A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design

Activity of Thioallyl Compounds From Garlic Against Giardia duodenalis Trophozoites and in Experimental Giardiasis

Fresh aqueous extracts (AGEs) and several thioallyl compounds (TACs) from garlic have an important antimicrobial activity that likely involves their interaction with exposed thiol groups at single aminoacids or target proteins. Since these groups are present in Giardia duodenalis trophozoites, in this work we evaluated the anti-giardial activity of AGE and several garlic's TACs. In vitro susceptibility assays showed that AGE affected trophozoite viability initially by a mechanism impairing cell integrity and oxidoreductase activities while diesterase activities were abrogated at higher AGE concentrations. The giardicidal activities of seven TACs were related to the molecular descriptor HOMO (Highest Occupied Molecular Orbital) energy and with their capacity to modify the –SH groups exposed in giardial proteins. Interestingly, the activity of several cysteine proteases in trophozoite lysates was inhibited by representative TACs as well as the cytopathic effect of the virulence factor giardipain-1. Of these, allicin showed the highest anti-giardial activity, the lower HOMO value, the highest thiol-modifying activity and the greatest inhibition of cysteine proteases. Allicin had a cytolytic mechanism in trophozoites with subsequent impairment of diesterase and oxidoreductase activities in a similar way to AGE. In addition, by electron microscopy a marked destruction of plasma membrane and endomembranes was observed in allicin-treated trophozoites while cytoskeletal elements were not affected. In further flow cytometry analyses pro-apoptotic effects of allicin concomitant to partial cell cycle arrest at G2 phase with the absence of oxidative stress were observed. In experimental infections of gerbils, the intragastric administration of AGE or allicin decreased parasite numbers and eliminated trophozoites in experimentally infected animals, respectively. These data suggest a potential use of TACs from garlic against G. duodenalis and in the treatment of giardiasis along with their additional benefits in the host's health

Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens—Factors Linked to Virulence and Pathogenicity

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens’ virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets

Trichinella spiralis : Stimulation of mast cells by TSL-1 antigens trigger cytokine mRNA expression and release of IL-4 and TNF through an Ig-independent pathway

International audiencePrevious in vitro studies have demonstrated that mast cells (MC) can be directly activated by Trichinella spiralis larvae 1 (TSL-1) antigens. To characterize even more this activation of MC and their possible role on induction and regulation of the Type 2 response generated against T spiralis infection, we studied the interaction between a hybrid rat MC line (HRMC), murine bone marrow MC (mBMMC), and TSL-1 antigens. Immunofluorescence staining and flow cytometry analysis showed that TSL-1 antigens bound to the surface of HRMC cells, resulting in the transcriptional induction and in the release of TNF and IL-4. Besides, an increase of IL-4 intracellular expression was also observed in mBMMC. This Suggests that MC may play an important role in the early immune response against T spiralis and may be a source of cytokines, that regulate the final onset of the immune mechanisms which determine the course of the infection

Genetic homogeneity of axenic isolates of Giardia intestinalis derived from acute and chronically infected individuals in Mexico

Cedillo-Rivera, Roberto ; Darby, Jocelyn M. ; Enciso-Moreno, J. Antonio ; Ortega-Pierres, Guadalupe ; Ey, Peter L

Expression of the Plasmodium falciparum Immunodominant Epitope (NANP)(4) on the Surface of Salmonella enterica Using the Autotransporter MisL

Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by the sec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 of Salmonella enterica and classified as an autotransporter due to its high homology to Escherichia coli AIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) from Plasmodium falciparum on the surface of Salmonella enterica serovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL β domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind the Escherichia coli heat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically inducible nirB promoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP in P. falciparum sporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria