intact cell monolayers not exposed to E. histolytica,which were considered a control (0% damage). For the adhesion assay, E. histolytica for each condition waslabeled with 8 �M CellTrace carboxyfluorescein succinimidyl ester (CFSE) for 15 min and washed twice withPBS prior to exposure to the cell monolayer. After 30 min of incubation at 37°C, nonattached E. histolyticaorganisms were gently washed with warm PBS, and the plate was read in a fluorometer at an excita-tion wavelength of 492 nm and an emission wavelength of 517 nm.

Zymogram of E. histolytica proteases and degradation of synthetic peptide substrates. E.histolytica lysed for each condition was obtained by three cycles of freeze-thawing. Two micrograms ofE. histolytica lysate freed from debris by centrifugation at 2,000 � g was loaded in 1% gelatin–12%polyacrylamide gels, and the electrophoresis was developed at 70 V for 15 min and 80 V for 2 h at 4°C.The gel was washed twice for 15 min with shaking in 2.5% Triton X-100 solution, incubated overnight at37°C in activation buffer at pH 7 (Tris-OH solution at 100 mM), washed with distilled water, and stainedwith Coomassie blue. The clear areas in the gels revealed the digestion of the gelatin by the cysteineproteinase activity. The gels were scanned with the SigmaGel program. The densitometry analysis of thebands was done using the software ImageJ (http://rsb.info.nih.gov/nih-image/). Proteolytic activity in E.histolytica lysates was evaluated by the degradation of the synthetic peptide substrate Z-Arg-Arg-pNA(Bachem), which is degraded by EhCP-A1/5, and the specific substrate for EhCP4, Z-Val-Val-Arg-AMC (21)(Enzo Life Sciences, NY). The reaction mixture consisted of a 0.1 mM concentration of the substrate in thereaction buffer (5 mM EDTA, 50 mM NaCl [pH 7]) followed by E. histolytica proteins (25 �g). The releaserate of pNA was measured by absorbance at 405 nm, while the release of AMC was evaluated in afluorometer with excitation at 365 nm and emission at 440 nm, every 2 min for 20 min at roomtemperature, for both substrates. One unit of enzymatic activity was defined as the number of micro-moles of digested substrate per minute per milligram of protein.

Experimental hepatic amebiasis. Two-month-old male hamsters (Mesocricetus auratus) weighing 80to 100 g were fasted for 24 h prior to surgery. Subsequently, they were anesthetized initially with 3%isoflurane and then with 1.5% of the same anesthetic during the surgical procedure. The abdominalsurface of the hamster was shaved and a longitudinal incision of the abdominal wall was made in twoplanes, skin and muscle wall, to expose the liver. E. histolytica (1 � 106 in 200 �l of TYI-S-33 without ABS)of each group (axenic E. histolytica, E. histolytica-E. coli DH5�, and E. histolytica-EPEC after three washeswith PBS plus gentamicin) was then directly inoculated in the left lobe using a tuberculin syringe. Theabdominal incision was sutured in two planes with 2/0 silk thread. After 3, 12, 24, 72, and 96 h ofinoculation, the hamsters were sacrificed with an overdose of anesthetic; the whole liver was firstweighed, and then only the liver lesion was dissected and weighed to calculate the percentage of thedamaged liver in relation to the total liver weight (percent ALA). Representative tissue fragments wereobtained for RNA extraction, and other fragments of the same section were fixed with 4% paraformal-dehyde (pH 7.2) for histological analysis.

Experimental intestinal amebiasis. Mice used were 10 to 12 weeks old and kept in cages withfilter-sterilized tops. C57BL/6 mice and Math1GFP mice (strain 013593) with a C57Bl/6 genetic background

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(Jackson Laboratory, Bar Harbor, ME) were used for colonic loop infections with E. histolytica. To inoculatemice, a laparotomy was performed on anesthetized mice to expose the proximal colon; a colonic loopwas created by suturing from the cecum-colon junction to 3 cm down the length of the colon. Mice werethen inoculated with 1 � 106 log-phase E. histolytica organisms that interacted or not with E. coli DH5�

or EPEC for 2.5 h, washed twice with PBS, and suspended in 100 �l of PBS. As controls, 100 �l of PBS andE. coli DH5� and EPEC cultures were used. Infections were carried out for 3 h and the colonic loop wasexcised. The intestinal content of the colonic loop was recovered in a 1.5-ml Eppendorf tube, homog-enized, and centrifuged at 3,000 � g to remove debris; the supernatant was recovered and normalizedto 1 mg/ml. Cytokine release in the intestinal lumen was evaluated by Luminex addressable laserbead-based immunoassay (mouse focused 13-plex Discovery Assay; Eve Technologies, Calgary, AB,Canada).

Whole-colon imaging. To screen whole tissue ex vivo, the colons from naive or infected Math1-GFPmice were dissected and then visualized using an InVivo Xtreme 4MP whole-body imaging system(Bruker, Billerica, MA) as previously described (24, 61). The green channel was visualized using 470-nmexcitation and 535-nm emission wavelengths. The imaging protocol contained two steps: reflectance(black and white) imaging (2-s exposure time) and fluorescent imaging at the given wavelength (5-sexposure time). Images from the InVivo Xtreme were acquired and analyzed using the Bruker molecularimaging software MI SE (version 7.1.3.20550). The fluorescence intensity was quantified by measuring themean fluorescence signal intensity (corrected by background) in a constant region of interest for eachindividual organ.

RT-qPCR for virulence factors in E. histolytica and host cytokines. The TRIzol method (InvitrogenLife Technologies, Carlsbad, CA) was used for RNA extraction. For E. histolytica, 800 �l of TRIzol wasused for 5 � 106 parasites, homogenized by repetitive pipetting and incubated for 5 min at roomtemperature. For tissue samples, 100 to 200 �g was used, 1 ml of TRIzol was added, and the tissues werehomogenized. For all samples, RNA extraction was performed according to the manufacturer’s instruc-tions. For reverse transcription-quantitative PCR (RT-qPCR), the First Strand cDNA synthesis retrotrans-cription kit (FERMENTAS, Thermo Scientific, Waltham, MA) was used, based on oligo(dT) sequences asprimers. For quantitative amplification by reverse transcription, the reactions were performed in aRotor-Gene 3000 real-time PCR system (Nainital, Uttarakhand, India). Each reaction mixture (total, 20 �l)contained 100 ng of cDNA, 10 �l of a master mix for 2� SYBR green qPCR (catalog number 204072;Qiagen, Venlo, Netherlands), and primers at 1 �M (2 �M). The amplification conditions were as follows:40 cycles of 95°C for 15 s, melting temperature for 20 s, 68°C for 20 s, and an adjacent melting step (67to 95°C). The relative differences in gene expression were calculated using the threshold cycle (2�ΔΔCT)methods with the Rotor-Gene software. For E. histolytica gene expression, E. histolytica ribosomal DNA(rDNA) was used as a control, and for hamster and mouse tissues the glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and �-actin genes, respectively, were used as housekeeping genes. Primersused for the detection of different cytokines and virulence factors are shown in Table S1.

Statistical analysis. All statistical analyses were performed using GraphPad Prism version 5.01(GraphPad Software, San Diego, CA). Student’s t test and two-way analysis of variance (ANOVA) withBonferroni post-t tests were used. Significant differences between groups were assessed at a P value of�0.05. Results are presented as the means of three independent experiments, along with standarddeviation (�SD), unless otherwise indicated.

Ethics statement. The Centre for Research and Advanced Studies (CINVESTAV) fulfills the standardof the Mexican Official Norm (NOM-062-ZOO-1999) Technical Specifications for the Care and Use ofLaboratory Animals. The Institutional Animal Care and Use Committee (IACUC/ethics committee) fromCINVESTAV has reviewed and approved all animal experiments (protocol number 0505-12, CICUAL 001).Mouse studies were approved under the University of Calgary (Calgary, AB, Canada) Animal CareCommittee, which adheres to the principles and policies regarding the care and use of experimentalanimals of the Canadian Council on Animal Care.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/IAI

.00279-19.SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.

ACKNOWLEDGMENTSWe thank Enrique González for excellent technical support and Björn Petri from the

Mouse Phenomics Resources Laboratory and the Live Cell Imaging Facility in the SnyderInstitute at the University of Calgary for imaging.

This work was funded in part by the Miguel Aleman Foundation (2016, 2017) and byPRODEP, Program for Professional Development, Secretary of Education, Mexico, inawards to V.T. and by a discovery grant (RGPIN-2019-04136) from the Natural Sciencesand Engineering Research Council of Canada awarded to K.C. L.A.F.-L. and A.L.-C.received scholarship support from the National Council of Science of Technology,Mexico, during their Ph.D. studies (321615 and 314101, respectively).

We declare no conflict of interest.

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