A phagocytosis mutant of Entamoeba histolytica is less virulent due to deficient proteinase expression and release

Experimental Parasitology 115 (2007) 192–199 www.elsevier.com/locate/yexpr

A phagocytosis mutant of Entamoeba histolytica is less virulent due to deWcient proteinase expression and release Ken K. Hirata a, Xuchu Que a, Samuel G. Melendez-Lopez a, Anjan Debnath b, Simona Myers a,1, D. Scott Herdman a, Esther Orozco c, Alok Bhattacharya d, James H. McKerrow b, Sharon L. Reed a,¤ a Departments of Pathology and Medicine, University of California, San Diego, CA 92103-8416, USA Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, CA 94158, USA c Departamento de Patologia Experimental, Centro de Investigacion y de Estudios Avanzados del, I.P.N., Mexico City, Mexico 07000, Mexico d School of Life Sciences, Jawaharlal Nehru University, New Delhi 10067, India b

Received 21 April 2006; received in revised form 24 June 2006; accepted 2 August 2006 Available online 20 September 2006

Abstract Cysteine proteinases are key virulence factors of Entamoeba histolytica that are released during the process of invasion. We used a chemical mutant of E. histolytica strain HM-1:IMSS, clone L6, which is deWcient in virulence, phagocytosis, and cysteine proteinase activity to help deWne the mechanisms of cysteine proteinase release. All cysteine proteinase genes of wild type HM-1 were present in the L6 mutant genome, but three of the major expressed proteinases, ehcp1, ehcp2, and ehcp5 were both transcribed, translated, and released at lower levels in L6. We hypothesized that a central protein such as the calcium binding protein 1, EhCaBP1, which is required for both phagocytosis and exocytosis might be deWcient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These Wndings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Entamoeba histolytica; Amebae; Parasite; Phagocytosis; Cysteine proteinases

1. Introduction Cysteine proteinases are key virulence factors of Entamoeba histolytica. Studies using native and recombinant proteinases have shown that they play a role in invasion through the degradation of extracellular matrix and epithelial cell basement membrane (Keene et al., 1986). Cysteine proteinases also have important interactions with the host immune system, including activation of complement (Reed et al., 1986) and degradation of the anaphylatoxins C3a and C5a (Reed et al., 1995), IgG (Tran et al., 1998), IgA (Kelsall and Ravdin, 1993), and pro-IL-18 (Que et al., 2003). The *

Corresponding author. Fax: +1 619 543 6614. Present address: Department of Obstetrics and Gynecology, University of California, Davis, Sacramento, CA, USA. 1

0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.08.004

E. histolytica genome project revealed that »40 genes encode cysteine proteinases (Loftus et al., 2005), but only a minority were expressed in cultured trophozoites (Bruchhaus et al., 2003). A recent study of the transcriptome of E. histolytica trophozoites during intestinal colonization found that the main up-regulated cysteine proteinases were EhCP4 and EhCP6 (Gilchrist et al., 2006), proteinases that are not expressed in culture (Bruchhaus et al., 2003). The study of the localization and activity of individual proteinases has been aided by the puriWcation of native and active, recombinant enzymes (Keene et al., 1986; Jacobs et al., 1998; Li et al., 1995; Que et al., 2002). At least three proteinases, EhCP2 (ACP2), EhCP5, and CP112 localize to the plasma membrane (Jacobs et al., 1998; Que et al., 2002; Garcia-Rivera et al., 1999), and EhCP3 (ACP1) is cytoplasmic (Que et al., 2002). Studies of the role of cysteine

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proteinases in the pathogenesis of other parasites have been greatly aided by gene knock-outs (reviewed in Rosenthal, 1999; Sajid and McKerrow, 2002), but homologous recombination has yet to be achieved in E. histolytica. In vivo studies using cysteine proteinase inhibitors (Stanley et al., 1995) and antisense constructs (Ankri et al., 1999) showed that loss of cysteine protease activity by amebae lead to reduced liver abscess formation in SCID mice and hamsters. However, cysteine proteinase inhibitors used to date are active against all of the amebic cathepsins tested (Que et al., 2002), and the EhCP5-antisense construct likely aVects multiple genes as well (Ankri et al., 1999). The only stable proteinase deWcient strain of E. histolytica, L6, was initially isolated as a phagocytosis deWcient clone (Orozco et al., 1983), and the basis for its decreased virulence has not been completely understood. We now show that this mutant also has decreased passive release of multiple cysteine proteinases, which is due to lower levels of calcium binding protein 1 (EhCaBP1), a central protein controlling phagocytosis and exocytosis. 2. Materials and methods 2.1. Entamoeba strains Entamoeba histolytica strain HM1:IMSS and the clone L6 (Orozco et al., 1983), were grown axenically in TYI-S-33 medium and subcultured every 48–72 h. Monoxenic Entamoeba dispar, SAW 1734, was grown in YI supplemented with Crithidia. 2.2. Cysteine proteinase activity Cysteine proteinase activity was measured in supernatants and lysates of trophozoites (5£106/ml) in PBS-Cys++ (PBS supplemented with 20 mM cysteine, 0.15 mM CaCl2, and 0.5 mM MgCl2) for 2 h at 37 °C as previously described (Reed et al., 1993). The liberation of the Xuorescent leaving group, 7-amino-4-methyl coumarin (AMC) from the peptide substrate, benzyloxycarbonyl-Arg-Arg-AMC (Enzyme Systems Products, Livermore, CA) was determined and calculated as the initial velocity of cleavage as nM/ml/min (Reed et al., 1993).

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oligo (dT) (Stratagene, La Jolla, CA). PCRs were performed in triplicate using SYBR Green PCR Core Reagents (Perkin-Elmer). The PCR product was continually monitored in a Perkin-Elmer Gene Amp 5700 Sequence Detection system with Quantitation software, which analyzes the ampliWcation curves derived from the incorporation of SYBR Green into the double stranded DNA product. The relative amount of product generated was measured by the threshold cycle (CT), when the level of speciWc PCR product increases exponentially and crosses the threshold of a passive reference dye in each sample. Individual CT values were based on three separate measurements. Each experimental mRNA level was normalized to an equivalent expression level of glyceraldehyde-3-phosphate dehydrogenase (GenBank Accession No. M89790). The speciWcity of the PCR ampliWcation was veriWed by melting curves of the Wnal amplicons. 2.5. Microarray hybridizations Total RNA was isolated from HM-1 and L6 trophozoites using TRIZOL (Invitrogen). cDNA synthesis, microarray hybridization, and microarray scanning were performed as described (Debnath et al., 2004). BrieXy, cDNA was synthesized from total RNA extracted from HM-1:IMSS or L6, coupled to CyScribe Cy3 or Cy5 (Amersham), and hybridized to the shotgun DNA microarray constructed by use of 6144 random inserts from an E. histolytica genomic DNA library (Debnath et al., 2004). Four hybridizations were performed with HM-1 trophozoite RNA labeled with Cy3 and L6 trophozoite RNA labeled with Cy5 (EhCS1p1-095, EhCS1p1-096, EhCS1p1102, and EhCS1p1-103); one hybridization with HM-1 trophozoite RNA labeled with Cy5 and L6 trophozoite RNA labeled with Cy3 (EhCS1p1-099). Scanning and data analysis were performed as previously described (Debnath et al., 2004). Only spots in which almost more than 70% of the pixels had a signal of at least twice the standard deviation (SD) of the local background were considered in subsequent data analysis. Clones whose expression decreased at least 4-fold in L6 relative to HM-1 were selected for further sequencing analysis. 2.6. Cysteine proteinase ELISA

2.3. PCR ampliWcation of cysteine proteinase genes Genomic DNA was puriWed from E. histolytica, L-6 and E. dispar trophozoite nuclei using the Wizard DNA puriWcation kit (Promega). Oligonucleotide primers were speciWcally designed to amplify each of the 40 cysteine proteinase genes (Bruchhaus et al., 2003). Products were visualized by ethidium bromide staining of agarose gels. 2.4. Kinetic RT-PCR Total RNA was extracted from trophozoites using the RNeasy Mini Kit (Qiagen) and cDNA synthesized with

To develop an ELISA to quantify the amount of proteinases released into media, EhCP1, EhCP2, EhCP3, and EhCP5 were all expressed as recombinant proteins as previously described (Que et al., 2002). Monoclonal antibody to EhCP3 (ACP1) and polyclonal anti-peptide antibody to EhCP2 (ACP2) were made as previously described (Que et al., 2002). Polyclonal antibodies were raised against recombinant EhCP1 (expressed in pRSETA, Invitrogen) and EhCP5 (in pBAD/Thio-TOPO, Invitrogen) and puriWed by nickel chromatography. Rabbits were injected subcutaneously three times with 100 g of each recombinant proteinase with Titermax (Sigma), and the IgG fraction was

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puriWed using Protein A chromatography (Pharmacia). To test for speciWcity of the antibodies, 1.0 g of each recombinant antigen (rEhCP1, 2, 3, and 5) was electrophoresed on 12% gels, transferred to nitrocellulose, reacted with a 1–5 g/ml dilution of each antibody, and detected with goat anti-mouse or anti-rabbit IgG alkaline phosphatase and BCIP/NBT (5-bromo, 4-chloro, 3-indolylphosphate/nitroblue tetrazolium, Zymed, San Francisco, CA). To quantify the amount of released proteinases, standard curves were generated with 6.5–50 ng of each recombinant protein. Released proteinases were collected as above (2.2), and a 1:100 dilution of the media (50 l) was used to coat 96 well plates in duplicate overnight at 4 °C and blocked with PBS-3% bovine serum albumin. Plates were extensively washed with PBS-0.05% Tween between each step and sequentially incubated with an »1.0 g/ml dilution of the polyclonal or monoclonal antibodies, followed by goat anti-mouse or anti-rabbit IgG horseradish peroxidase conjugates. Plates were developed with peroxidase substrate SureBlue, KPL, Gaithesburg, Md), 100 l Stop solution was added, and the absorbance read at 450 nm. 2.7. Phagocytosis assay Phagocytosis was measured by incubating trophozoites (1 £ 104) in TY-I-S33 without serum with Xuorescent Nile Red Sulfate polystyrene latex beads (2 £ 107, Interfacial Dynamics, Portland, Ore.) for 30 min at 37 °C. The extracellular beads were removed by washing, trophozoites Wxed for 10 min in 4% formaldehyde at 4 °C, and the number of phagocytosed beads determined in aliquots of trophozoites by Xuorescent microscopy.

Trophozoites were allowed to attach to chamber slides (Lab Tek, Nunc), phagocytose human erythrocytes for 10 min, and Wxed for immunoXuoresence labeling as previously described (Sahoo et al., 2004) with 3.7% warmed paraformaldehyde and permeabilized with 0.1% Triton X-100/PBS. EhCaBP1 polyclonal antibody was used at a 1:50 dilution, anti-rabbit Alexa 488 (Molecular Probes) at 1:500 and phalloidin (Sigma) at 1:500. Imaging was performed on a Nikon Optiphot Xuorescent microscope. 3. Results 3.1. L6 has less total cysteine proteinase activity than E. histolytica or E. dispar To deWne the defect in L6, we Wrst compared the amount of cysteine proteinase activity in lysates and released in media by E. histolytica, L6, and E. dispar. E. histolytica lysates had signiWcantly more cysteine proteinase activity than L6 or E. dispar (p < 0.001 by Student’s paired t test, Fig. 1). E. histolytica (HM-1:IMSS) also released signiWcantly more proteinase into the media than E. dispar or L6 (p < 0.001 by Student’s paired t test, Fig. 1). 3.2. All cysteine proteinase genes are present in L6 To determine if chemical mutagenesis in L6 resulted in full or partial deletions of cysteine proteinase genes, we ampliWed products containing the translated regions of all forty cysteine proteinases. All genes were detected in the L6 genome (data not shown).

2.8. EhCaBP1 immunoblots and Xuorescence microscopy The levels of EhCaBP1 in HM-1 and L6 were compared in lysates by immunoblots. Freshly harvested Entamoeba cells were resuspended at 106/ml in lysis buVer (50 mM Tris, 2 mM EDTA, pH 7.4, 0.1% NP-40 with inhibitors (Complete Mini, Roche) plus additional E64 to a Wnal concentration of 100 M). The cell lysates were vortexed and frozen at ¡70 °C. After two freeze thaw cycles and a 15 s sonication, the lysates were centrifuged at 10,000g for 5 mins at 4 °C, and the cleared supernatants were assayed for protein concentration. Equal amounts of the lysates (400 ng) were electrophoresed (4–20% gradient gels; Novex) and transferred to PVDF membranes. Following blocking with 5% milk/TBS/0.05% Tween 20, the blots were probed with rabbit anti-EhCaBP1 serum (Sahoo et al., 2004) (1:2000 dilution) or monoclonal antibodies to the peroxiredoxin (FP-10,11, and 19, 0.1 g/ml Wnal concentration, Choi et al., 2005), followed by Zymax™ goat anti-rabbit or goat-anti mouse IgG HRP conjugate. The blots were developed with SuperSignal Chemiluminescent Substrate (Pierce). After imaging the developed blots, band densities were determined using Quantity One software (Bio-Rad).

Fig. 1. L6 has less intracellular and released cysteine proteinase activity. The total cysteine proteinase activity in lysates and released into media by E. histolytica, E. dispar, and L6 was measured by the initial velocity of the cleavage of a Xuorescent peptide substrate (Z-Arg-Arg-AMC). Values represent the means § SE of 17 determinations.

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3.3. Cysteine proteinases genes are transcribed at lower levels in L6 We next evaluated the transcription levels of the Wve major expressed cysteine proteinase genes using real-time (RT) PCR. We found that all Wve genes were transcribed in L6, but ehcp1, ehcp2, and ehcp5 were all transcribed at 4- to 8-fold lower levels than E. histolytica (Table 1). The levels of EhCP3 and EhCP112 were unchanged. 3.4. Transcriptional diVerences between HM-1 and L6 To measure variation in gene expression between HM-1 and L6, the arrays were probed with diVerentially labeled cDNAs, prepared from total RNA isolated from HM-1 and L6. Through diVerential hybridization, genes coding for a cysteine proteinase (clone AEC 3223), proteins implicated in cytoskeletal reorganization (clone AEC 1293), and the Gal/GalNAc lectin (clone AEC 2837) were found to be down-regulated in L6 compared to wild type HM1. A clone (AEC 3223) showing homology (at the amino acid level) with E. histolytica cysteine proteinase, EhCP1 (99%), EhCP2 (79%) and EhCP8 (68%) was 6- to 10-fold downregulated in L6 in diVerent hybridizations. A histogram showing the distribution of the signal for each Xuorophore over the entire array indicated that at least 1500 clones were expressed at signiWcantly lower levels (64-fold) in L6 compared with HM1 (Fig. 2). The microarray hybridization data used in this report are available at http:// www.ucsf.edu/mckerrow/amoebaarray.html. 3.5. L6 releases less cysteine proteinases by ELISA To measure the actual release of individual proteinases, we expressed recombinant antigens of EhCP1, EhCP2, EhCP3, and EhCP5 and produced monoclonal or polyclonal antibodies, which do not cross-react (Fig. 3). E. histolytica (HM-1:IMSS),L6,or E. dispar trophozoites were incubated in media for 2 h and the amount of each proteinase released determined by comparison with standard curves. EhCP2 was the major released proteinase, followed by EhCP5 > EhCP1 > EhCP3. The same order of release was found in E. dispar and L6, but the magnitude was greatly decreased (Fig. 4). Interestingly, the level of release did not correlate directly with the

Fig. 2. HM1 vs. L6 gene expression. Histogram of the distribution of HM1-speciWc and L6-speciWc signals. The log transformation values of the ratio of medians (635/532) were plotted on the X-axis and number of spots on the array was plotted on the Y-axis. The number of clones upregulated in L6 are shown in black and the number of clones down-regulated in L6 in gray.

transcription level for the same genes, which was EhCP1 > EhCP2 > EhCP5 > EhCP3. 3.6. L6 is deWcient in phagocytosis and passive cysteine proteinase release To evaluate a potential link between phagocytosis and proteinase release, we Wrst compared the ability of E. histolytica (HM-1:IMSS),L6, and E. dispar to phagocytose Xuorescent beads. E. histolytica (HM-1:IMSS) was highly phagocytic with >80% of trophozoites phagocytosing more than one bead (p < 0.002 vs. E. dispar, p < 0.001 vs. L6, Fig. 5). Only 13% of L6 trophozoites phagocytosed a single bead. Similar results were found with GFP-labeled bacteria (results not shown). These Wndings were not due to delayed phagocytosis by L6 as the same diVerences in phagocytosis were found at 24 h (data not shown). We hypothesized that cysteine proteinase might be passively released or exocytosed during the process of phagocytosis. To test this, we allowed E. histolytica (HM-1:IMSS) trophozoites to phagocytose beads for 30 min and then determined the cysteine proteinase activity released in the media. E. histolytica (HM-1:IMSS) trophozoites increased

Table 1 Quantitative RT-PCR analysis of gene expression Gene

mRNA copy number § SE HM-1:IMSS

mRNA copy number § SE L6

Fold decrease (from HM-1)

EhCP1 EhCP2 EhCP3 EhCP5 EhCP112 EhCaBP1

1236.8 § 58.8 912.6 § 39.8 15.7 § 1.9 47.8 § 15.3 51.7 § 6.0 177.4 § 30.7

161.7 § 19.8 170.6 § 18.8 26.6 § 3.1 12.8 § 3.8 61.0 § 7.0 24.2 § 3.8

7.7a 5.3a — 3.7a — 7.3a

Copy numbers of speciWc mRNA levels were normalized to levels of G3PDH. Values represent the means + SE of three determinations. a All values are signiWcantly diVerent from the L6 as tested by Student’s t test, with a p < 0.05.

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Fig. 3. SpeciWcity of antibodies to cysteine proteinases. Western blots of recombinant EhCP1, EhCP2, EhCP3, and EhCP5 reacted with monospeciWc rabbit antibody to EhCP1 (A), anti-peptide antibodies to EhCP2 (B), monoclonal antibody to EhCP3 (C), and monospeciWc rabbit antibody to EhCP5 (D). The recombinant proteinases are expressed as fusion proteins to CheY (rACP1) or thioredoxin (rEhCP2, rEhCP3, and rEhCP5). rEhCP5 forms multimers.

the release of cysteine proteinases by 66% while no increase was detected with E. dispar or L6 (Fig. 6, p < 0.05 by Student’s t test). 3.7. Less EhCaBP1is expressed in L6 Since cysteine proteinase release appeared to be related to phagocytosis, we evaluated potential causes of defects in phagocytosis. Calcium Binding Protein 1 (EhCaBP1) was recently shown to be a central protein in control of remodeling of membranes for phagocytosis and endocytosis (Sahoo et al., 2004). When we compared the mRNA levels for EhCaBP1 in E. histolytica and L6, we found it was decreased 7-fold in L6 (Table 1). To conWrm that this resulted in lower expression of EhCaBP1 protein, we compared protein levels in lysates of E. histolytica and L6 trophozoites. On immunoblots of lysates corrected for protein concentrations, we found that EhCaBP1 was reduced 70% in L6 trophozoites (Fig. 7). When the co-localization of EhCaBP1 and polymerized actin was compared in E. histolytica and L6 trophozoites, less EhCaBP1 was present in L6 trophozoites, but no diVerences in the co-localization with actin could be detected (data not shown).

Fig. 5. L6 phagocytoses fewer beads than E. histolytica or E. dispar. The number of Xuorescent beads phagocytosed by E. histolytica, E. dispar, and L6 trophozoites under serum free conditions in 30 min was determined by Xuorescent microscopy. Each value represents the mean of at least three experiments § SE.

4. Discussion A number of virulence factors are required for E. histolytica to successfully invade the bowel. Before trophozoites

Fig. 4. Extracellular release of cysteine proteinases. The amount of EhCP1, EhCP2, EhCP3, or EhCP5 released into the media was measured by ELISA. Standard curves were generated using recombinant antigen of each of the four proteinases (6.5–50 ng) and detected with monoclonal antibodies to EhCP3 and polyclonal antibodies to EhCP1, 2, and 5. Released proteinases were measured in 50 l of media in triplicate, and the amount of each proteinase released (nanograms)/l of media was determined from the standard curves run with each experiment. The decrease in proteinase release by L6 vs. HM-1 was signiWcant for EhCP1 (p D 0.003), EhCP2 (p D 0.02), and EhCP5 (p D 0.05 by Student’s t test).

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even encounter the epithelium, the cellular junctions begin to separate (Takeuchi and Phillips, 1975), likely the result of released cysteine proteinases on components of the epithelial desmosomes and basement membrane (Keene et al., 1986). Attachment to epithelial cells occurs via the GalGalNac lectin (Dodson et al., 1999), and host cells are killed through the action of an amebapore (Leippe et al., 1991). E. histolytica trophozoites are actively phagocytic in the bowel and during invasion, ingesting bacteria and host cell debris. One of the major questions in amebic pathogenesis is how

Fig. 6. Entamoeba histolytica increases release of cysteine proteinase activity after phagocytosis, but E. dispar and L6 do not. To evaluate the passive release of cysteine proteinases during phagocytosis, the total cysteine proteinase activity released in the media was compared in trophozoites of E. histolytica, E. dispar, and L6 incubated in serum free growth media for 30 min, with or without latex beads. The % increase in cysteine proteinase release following phagocytosis of latex beads was determined. Values represent the mean of at least three determinations § SE.

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E. histolytica can invade while E. dispar, which also has genes encoding galactose-inhibitable lectins, amebapores, and all but two of the cysteine proteinase genes, cannot. To further understand the link between phagocytosis and cysteine proteinase release, we took advantage of the L6 clone. This unique mutant of E. histolytica strain HM-1 was selected for deWcient phagocytosis by ingestion of BUdR-labeled bacteria (Orozco et al., 1983). Coincidentally, the clones were also found to be deWcient in cysteine proteinase activity (Keene et al., 1990), however the mechanisms were not understood. Although chemical mutants of E. histolytica have not been identiWed in vivo, this clone provided a unique opportunity to study some of the key requirements for expression and release of cysteine proteinases. We conWrmed that L6 has less cysteine proteinase activity in lysates and media than even noninvasive E. dispar (Fig. 1). This defect was not attributable to deletions of any cysteine proteinase genes, although it is possible that point mutations within the coding or regulatory regions of the cysteine proteinase genes might aVect their transcription or translation and will be the focus of future studies. When levels of mRNA of the major released proteinases were evaluated by RT-PCR, EhCP1, 2, and 5 were signiWcantly reduced in L6 (Table 1). Microarray analysis conWrmed a 6- to 10-fold lower level of EhCP1 mRNA in L6. All of the proteinases measured by a sensitive ELISA (Fig. 4) were released in lower amounts in L6, although in the same relative order. L6 lysates also have less EhCP112 as part of a complex with a surface adhesin (Ocadiz et al., 2005). This suggests that the same proteinases are released passively through phagocytosis, but transport and active

Fig. 7. L6 trophozoites express less EhCaBP1. E. histolytica and L6 trophozoite lysates (400 ng protein) were electrophoresed by SDS–PAGE and the resulting immunoblots reacted with polyclonal antisera to EhCaBP1. Equivalent bands are seen in a loading control reacted with monoclonal antibodies to the amebic peroxiredoxin (which forms dimers).

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release of the cysteine proteinases may diVer, an area we are actively investigating. L6 trophozoites had signiWcantly reduced phagocytosis, even less than noninvasive E. dispar (Fig. 5). Cysteine proteinase activity might be co-regulated in some fashion with the process of phagocytosis, either involving passive release or exocytosis (see below) (Fig. 6). There was no increased release of cysteine proteinases into the media with the poorly phagocytic E. dispar or L6, while E. histolytica released 66% more cysteine proteinase activity. Phagocytosis is an active process in E. histolytica, involving actin polymerization controlled by a number of proteins. Overexpression of the mutant p21racA-V12, which forces GTPase to be Wxed in the active state, caused accumulation of actin under the plasma membrane and markedly decreased phagocytosis (Ghosh and Samuelson, 1997). The Rab proteins are also important regulators of vesicular transport, including cysteine proteinases (Temesvari et al., 1999; Saito-Nakano et al., 2004; Nakada-Tsukui et al., 2005). RabB was overexpressed in L6, but the complex remained in cytoplasmic vesicles and was ineYciently translocated to phagocytic vesicles (GuzmanMedrano et al., 2005). Both EhRab5 and Rab7 have been linked with the transport of cysteine proteinases via a prephagosomal vacuole, but the pathways controlling speciWc cysteine proteinases remain to be determined (Saito-Nakano et al., 2004; Nakada-Tsukui et al., 2005; Okada et al., 2005). Because both phagocytosis and the passive release of multiple cysteine proteinases were disrupted in this clone, we hypothesized that a central control protein was involved. A potential candidate is EhCaBP1, which colocalizes with polymerized F-actin and inhibition by antisense expression impaired both processes (Sahoo et al., 2004).We found that less EhCaBP1 mRNA expression (Table 1) and less EhCaBP1 protein were detected in L6 (Fig. 7). Calcium Xuxes have recently been linked with membrane repair and lysosomal exocytosis, manifest as genetic defects in beige-J mice and the human recessive disorder, Chediak-Higashi syndrome (Huynh et al., 2004). The disruption of calcium homeostasis mediated by EhCaBP1 in the L6 mutant lends further support to this link in a lower eukaryote, leading to the defective phagocytosis, proteinase release, and virulence in this mutant. Acknowledgments This work was presented in part at the XIV Seminar on Amebiasis, Mexico City, November 27–30, 2000 (Abstract S237). The work was supported by grants from the United States Public Health Service Grant RO1AI49531 (S.R.), DK-35108 (S.R.), and AI35707 (J.M.), and the Sandler Center for Basic Research in Parasitic Diseases. S.G.M.L was a recipient of a PROMEP-UABC Scholarship (UABC-123, Clave P/PROMEP: UABC-2000-12-01; Ministry of Public Education, México.

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