Michelle A.R. Freitas

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.

Characterization of the presence and distribution of Foxp3(+) cells in chagasic patients with and without megacolon.

Patients with Chagass disease in the chronic phase regularly present with the chagasic megacolon. This form is characterized by inflammation, neuronal destruction, and organ dilatation. Chagasic patients with megacolon always present with inflammatory process near the enteric plexuses of the colon, as previously demonstrated. The aim of this study is to characterize the presence and distribution of Foxp3(+) cells in the muscle layers and neuronal plexuses area of the colon from chagasic patients with and without megacolon. Our results demonstrated that chagasic patients without megacolon presented with an increased concentration of Foxp3(+) cells in all colon layers compared with chagasic patients with megacolon and noninfected individuals. These cells were situated mainly near the blood vessels and rarely were associated with the inflammatory foci. We believe that the presence of Foxp3(+) cells may help to control the inflammatory process through the management of lymphocyte migration and, consequently, prevent neuronal destruction and chagasic megacolon development.

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.

Characterization of enteroglial cells and denervation process in chagasic patients with and without megaesophagus

Chagas disease is caused by infestation with the parasite Trypanosoma cruzi, and some patients who are serologically positive develop chronic megaesophagus, whereas others are symptom-free. Gastrointestinal form of Chagas disease involves an inflammatory invasion of the enteric plexuses and degeneration of enteric neurons and previous works related that enteroglial cells would be involved in enteric inflammatory responses. Because of this, the aims of this study were to determine the relation of enteroglial cells with the denervation process in chagasic patients with and without megaesophagus and seronegative individuals. Our results indicated that the innervation of the esophageal muscle was substantially reduced in patients with megaesophagus, but asymptomatic seropositive subjects were not different to seronegative controls. Besides, patients with megaesophagus had significant decreased of enteroglial cells labeled with S-100 and glial fibrillary acidic protein, whereas patients without megaesophagus presented an increased of both labels. We believe that enteroglial cells would operate a mechanism of defense in the enteric nervous system against the Trypanosoma cruzi infection, which could prevent the organ denervation and preserve the esophagus function.

Entamoeba histolytica: cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels.

Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis.

Glycogen as a carbohydrate energy reserve in trophozoites of Giardia lamblia

Although there is indirect evidence to suggest that glycogen is present in G. lamblia, to date it has not been purified and identified from this organism. In this study, a high molecular weight carbohydrate was purified and characterized and its physiological role as an energetic reserve was established. The monosaccharide constituents of the carbohydrate reserve were identified as glucose by two independent methods: thin layer chromatography and an enzymatic assay. The degree of branching of the molecule was evaluated by comparing its absorbance spectrum in the presence of lugol with spectra of standard solutions of glycogen and starch under the same conditions. The results strongly suggest that glycogen is present in G. lamblia and acts as an energy reserve in trophozoites of this organism.

Substance P and NK1 receptor expression in the enteric nervous system is related to the development of chagasic megacolon

Chagasic megacolon is one of the most important forms of Chagas disease. This form is characterized by inflammation, neuronal destruction and organ dilatation. The aim of this study is to characterize the expression of substance P and its main receptor, NK1 receptor, in dilated and non-dilated samples of colon from chagasic patients with megacolon. Our results demonstrate that dilated portions of colon present high levels of substance P and low levels of NK1 receptor, whereas non-dilated portions and samples from non-infected individuals present low levels of substance P and high levels of NK1 receptor. We believe that this may indicate a neuro-immune relationship that occurs in Chagas disease.

Relation between mast cells concentration and serotonin expression in chagasic megacolon development

Chagas’ disease is still reaching about 10 million people in the world. In South America, one of the most severe forms of this disease is the megacolon, characterized by severe constipation, dilated sigmoid colon and rectum and severe malnutrition. Previous data suggested that mast cells and serotonin (5‐hydroxytryptamine [5‐HT]) expression could be involved in intestinal homeostasis control, avoiding the chagasic megacolon development. The aim at this study was to characterize the presence of mast cells and expression of serotonin in chagasic patients with and without megacolon and evaluate the relation between mast cells, serotonin and megacolon development. Our results demonstrated that patients without megacolon feature a large amount of serotonin and few mast cells, while patients with megacolon feature low serotonin expression and a lot of mast cells. We believe that serotonin may be involved in the inflammatory process control, triggered by mast cells, and the presence of this substance in large quantities of the intestine could represent a mechanism of megacolon prevention.

Expression of caspase-3 in enteric cells is related to development of chagasic megacolon

Chagas disease is endemic in many regions of Brazil as in most countries of Latin America. The World Health Organization estimates that there are 11 million infected people in Latin America and 2 to 3 million patients with chronic complications of the disease. Microscopically, a common observation in chagasic megacolon is the neuronal loss in both plexuses [1]. We believe that the denervation observed in chagasic megacolon can be caused by both parasite injury and by apoptosis. Apoptosis is a form of programmed cell death and involves a series of biochemical events leading to characteristic cell morphology and death. The process of apoptosis is controlled by a diverse range of cell signals, which may originate in either extracellular or intracellular areas. The apoptosis cascade is always initiated by activation of caspase-3. This protein is activated in apoptotic conditions and executes the apoptosis process [2]. In the digestive form of Chagas disease, as in the immune system, the parasite has a role in neuronal death [3]. Our hypothesis is that the apoptosis mechanisms may be involved in the development of chagasic megacolon. To evaluate this, our group used a peroxidase immunohistochemistry technique to analyze caspase-3 expression in colon samples from full-thickness colon wall tissue obtained from 12 chagasic patients with megacolon, 12 chagasic patients without megacolon, and 14 control individuals submitted to necropsy or surgical procedures at Universidade Federal de Goiás (Goiânia, Minas Gerais, Brazil). Patients did not receive any parasite-specific treatment. Informed consent was obtained from the patient or family members prior to tissue procurement, and this work was approved by the UFMG Research Ethics Committee (ETIC no 127/03). Tissue samples were prepared according to the methods described in da Silveira et al 2008 [4]. To localize the apoptotic cells, the antihuman caspase-3 (IMGENEX, Cod. IMG-144A, San Diego, CA) was used. The immunohistochemistry protocol and the data analysis methods were performed according to the methods described in da Silveira et al 2008 [4]. The hematoxylin-eosin staining technique was applied in each case to evaluate the inflammatory process.

Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization

Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.