Cell surface proteins of Entamoeba histolytica

To ask what is new in Entamoeba histolytica research, one need look no further than the surface of this protozoan parasite. In the past year the cloning and partial characterization of five different surface antigens have been reported, a remarkable result of international research efforts against amebiosis. One of these proteins is the first protective immunogen identified in the animal model of amebic liver abscess. Barbara Mann and William Petri review these recent results, propose a nomenclature for the gene family of E. histolytica galactose lectins and discuss the roles of the different surface proteins in adhesion.     

Diversity of phosphoinositide binding proteins in Entamoeba histolytica

Phosphatidylinositol phosphates (PIPs, phosphoinositides) are localized to the membranes of all cellular compartments, and play pivotal roles in multiple cellular events. To fulfill their functions, PIPs that are located to specific organelles or membrane domains bind to and recruit various proteins in spatiotemporal specific manner via protein domains that selectively bind to either a single or an array of PIPs. In Entamoeba histolytica, the human intestinal protozoan parasite, PIPs and PIP-binding proteins have been shown to be involved in their virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis. In silico search of the domains and the signatures implicated in PIP binding in the E. histolytica proteome allows identification of dozens of potential PIP-binding proteins. However, such analysis is often misleading unless the protein domain used as query is cautiously selected and the binding specificity of the proteins are experimentally validated. This is because all the domains initially presumed to bind PIPs in other systems are not always capable of PIP binding, but rather involved in other biological roles. In this review, we carried out in silico survey of proteins which have PIP-binding domains in the E. histolytica genome by utilizing only validated PIP-binding domains that had been experimentally proven to be faithful PIP-binding bioprobes. Our survey has identified that FYVE (Fab1, YOTB1, Vac1, EEA1) and PH (pleckstrin homology) domain containing proteins are the most expanded families in E. histolytica. A few FYVE domain-containing proteins (EhFP4 and 10) and phox homology (PX) domain containing proteins (EhSNX1 and 2) were previously studied in depth in E. histolytica. Furthermore, most of the identified PH domain-containing proteins are annotated as protein kinases and possess protein kinase domains. Overall, PIP-binding domain-containing proteins that can be identified by in silico survey of the genome using the domains from well characterized bioprobes are limited in E. histolytica. However, their domain architectures are often unique, suggesting unique evolution of PIP-binding domain-containing proteins in this organism.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

The plasma membrane of Entamoeba histolytica: structure and dynamics.

The plasma membrane components of the parasitic protozoan Entamoeba histolytica, the causative agent of human invasive amebiasis, have been biochemically and immunologically characterized during the last decade. In addition, genes coding for certain surface proteins have been cloned. In spite of these advances, a unified characterization of plasma membrane antigenic components of the parasite is still required for badly needed advancements in the design of useful diagnostic, epidemiologic, and immunoprophylactic tools. Here we review current knowledge on this issue and address the problem of the considerable variation in the electrophoretic profiles of plasma membrane proteins obtained by different groups. In addition, the differences in the degree of recognition of reported membrane antigens with human immune sera, and the diverse interpretations concerning the possible functions of the surface molecules characterized are discussed. A comparative analysis of plasma membrane proteins of E histolytica trophozoites using three different isolation methods revealed that it is possible to select for specific membrane proteins, depending on the lysis conditions. In our view, the method of Calderón and Avila preserves more proteins than other methods tested. Using sera from recent cases of invasive amebiasis studied by several laboratories in various geographical areas, a basic antigenic pattern of 11 principal proteins with molecular weights of 220, 170, 150, 125, 97, 80, 60, 45, 20 and 9 kDA was established for the pathogenic E histolytica strain HM1:IMSS, used by most research groups.     

Immunosuppressive effect of Entamoeba histolytica extract on hamsters

The immune response to sheep red blood cells (SRBC) in mice and hamsters injected with an extract of entamoeba histolytica was studied. Both the primary and secondary immune response, measured by anti-SRBC antibody titers, were unaltered in the mouse, while a significant depression of the primary, but not the secondary, response was observed in the hamster. The effect was greatest when the amebic extract (AE) and SRBC were injected on the same day. The number of anti-SRBC rosettes formed in the spleen cells of hamsters treated with both AE and SRBC on day 0 was measured from days 1-16. The response peaked on day 13, while cells from animals injected with SRBC alone gave a maximal response on day 5. The formation of anti-SRBC rosettes in T-lymphocyte-enriched spleen cells treated with anti-gamma globulin serum and complement was almost abolished for the duration of the experiment. It is suggested that the mechanism responsible for this immunosuppressive phenomenon could involve early interference in the afferent limb of the immune response.     

Entamoeba histolytica: cyst-like structures in vitro induction

The cyst of Entamoeba histolytica is responsible for amebiasis infection. However, no axenic in vitro system exists that promotes mass encystation for studying this process of this human-infecting parasite. Cyst-like structures of E. histolytica obtained in this work were induced using TYI-S-33 media in combination with enterobacterias Escherichia coli and Enterococcus faecalis conditioned media, high CO2 tension and histamine. Cyst-like structures showed the same characteristics of a typical E. histolytica cyst: aggregation, resistance to 0.15% sarcosyl for 10 min, high signal of fluorescence under UV light when stained with 10% calcofluor M2r and the surface topology showed a wrinkled wall. In addition these structures are multinucleated with condensed chromatin attached to nuclear membrane, contain big vacuoles and ribonucleoproteic helices in the cytoplasm and also present a thin cell wall. Last all characteristics are all the same as a typical of E. histolytica cyst.     

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca²⁺ plays a pivotal role in amoebic pathogenesis, and Ca²⁺-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca²⁺ signaling cascade. E. histolytica CaBPs share (29–47%) sequence identity with ubiquitous Ca²⁺-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca²⁺, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.     

Codon usage in Entamoeba histolytica.

The codon usage of 10 E. histolytica genes comprising 4455 codons was analysed. The codon usage revealed an extremely biased use of synonymous codons with a preference for NNU (44%) and NNA (41.4%) codons. Codons CGG (arg), AGG (arg) and CCG (pro) were absent in the E. histolytica genes examined. The codon usage of E. histolytica resembled that of Plasmodium falciparum.     

Pyruvate-to-ethanol pathway in Entamoeba histolytica.

The pyruvate-to-ethanol pathway in Entamoeba histolytica is unusual when compared with most investigated organisms. Pyruvate decarboxylase (EC 4.1.1.1), a key enzyme for ethanol production, is not found. Pyruvate is converted into acetyl-CoA and CO2 by the enzyme pyruvate synthase (EC 1.2.7.1), which has been demonstrated previously in this parasitic amoeba. Acetyl-CoA is reduced to acetaldehyde and CoA by the enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10) at an enzyme activity of 9 units per g of fresh cells with NADH as a reductant. Acetaldehyde is further reduced by either a previously identified NADP+-linked alcohol dehydrogenase or by a newly found NAD+-linked alcohol dehydrogenase at an enzyme activity of 136 units per g of fresh cells. Ethanol is identified as the product of soluble enzymes of amoeba acting on pyruvate or acetyl-CoA. This result is confirmed by radioactive isotopic, spectrophotometric and gas-chromatographic methods.     

Virulence factors of Entamoeba histolytica.

Recent studies have increased our knowledge of Entamoeba histolytica cell biology and gene regulation. In the ameba, dominant-negative mutations in the Gal/GalNAc lectin affect adhesion and cytolysis, whereas mutations in meromyosin affect cytoskeletal function. Studying these mutant proteins has improved our understanding of the role of these proteins in E. histolytica virulence. The characterization of the CP5 cysteine protease and the induction of apoptosis in host target cells has led to a better comprehension of the mechanisms by which trophozoites can lyse target cells.