Entamoeba histolytica: cytopathogenicity and lectin activity of avirulent mutants

Three clones of Entamoeba histolytica (L-6, C93, C919) were isolated by mutagenesis with ethyl methanesulfonate from the axenic strain HM1:IMSS and were studied for adherence, cytolytic, and soluble galactose inhibitable lectin activity. Avirulent clones adhered to and killed fewer Chinese hamster ovary cells than HM1:IMSS (P less than 0.01). However, only C919 was deficient in adherence to red blood cells. Galactose (1.0 g) completely inhibited adherence of all the mutants to Chinese hamster ovary cells; however, adherence to erythrocytes was only partially inhibitable by galactose. Avirulent mutants were more susceptible to being killed by human neutrophils in vitro (P less than 0.01 compared to HM1:IMSS). Soluble protein preparations from all the avirulent mutants were markedly less mitogenic for human lymphocytes and had lower lectin activity for Chinese hamster ovary cells compared to the HM1:IMSS wild type (P less than 0.01 for each activity with each mutant). Indirect immunofluorescence with a monoclonal antibody (F-14) that recognizes the Gal/GalNAc lectin was positive for L-6 and C919. These findings utilizing avirulent mutants of E. histolytica further support a role for the amebic galactose inhibitable lectin in the in vivo pathogenesis of amebiasis.     

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family

Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.     

Detergent-resistant membrane domains are required for mast cell activation but dispensable for tyrosine phosphorylation upon aggregation of the high affinity receptor for IgE

Aggregation of the high affinity receptor for IgE (FceRI) on mast cells results in the rapid phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn, which initiates the signaling cascades leading to secretion of inflammatory mediators. The detergent-resistant membranes (DRMs) have been implicated in FcepsilonRI signaling because aggregated receptors emigrate to DRMs that are enriched in certain signaling components. We evaluated the role of DRMs in FcepsilonRI signaling by disruption of DRMs using a cholesterol-binding agent, methyl-beta-cyclodextrin (MBCD). While treatment of rat basophilic leukemia cells with MBCD inhibits degranulation and Ca(2+) mobilization upon aggregation of FcepsilonRI, MBCD hardly affects the aggregation-induced tyrosine phosphorylation of FcepsilonRI as well as other signaling molecules such as phospholipase C-gamma1 (PLC-gamma1). MBCD delocalizes phosphatidylinositol 4,5-bisphosphate from DRMs, which may prevent MBCD-treated cells from producing inositol 1,4,5-trisphosphate by means of activated PLC-gamma1. These data suggest an indispensable role for DRMs in the Ca(2+) response rather than tyrosine phosphorylation, and support a model of receptor phosphorylation in which aggregated FcepsilonRI is tyrosine phosphorylated outside DRMs by constitutively associated Src family kinase Lyn via a transphosphorylation mechanism.     

Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.The removal of apoptotic cells, known as efferocytosis, is a series of arranged events from the recruitment of phagocytes to sites where apoptotic cells are generated to the digestion of apoptotic cells by phagocytes.^{1, 2, 3} One of the key steps during efferocytosis is the recognition of dying cells by phagocytes. Phagocytes can detect apoptotic cells by the direct or indirect association of multiple receptors on phagocytes with ligands on apoptotic cells.^{4, 5, 6, 7, 8, 9} Some receptors on the surface of phagocytic cells not only bind to apoptotic cells but also transduce apoptotic cell recognition signals into phagocytes in order to mediate the ingestion of apoptotic cells. For instance, brain-specific angiogenesis inhibitor 1 (BAI1) and stabilin-2, which are phosphatidylserine (PtdSer) receptors, recognize PtdSer on apoptotic cells and relay signals to the Elmo-Dock-Rac module and Gulp, respectively, via their cytoplasmic tails.^{8, 10, 11} By contrast, it has been suggested that other receptors, called tethering receptors, merely tether apoptotic cells to phagocytes without mediating downstream signal transduction, following which the internalization of apoptotic cells is mediated by the association of these receptors with co-receptors or other engulfment receptors located nearby.^{12, 13, 14, 15, 16} However, it is unclear whether co-receptors for tethering receptors exist in tethering receptor-mediated phagocytosis of apoptotic cells, and, if they do, whether they are indispensable for this process.One intriguing characteristic of tethering receptors is that they have cytoplasmic tails lacking any signaling motifs or are anchored via glycophosphatidylinositol (GPI) to the outer leaflet of the plasma membrane. For example, Tim-4, a PtdSer receptor with a short cytoplasmic tail that promotes the engulfment of apoptotic cells by the binding of its IgV domain to PtdSer on apoptotic cells, lacks signaling motifs in its cytoplasmic tail. It has been known that neither the cytoplasmic tail nor the transmembrane region of Tim-4 is essential for Tim-4-mediated engulfment of apoptotic cells. Accordingly, it functions as a tethering receptor to secure apoptotic cells on phagocytes.^{9, 14} CD14 is located at the exofacial leaflet of the plasma membrane through its GPI anchor, which rules out the possibility that it mediates direct signal transduction into phagocytes after binding to apoptotic cells. Consequently, it is also considered to be a tethering receptor.¹⁵Phospholipids such as PtdSer and phosphatidylcholine (PtdCho) are unequally distributed between the inner and outer leaflets of the plasma membrane in the normal state. For instance, uncharged phospholipids such as PtdCho and sphingomyelin are primarily located in the outer leaflet, whereas positively or negatively charged phospholipids (such as phosphatidylethanolamine or PtdSer, respectively) are restricted to the inner leaflet facing the cytosol.^{17, 18, 19} However, this asymmetric distribution of phospholipids in the plasma membrane is disrupted during apoptosis. In the plasma membrane of apoptotic cells, PtdSer is exposed to the outer leaflet of the plasma membrane by the activity of scramblases and flippases.^{18, 20, 21} Thus, exposed PtdSer is a hallmark of apoptotic cells and is the best characterized ligand on apoptotic cells for efferocytosis. PtdSer on the surface of apoptotic cells can be recognized by various PtdSer-sensing membrane proteins on phagocytes, collectively called PtdSer receptors, including tethering receptors.Besides PtdSer receptors, many PtdSer-binding proteins have been identified. These proteins are involved in various biological processes such as blood coagulation, synaptic vesicle fusion, membrane scaffolding, and signal transduction.²² One of the best known proteins is annexin A5, which has been extensively studied as a PtdSer-binding protein. Annexin A5 belongs to the family of annexins, which are characterized by their Ca²⁺-dependent ability to bind to negatively charged phospholipids and share structural properties. Annexins are considered to be cytosolic proteins because they lack a 5′ leader sequence; however, some annexins, including annexin A5, are also found on the cell surface and in the circulation. This and related properties imply that annexins participate in diverse biological events from membrane dynamics to cell differentiation and migration.^{23, 24, 25} However, the physiological significance of this family is poorly understood. Among annexins, annexin A5 binds to PtdSer with high affinity. Because of this property, annexin A5 has been harnessed as a molecular probe to distinguish apoptotic cells from live cells both in vivo and in vitro for decades.^{25, 26}In this study, annexin A5 was expressed on the cell surface through a GPI anchor to delineate whether a tethering receptor without its co-receptor can promote efferocytosis. GPI-anchored annexin A5 (Anxa5-GPI) should not interact with any plasma membrane or extracellular protein, at least those involved in the engulfment of apoptotic cells. Thus, it is possible to exclude the effects of co-receptors on Anxa5-GPI-mediated phagocytosis of apoptotic cells. The expression of Anxa5-GPI in phagocytes promoted not only the binding but also the internalization of apoptotic cells. By contrast, phagocytosis of carboxylate beads and Escherichia coli was not affected by the expression of Anxa5-GPI in phagocytes. Anxa5-GPI-induced efferocytosis was not only partially dependent on a specific engulfment pathway but also relied on the generally known cytoskeletal engulfment machinery. Our observations suggest that co-receptors are dispensable for tethering receptor-mediated efferocytosis. In addition, tethering receptors could enhance efferocytosis through diverse engulfment machinery located nearby.     

Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence

Background

Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world.

Methodology/Principal Findings

Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants.

Conclusions/Significance

Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.     

Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica.

Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A-Sepharose, hydroxylapatite and DEAE-Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S, 3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylg uanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis.     

Entamoeba histolytica: cytopathogenicity of intact amebae and cell-free extracts; isolation and characterization of an intracellular toxin.

Toxicity assays on cell-free extracts of virulent and nonvirulent strains of Entamoeba histolytica were carried out in microtiter plates. These extracts had a cytopathogenic effect (CPE) on monolayers of baby hamster kidney cells. CPE was inhibited by normal human serum, fetal calf serum, and other sera, probably due to their IgG component. Using gel chromatography the toxic material, a protein, was found in a fraction with molecular weight between 35,000 and 45,000. This fraction contained a strong glycoprotein antigen. CPE caused by this toxin differs in several ways from the earlier described “contact lysis” caused by intact amebae. The possible significance of these two modes of toxicity of Entamoeba histolytica for the pathogenesis of amebiasis is discussed.     

Entamoeba histolytica: cytopathogenicity, including serum effects on contact-dependent and toxin-induced lysis of hamster kidney cell monolayers

Some properties of toxin, isolated from extracts of axenically cultivated Entamoeba histolytica or excreted by intact amoebae, were investigated using a toxicity assay in microplates with monolayers of baby hamster kidney cells. Preparative isoelectric focusing showed that the highest cytotoxic activity was present in a fraction of antigen containing protein bands with an isoelectric point between 4.5 and 5. Activity of the toxin was stable between pH 4 and 10. Nonimmune rabbit serum and concanavalin A, coupled to Sepharose beads, were able to bind a large amount of toxin. Cytotoxicity of antigen was inhibited by specific immune IgG and by unknown factors in nonimmune serum with a molecular weight between 50,000 and 100,000. The toxin was inactivated by trypsin, but not by trypsin inhibitor. Its activity was thiol dependent. Serum also had a marked inhibitory effect on contact lysis of BHK cells induced by intact trophozoites. A considerable reduction of both contactdependent and toxin-induced Cytopathogenicity was observed when Diamond's TP-S-1 medium was used in the assay, in which the TP broth had been autoclaved. It is suggested that Entamoeba histolytica exocytozes toxin, which acts on adjacent cells during close contact.     

The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.     

TAM receptors are dispensable in the phagocytosis and killing of bacteria

Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria. Tyro3, Axl, and Mertk (TAM) are important in the phagocytosis of apoptotic cells by macrophages. Animals lacking these receptors are hypersensitive to bacterial products. In this report, we examine whether the TAM receptors are involved in the phagocytosis of bacteria. We found that macrophages lacking Mertk, Axl, Tyro3 or all three receptors were equally efficient in the phagocytosis of Gram-negative E. coli. Similarly, the phagocytosis of E. coli and Gram-positive S. aureus bioparticles by macrophages lacking TAM receptors was equal to wild-type. In addition, we found that Mertk did not play a role in killing of extracellular E. coli or the replication status of intracellular Francisella tularensis. Thus, while TAM receptors may regulate signal transduction to bacterial components, they are not essential for the phagocytosis and killing of bacteria.     

Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan

Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers.     

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.     

Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity.

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.     

Purification, refolding and autoactivation of the recombinant cysteine proteinase EhCP112 from Entamoeba histolytica

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies.     

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica , we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.     

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

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 deficient in virulence, phagocytosis, and cysteine proteinase activity to help define 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 deficient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These findings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant.