Fernando Anaya-Velázquez

Trichomonas vaginalis: Ultrastructural Bases of the Cytopathic Effect

ABSTRACT. The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT‐10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT‐3 strain. However, the time course of cytolysis with GT‐3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.

Entamoeba histolytica: effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055.

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing(R) technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.

In Vitro Studies of Chromone-Tetrazoles against Pathogenic Protozoa, Bacteria, and Fungi

In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.

Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays

Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or β-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the β-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (β-galactosidase activity) studies of different xenobiotic substances and pollutants.

Entamoeba histolytica: A simplified method to quantify its cytotoxicity

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.

In vitro activity of nalidixic acid and its iron (III) complex on Entamoeba histolytica

The in vitro activity of the antibacterial agent nalidixic acid (HNal) and its iron (III) complex (FeNal) against Entamoeba histolytica HM1 strain trophozoites in axenic or monoxenic (associated with Clostridium symbiosum) cultures was investigated. Using a dilution test with TYI-S-33 medium, this protozoan was found to be susceptible to both drugs, but FeNal showed amoebicidal activity only at concentrations higher than those used with HNal.

Synthetic biology: Novel approaches for microbiology

In the past twenty years, molecular genetics has created powerful tools for genetic manipulation of living organisms. Whole genome sequencing has provided necessary information to assess knowledge on gene function and protein networks. In addition, new tools permit to modify organisms to perform desired tasks. Gene function analysis is speed up by novel approaches that couple both high throughput data generation and mining. Synthetic biology is an emerging field that uses tools for generating novel gene networks, whole genome synthesis and engineering. New applications in biotechnological, pharmaceutical and biomedical research are envisioned for synthetic biology. In recent years these new strategies have opened up the possibilities to study gene and genome editing, creation of novel tools for functional studies in virus, parasites and pathogenic bacteria. There is also the possibility to re-design organisms to generate vaccine subunits or produce new pharmaceuticals to combat multi-drug resistant pathogens. In this review we provide our opinion on the applicability of synthetic biology strategies for functional studies of pathogenic organisms and some applications such as genome editing and gene network studies to further comprehend virulence factors and determinants in pathogenic organisms. We also discuss what we consider important ethical issues for this field of molecular biology, especially for potential misuse of the new technologies.

Entamoeba histolytica: molecular cloning and characterization of a novel neutral sphingomyelinase.

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg(2+)-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn(2+) and partially inhibited by Zn(2+). nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn(2+), and abolished by 5mM Zn(2+). A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.

Recognition of Carbohydrate Epitopes Specific for Electron-Dense Granule Antigens from Entamoeba histolytica by Monoclonal Antibodies in the Cecal Content of Infected Hamsters

The pathogenic trophozoites of Entamoeba histolytica produce and secrete electron-dense granules (EDG) containing collagenase, considered a virulence factor. Two monoclonal antibodies (MAbs) (L7.1 and L1.1) anti-EDG antigens were raised. MAb L7.1 has been reported to recognize proteic EDG antigens and MAb L1.1 reacted with a carbohydrate epitope. These epitopes were present in axenic and xenic amoebas. To detect EDG antigens by a enzyme-linked immunosorbent assay (ELISA) in a experimental model of early intestinal amoebiasis, both MAbs were employed. E. histolytica HM1 axenic and monoxenic trophozoites were inoculated into the cecum according to the washed-closed cecal loop technique. The cecal content was recovered at 8, 24, and 48 h post-inoculation. Antigens from EDG in whole trophozoites and cell-free supernatants were detected. Our results indicate that it is possible to detect EDG antigens in the cecal content of hamsters in the early phase of the invasive amoebiasis.

Cytological and Immunological Methods to Identify Entamoeba histolytica and Entamoeba dispar Trophozoites in Patients from Guanajuato, Mexico

Two related amebas colonize the human large intestine: pathogenic Entamoeba histolytica , the causative agent of amebiasis, and noninvasive Entamoeba dispar. In Mexico, 8.4% of the population have serum antibodies to E. histolytica (1). In the Mexican state of Guanajuato, diagnosis of amebiasis is carried out by concentration methods and cytology, in view of their reliability and low cost. However, laboratories detect cysts that are morphologically similar in both species. Trophozoites also have the same morphology by bright-field microscopy but differ in their capability to phagocytose red blood cells, as erythrophagocytosis is greater in E. histolytica than in E. dispar (2). Recently, the World Health Organization (WHO) recommended the development of improved methods for the specific diagnosis of E. histolytica infection employing technologies appropriate for use in developing countries (3). Among the cytological techniques available to routine clinical laboratories, several methods could be useful to differentiate both species on the basis of erythrophagocytosis or presence of surface antigens. In this work, we compare the standard Trichrome stain, Wright’s stain (4), and an ELISA method produced by TechLab ® to distinguish E. histolytica from E. dispar trophozoites in samples of patients with suspected amebiasis. We found that all tested methods are useful, but that Wright’s stain is easier to perform and has a lower cost.