Maria Aparecida Gomes

The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes

BackgroundA number of studies have shown that peptidases and in particular cysteine peptidases constitute major pathogenicity factors in Entamoeba histolytica. Recent studies have suggested that a considerable number of genes coding for proteolytic enzymes are present within the E. histolytica genome and questions remain about the mode of expression of the various molecules.ResultsBy homology search within the recently published amoeba genome, we identified a total of 86 E. histolytica genes coding for putative peptidases, including 46 recently described peptidase genes. In total these comprise (i) 50 cysteine peptidases of different families but most of which belong to the C1 papain superfamily, (ii) 22 different metallo peptidases from at least 11 different families, (iii) 10 serine peptidases belonging to 3 different families, and (iv) 4 aspartic peptidases of only one family. Using an oligonucleotide microarray, peptidase gene expression patterns of 7 different E. histolytica isolates as well as of heat stressed cells were analysed. A total of 21 out of 79 amoeba peptidase genes analysed were found to be significantly expressed under standard axenic culture conditions whereas the remaining are not expressed or at very low levels only. In heat-stressed cells the expression of 2 and 3 peptidase genes, respectively, were either decreased or increased. Only minor differences were observed between the various isolates investigated, despite the fact that these isolates were originated from asymptomatic individuals or from patients with various forms of amoebic diseases.ConclusionEntamoeba histolytica possesses a large number of genes coding for proteolytic enzymes. Under standard culture conditions or upon heat-stress only a relatively small number of these genes is significantly expressed and only very few variations become apparent between various clinical E. histolytica isolates, calling into question the importance of these enzymes in E. histolytica pathogenicity. Further studies are required to define the precise role of most of the proteolytic enzyme for amoeba cell biology but in particular for E. histolytica virulence.

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.

The old and new therapeutic approaches to the treatment of giardiasis: where are we?

Giardia lamblia is the causative agent of giardiasis, one of the most common parasitic infections of the human intestinal tract. This disease most frequently affects children causing abdominal pain, nausea, vomiting, acute or chronic diarrhea, and malabsorption syndrome. In undernourished children, giardiasis is a determining factor in retarded physical and mental development. Antigiardial chemotherapy focuses on the trophozoite stage. Metronidazole and other nitroimidazoles have been used for decades as the therapy of choice against giardiasis. In recent years many other drugs have been proposed for the treatment of giardiasis. Therefore, several synthetic and natural substances have been tested in search of new giardicidal compounds. This study is a review of drugs used in in vitro and in vivo tests, and also drugs tested in clinical trials (nonrandomized and randomized).

Phagocytosis of Giardia lamblia trophozoites by human colostral leukocytes.

Aim: To determine the phagocytic activity of the polymorphonuclear and mononuclear cells present in human colostrum, and to verify the influence of opsonins in the adherence, ingestion and killing of Giardia lamblia trophozoites. Methods: Polymorphonuclear and mononuclear phagocytes were incubated with G. lamblia trophozoites, in the presence as well as the absence of supernatant of human colostrum (the source of opsonins) for 30, 60 and 120 min. The trophozoites/phagocytes ratio was 1:1, and the percentage of phagocytosed trophozoites was determined by microscopic examination of acridine orange‐stained cells. Results: The mononuclear phagocytes presented more functional activity than the polymorphonuclear. The highest indexes of adherence (77.6±5.1), ingestion (68.9±5.5) and killing (48.5±4.9) were obtained through the incubation of mononuclear cells in the presence of colostrum supernatant for 120 min.

Melatonin reduces the severity of experimental amoebiasis

BackgroundMelatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites.MethodsThe effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites.ResultsThe areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae.ConclusionsThese results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.

An improved method to distinguish Entamoeba histolytica and Entamoeba dispar

A 482 base pair gene fragment from samples of amoebae E. histolytica and E. dispar was amplified by PCR. The amplification products of fragments from the 2 species of amoebae presented differences in mobility in non-denaturing polyacrylamide gel, probably due to sequence-dependent conformational alterations in the DNA fragments. The method described here permits E. histolytica and E. dispar to be distinguished with greater sensitivity and rapidity.

Effect of metronidazole analogues on Giardia lamblia cultures

We comparatively evaluate the effect of metronidazole (MTZ) and its five analogues on trophozoites of Giardia lamblia axenically growing. The compounds MTZ-Ms, MTZ-I, MTZ-Br, MTZ-N3, and MTZ-NH3Cl were obtained by molecular modification of the side chain of MTZ. Four of them presented higher giardicidal activity when compared with MTZ. Among them, MTZ-Br and MTZ-I were the most active, without cytotoxic effects against mitogen-activated human peripheral blood mononuclear cells (PBMC). The alteration of MTZ side chain constitutes a fruitful field to develop new drugs for the treatment not only of giardiasis but also of other diseases and signalize that metronidazole analogues are promising candidates as giardicidal and should be further evaluated.

Histopathological and immunohistochemical study of the hepatic lesions experimentally induced by Entamoeba dispar

The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time the experimental lesions produced by E. dispar were described in details, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to Entamoeba histolytica (E. histolytica). Variation in the virulence is therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to better understand the disease.

A single step duplex PCR to distinguish Entamoeba histolytica from Entamoeba dispar

In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica.

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.