The Genomes of Two Strains of Taenia crassiceps the Animal Model for the Study of Human Cysticercosis.

Human cysticercosis by Taenia solium is the major cause of neurological illness in countries of Africa, Southeast Asia, and the Americas. Publication of four cestode genomes (T. solium, Echinococcus multilocularis, E. granulosus and Hymenolepis microstoma) in the last decade, marked the advent of novel approaches on the study of the host-parasite molecular crosstalk for cestode parasites of importance for human and animal health. Taenia crassiceps is another cestode parasite, closely related to T. solium, which has been used in numerous studies as an animal model for human cysticercosis. Therefore, characterization of the T. crassiceps genome will also contribute to the understanding of the human infection. Here, we report the genome of T. crassiceps WFU strain, reconstructed to a noncontiguous finished resolution and performed a genomic and differential expression comparison analysis against ORF strain. Both strain genomes were sequenced using Oxford Nanopore (MinION) and Illumina technologies, achieving high quality assemblies of about 107 Mb for both strains. Dotplot comparison between WFU and ORF demonstrated that both genomes were extremely similar. Additionally, karyotyping results for both strains failed to demonstrate a difference in chromosome composition. Therefore, our results strongly support the concept that the absence of scolex in the ORF strain of T. crassiceps was not the result of a chromosomal loss as proposed elsewhere. Instead, it appears to be the result of subtle and extensive differences in the regulation of gene expression. Analysis of variants between the two strains identified 2,487 sites with changes distributed in 31 of 65 scaffolds. The differential expression analysis revealed that genes related to development and morphogenesis in the ORF strain might be involved in the lack of scolex formation.

Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica (E. histolytica), an extracellular enteric protozoan. This infection mainly affects people from developing countries with limited hygiene conditions, where it is endemic. Infective cysts are transmitted by the fecal-oral route, excysting in the terminal ileum and producing invasive trophozoites (amoebae). E. histolytica mainly lives in the large intestine without causing symptoms; however, possibly as a result of so far unknown signals, the amoebae invade the mucosa and epithelium causing intestinal amoebiasis. E. histolytica possesses different mechanisms of pathogenicity for the adherence to the intestinal epithelium and for degrading extracellular matrix proteins, producing tissue lesions that progress to abscesses and a host acute inflammatory response. Much information has been obtained regarding the virulence factors, metabolism, mechanisms of pathogenicity, and the host immune response against this parasite; in addition, alternative treatments to metronidazole are continually emerging. An accesible and low-cost diagnostic method that can distinguish E. histolytica from the most nonpathogenic amoebae and an effective vaccine are necessary for protecting against amoebiasis. However, research about the disease and its prevention has been a challenge due to the relationship between E. histolytica and the host during the distinct stages of the disease is multifaceted. In this review, we analyze the interaction between the parasite, the human host, and the colon microbiota or pathogenic microorganisms, which together give rise to intestinal amoebiasis.

Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice.

Amoebiasis caused by the protozoan parasite Entamoeba histolytica remains a public health problem in developing countries, making the identification of new anti-amoebic compounds a continuing priority. Previously, we have shown that lactoferrin (Lf) and several Lf-derived peptides exhibit in vitro anti-amoebic activity independently of their iron-binding activity. Here, we evaluated the amoebicidal effect of synthetic Lf-derived peptides Lfcin-B, Lfcin 17-30, and Lfampin, analyzed the mechanism of death induced by the peptides and determined their therapeutic effects on murine intestinal amoebiasis. MTT assays in trophozoite cultures of E. histolytica exposed to each peptide (1-1000 µM) showed that Lfampin is far more amoebicidal than Lfcins. Lfampin killed 80% of trophozoites at doses higher than 100 µM in 24 h, and FACs analysis using Annexin V/propidium iodide showed that death occurred mainly by necrosis. In contrast, Lfcin-B and Lfcin 17-30 appeared to have no significant effect on amoebic viability. FACs and confocal microscopy analysis using FITC-labeled peptides showed that all three peptides are internalized by the amoeba mainly using receptor (PI3K signaling) and actin-dependent pathways but independent of clathrin. Docking studies identified cholesterol in the amoeba's plasma membrane as a possible target of Lfampin. Oral treatment of intracecally infected mice with the abovementioned peptides at 10 mg/kg for 4 days showed that Lfampin resolved 100% of the cases of intestinal amoebiasis, whereas Lfcin 17-30 and Lfcin-B were effective in resolving infection in 80 and 70% of cases, respectively. These data show that although synthetic bovine Lf-derived peptides exhibit varying amoebicidal potentials in vitro, they do resolve murine intestinal amoebiasis efficiently, suggesting that they may be useful as a therapeutic treatment.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures.

The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness.

Transient transgenesis of the tapeworm Taenia crassiceps.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). Infestation of the human brain, also known as neurocysticercosis, is the most common parasite disease of the central nervous system worldwide. Significant advances in the understanding of the disease have been achieved using the Taenia crassiceps murine model. We describe here a successful transfection protocol of T. crassiceps cysticerci as the first step to approach a number of currently inaccessible biological questions on cysticercosis. T. crassiceps cysticerci (ORF strain) were microinjected with the plasmid pcDNA3.1/NT-GFP-TOPO, encoding the green fluorescent protein (GFP) driven by a cytomegalovirus promoter (CMV). Twelve hours after the microinjection, GFP fluorescence gradually developed in patches associated to bud structures in the bladder wall of cysts. Fluorescence reached a peak at 24-48 h and lasted up to 72 h after the microinjection. Immunohistochemical studies on tissue sections of transfected cysts using an anti-GFP antibody, demonstrated co-localization of the antibody and the GFP fluorescence in the tegumentary cytoplasm and subtegumentary cytons. To validate at the mRNA level the expression of GFP, we carried out RT-PCR using two pairs of nested primers. Results showed expression of GFP-mRNA at 24 h post-transfection. Moreover, western blot assays of crude extracts of transfected cysts, carried out using the anti-GFP specific antibody, showed the expected protein band of 27 kDa, demonstrating that the GFP expression started at 24 after plasmid microinjection and was maintained up to 72 h. These findings will facilitate the development of functional genomics approaches applied to this model of cysticercosis.

The role of chemokines in breast cancer pathology and its possible use as therapeutic targets.

Chemokines are small proteins that primarily regulate the traffic of leukocytes under homeostatic conditions and during specific immune responses. The chemokine-chemokine receptor system comprises almost 50 chemokines and approximately 20 chemokine receptors; thus, there is no unique ligand for each receptor and the binding of different chemokines to the same receptor might have disparate effects. Complicating the system further, these effects depend on the cellular milieu. In cancer, although chemokines are associated primarily with the generation of a protumoral microenvironment and organ-directed metastasis, they also mediate other phenomena related to disease progression, such as angiogenesis and even chemoresistance. Therefore, the chemokine system is becoming a target in cancer therapeutics. We review the emerging data and correlations between chemokines/chemokine receptors and breast cancer, their implications in cancer progression, and possible therapeutic strategies that exploit the chemokine system.

Reciprocal interaction between the suprachiasmatic nucleus and the immune system tunes down the inflammatory response to lipopolysaccharide.

Several studies have shown circadian variations in the response of the immune system suggesting a role of the suprachiasmatic nucleus (SCN). Here we show that lipopolysaccharide (LPS) administration in the beginning of the active period induced more severe responses in temperature and cytokines than LPS given in the rest period. Moreover night administered LPS increased SCN basal neuronal activity indicating a direct influence of inflammation on the SCN. Bilateral lesions of the SCN resulted in an increased inflammatory response to LPS demonstrating that an interaction between the SCN and the immune system modulates the intensity of the inflammatory response.

Molecular characterization, functional expression, tissue localization and protective potential of a Taenia solium fatty acid-binding protein.

The fatty acid-binding proteins (FABPs) comprise a family of proteins that are widely expressed in animal cells and perform a variety of vital functions. Here, we report the identification, characterization, recombinant expression, tissue localization and protective potential of a Taenia solium FABP (TsFABP1). The TsFABP1 primary structure showed all the conserved residues characteristic of the subfamily iv of the intracellular Lipid-Binding Proteins (iLBPs), including those involved in the binding stabilization of the fatty acid molecule. Through a competitive binding assay we found that TsFABP1 is able to bind at least six different fatty acids with preference toward palmitic and stearic acid, suggesting that TsFABP1 is a member of the iLBP subfamily iv. Immunolocalization assays carried out on larval and adult tissues of four species of taeniids using anti-TsFABP1 hyperimmune sera produced in mice and rabbit, showed intense labeling in the tegument of the spiral canal and in subtegumental cytons of the larvae. These findings suggest that the spiral canal might be a major place for FA uptake in the developing scolex. In contrast, only subtegumental cytons in the adult worms stained positive. We propose that TsFABP1 is involved in the mechanism to mobilize fatty acids between compartments in the extensive syncytial tissue of taeniids. Protection assays carried out in a murine model of cysticercosis showed that subcutaneous immunization with TsFABP1 resulted in about 45% reduction of parasite load against an intraperitoneal challenge with Taenia crassiceps cysts. This reduction in parasite load correlated with the level of cellular and humoral immune responses against TsFABP1, as determined in spleen lymphocyte proliferation and ELISA testing.

In vitro induction of Entamoeba histolytica cyst-like structures from trophozoites.

Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment.

The genome project of Taenia solium.

We have constituted a consortium of key laboratories at the National Autonomous University of Mexico to carry out a genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis, and, in conjunction with the Echinococcus granulosus and Echinococcus multilocularis genome project of expressed sequence tags (ESTs), will mark the advent of genomics for cestode parasites. Our project is planned in two consecutive stages. The first stage is being carried out to determine some basic parameters of the T. solium genome. Afterwards, we will evaluate the best strategy for the second stage, a full blown genome project. We have estimated the T. solium genome size by two different approaches: cytofluorometry on isolated cyton nuclei, as well as a probabilistic calculation based on approximately 2000 sequenced genomic clones, approximately 3000 ESTs, resulting in size estimates of 270 and 251 Mb, respectively. In terms of sequencing, our goal for the first stage is to characterize several thousand EST's (from adult worm and cysticerci cDNA libraries) and genomic clones. Results obtained so far from about 16,000 sequenced ESTs from the adult stage, show that only about 40% of the T. solium coding sequences have a previously sequenced homologue. Many of the best hits are found with mammalian genes, especially with humans. However, 1.5% of the hits lack homologues in humans, making these genes immediate candidates for investigation on pharmaco-therapy, diagnostics and vaccination. Most T. solium ESTs are related to gene regulation, and signal transduction. Other important functions are housekeeping, metabolism, cell division, cytoskeleton, proteases, vacuolar transport, hormone response, and extracellular matrix activities. Preliminary results also suggest that the genome of T. solium is not highly repetitive.

Antigenic proteins associated with calcareous corpuscules of taenia solium: partial characterization of a calcium-binding protein.

BACKGROUND: A protein fraction was isolated from calcareous corpuscles of Taenia solium cysticerci. The antigens in this fraction were recognized in ELISA and Western blot assays by all sera from a group of patients with active neurocysticercosis (NC) and were not recognized by the sera from patients with other neurological disorders. Western blot analysis also showed that several high molecular weight proteins were strongly recognized by antibodies in all the neurocysticercotic patient sera, suggesting a potential for serological diagnosis of neurocysticercosis. METHODS: In order to characterize these antigenic proteins, we used a monoclonal antibody raised against a high MW calcium-binding protein associated with calcareous corpuscles of Echinococcus granulosus (EgCaBP1). RESULTS: Western blot assays revealed the recognition of a protein band of about 260 kDa, appearing within the range of the high MW antigens recognized by the NC sera. Several cDNA clones were isolated through screening of a T. solium metacestode library with a DNA probe for EgCaBP1, containing partial coding sequences showing about 88% identity with the protein of E. granulosus. Moreover, a recombinant product expressed in bacteria from the partial coding sequence of T. solium showed the ability to bind Ca2+ and was recognized by the monoclonal antibody. This recombinant calcium-binding protein of T. solium was not recognized by the NC patient sera by ELISA and Western blot. CONCLUSIONS: Antigenic proteins in the calcareous corpuscles of T. solium metacestodes deserve further analysis as candidates in the development of diagnostic tools for neurocysticercosis.