PtdIns(4,5)P2 is generated by a novel phosphatidylinositol 4-phosphate 5-kinase in the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal protist parasite that causes amoebiasis, a major source of morbidity and mortality in developing countries. Phosphoinositides are involved in signalling systems that have a role in invasion and pathogenesis of this parasite. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyses the generation of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2 ), a key species of phosphoinositide that regulates various cellular processes. However, phosphatidylinositol phosphate kinase (PIPK) family of enzymes have not been characterized in E. histolytica. Here, we report the identification and characterization of type I PIPK (EhPIPKI) of E. histolytica. Computational analysis revealed homologs of type I and III PIPK family in E. histolytica and the absence of type II PIPK. In spite of low overall sequence identity, the kinase domain was found to be highly conserved. Interestingly, a unique insertion of a tandem repeat motif was observed in EhPIPKI distinguishing it from existing PIPKs of other organisms. Substrate profiling showed that EhPIPKI could phosphorylate at third and fifth hydroxyl positions of phosphatidylinositols, though the predominant substrate was phosphatidylinositol 4-phosphate (PtdIns(4)P). Furthermore, EhPIPKI underwent intracellular cleavage close to the amino-terminal, generating two distinct fragments Nter-EhPIPKI (27p) and Cter-EhPIPKI (47p). Immunofluorescence and cellular fractionation revealed that the full-length EhPIPKI and the Cter-EhPIPKI containing carboxyl-terminal activation loop were present in the plasma membrane while the Nter-EhPIPKI was observed in the cytosolic region. In conclusion, E. histolytica has a single EhPIPKI gene that displays novel properties of post-translational processing, the presence of a repeat domain and substrate specificity not observed in any PIPK enzyme so far.

The Activity of Matrix Metalloproteinases (MMP-2, MMP-9) and Their Tissue Inhibitors (TIMP-1, TIMP-3) in the Cerebral Cortex and Hippocampus in Experimental Acanthamoebiasis.

The pathological process occurring within the central nervous system (CNS) as a result of the infection by Acanthamoeba spp. is not fully understood. Therefore, the aim of this study was to determine whether Acanthamoeba spp. may affect the levels of matrix metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-3) and MMP-9/TIMP-1, MMP-2/TIMP-3 ratios in the cerebral cortex and hippocampus, in relation to the host's immunological status. Our results showed that Acanthamoeba spp. infection can change the levels of MMP and TIMP in the CNS and may be amenable targets for limiting amoebic encephalitis. The increase in the activity of matrix metalloproteinases during acanthamoebiasis may be primarily the result of inflammation process, probably an increased activity of proteolytic processes, but also (to a lesser extent) a defense mechanism preventing the processes of neurodegeneration.

Differential expression and immunolocalization of antioxidant enzymes in Entamoeba histolytica isolates during metronidazole stress.

Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress.

Reevaluating the role of Acanthamoeba proteases in tissue invasion: observation of cytopathogenic mechanisms on MDCK cell monolayers and hamster corneal cells.

The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.

Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE.

We purified and characterized a serine proteinase secreted by Acanthamoeba healyi to evaluate it as a possible virulence factor in the pathogenesis of granulomatous amoebic encephalitis (GAE). Ammonium sulfate precipitated culture supernatant of A. healyi OC-3A strain was purified by chromatography on CM-Sepharose, Sephacryl-S200, and Q-2 anion-exchange columns. The purified 33-kDa enzyme had a pH optimum of 8.0 and a temperature optimum of 40 C. Phenylmethylsulfonylfluoride and diisopropyl fluorophosphate, serine proteinase inhibitors, diminished activity of the enzyme to near zero. In addition to types I and IV collagen and fibronectin, the main components of the extracellular matrix, other proteins such as fibrinogen, IgG, IgA, albumin, and hemoglobin were also degraded by the enzyme. The broad substrate specificity of this secreted serine proteinase suggests that it may play an important role in pathogenesis of GAE by A. healyi.

Energy production in Entamoeba histolytica: new perspectives in rational drugs design

The amebicidal action of metronidazol is activated when the enzyme pyruvate: ferredoxin oxido reductase transfers reducing equivalents to the nitro group of the drug. The enzyme is present in Entanoeba histolytica and other anaerobic parasites like Giardia and Trichomonas that lack mitochondria. The selectivity of the drug can be ascribed to the absence of the reductasa in the human host. E. histolytica possesses other enzymes involved in glucose catabolism that are interesting for the rational desing of new drugs. It has glycolytic enzymes that are important for the production of energy like phosphofructokinase, pyruvate phosphate dikinase, phosphoenolpyruvate carboxytransphosphorylase and thiokinase, which use pyrophosphate as a phosphate donor and have no human counterparts. The first part of this article describes the reactions by which E. histolytica obtains energy from glucose degradation, and includes recent advances in thecloning of genes for the various participating enzymes. The second part shows an alternative view for the study of target enzymes that are unique to the parasite, and indicates their importance in therapautic research

The zymodemes of Entamoeba histolytica in New Delhi, India.

The prevalence of various pathogenic and non-pathogenic zymodemes of Entamoeba histolytica in New Delhi, India was studied by characterising its isoenzyme patterns. Non-pathogenic zymodeme I was found to be most common and was detected in 39 of 49 infections. The other non-pathogenic zymodemes detected were XVII and XVIII in two cases each. The pathogenic zymodemes detected were II (four cases), II alpha- (alpha) (one case) and XIV (one case). Approximately 72% of those harbouring non-pathogenic zymodemes and 33% of those harbouring pathogenic zymodemes were asymptomatic. All the symptomatic individuals harbouring non-pathogenic zymodemes were seronegative for amoebiasis. However, subjects harbouring pathogenic zymodeme but asymptomatic were seropositive for amoebiasis at a titre of greater than 1:80, indicating a past infection or subclinical tissue invasion by the parasite. The new zymodeme isolated from a case of acute dysentery was designated II alpha- on the basis of the banding pattern of the enzyme glucose phosphate isomerase.

Isoenzyme analysis of Entamoeba histolytica for evaluation of pathogenicity: detection of zymodeme XIX in South America.

Authors report the results of the isoenzyme analysis of strains of Entamoeba histolytica isolated from international travellers. This recently developed method allows the detection of pathogenic strains by evaluating the electrophoretic mobility of cytoplasmic enzymes and was proved to be more reliable and quickly feasible than previous ones. The experience reported refers to three strains isolated from travellers coming from Ecuador, Brazil and Indonesia, respectively; the zymodeme XIX (in accordance with the Sargeaunt's classification) was identified in all the three cases. This zymodeme has been first detected in 1981 and should currently be considered rare; moreover, it has never been previously reported from the Americas.

Soil ameba infection. Specific indirect immunoenzymatic (peroxidase) staining of formalin-fixed paraffin sections.

This report describes the use of the immunoenzymatic (peroxidase) method to identify the species and to stain distinctively the amebas in formalin-fixed paraffin-mounted sections. This permits the use of hematoxylin and eosin counterstaining. The method, now well developed by others for many purposes, is an alternative to immunofluorescence and seems to offer a number of advantages and a lesser number of disadvantages.