Interferon-gamma-activated macrophages infected with Burkholderia cenocepacia process and present bacterial antigens to T-cells by class I and II major histocompatibility complex molecules.

Burkholderia cenocepacia is an emerging opportunistic pathogen for people with cystic fibrosis and chronic granulomatous disease. Intracellular survival in macrophages within a membrane-bound vacuole (BcCV) that delays acidification and maturation into lysosomes is a hallmark of B. cenocepacia infection. Intracellular B. cenocepacia induce an inflammatory response leading to macrophage cell death by pyroptosis through the secretion of a bacterial deamidase that results in the activation of the pyrin inflammasome. However, how or whether infected macrophages can process and present B. cenocepacia antigens to activate T-cells has not been explored. Engulfed bacterial protein antigens are cleaved into small peptides in the late endosomal major histocompatibility class II complex (MHC) compartment (MIIC). Here, we demonstrate that BcCVs and MIICs have overlapping features and that interferon-gamma-activated macrophages infected with B. cenocepacia can process bacterial antigens for presentation by class II MHC molecules to CD4+ T-cells and by class I MHC molecules to CD8+ T-cells. Infected macrophages also release processed bacterial peptides into the extracellular medium, stabilizing empty class I MHC molecules of bystander cells. Together, we conclude that BcCVs acquire MIIC characteristics, supporting the notion that macrophages infected with B. cenocepacia contribute to establishing an adaptive immune response against the pathogen.

Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Growth hormone ameliorates high glucose-induced steatosis on in vitro cultured human HepG2 hepatocytes by inhibiting de novo lipogenesis via ChREBP and FAS suppression.

OBJECTIVE: Growth hormone (GH) deficiency has been associated with increased steatosis but the molecular mechanism has not been fully elucidated. We investigated the effect of GH on lipid accumulation of HepG2 cells cultured on an in vitro steatosis model and examined the potential involvement of insulin-like growth factor 1 (IGF-1) as well as lipogenic and lipolytic molecules. METHODS: Control and steatosis conditions were induced by culturing HepG2 cells with 5.5 or 25 mmol/l glucose for 24 h, respectively. Afterward, cells were exposed to 0, 5, 10 or 20 ng/ml GH for another 24 h. Lipid content was quantified as well as mRNA and protein levels of IGF-1, carbohydrate responsive element-binding protein (ChREBP), sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS), carnitine palmitoyltransferase 1A (CPT1A), and peroxisome proliferator-activated receptor alpha (PPAR-alpha) by qPCR and western blot, respectively. Data were analyzed by one-way ANOVA and the Games-Howell post-hoc test. RESULTS: In the steatosis model, HepG2 hepatocytes showed a significant 2-fold increase in lipid amount as compared to control cells. IGF-1 mRNA and protein levels were significantly increased in control cells exposed to 10 ng/ml GH, whereas high glucose abolished this effect. High glucose also significantly increased both mRNA and protein of ChREBP and FAS without having effect on SREBP1c, CPT1A and PPAR-alpha. However, GH inhibited ChREBP and FAS production, even in HepG2 hepatocytes cultured under steatosis conditions. CONCLUSIONS: Growth hormone ameliorates high glucose-induced steatosis in HepG2 cells by suppressing de novo lipogenesis via ChREBP and FAS down-regulation.

Antigenic membrane proteins of virulent variant of Entamoeba histolytica HM-1:IMSS.

Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.

Neutrophil extracellular traps and MPO in models of susceptibility and resistance against Entamoeba histolytica.

The main effector mechanisms of neutrophils are the release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO). In this work, we evaluated the role of NETs and the activity of MPO in the interactions of rodent neutrophils with amoebae and in amoebic liver abscess (ALA)-resistant and ALA-susceptible models. We showed with in vitro assays that mice produced greater amounts of NETs and MPO than did hamsters, and the elastase activity was high in both models. However, the inhibition of NETs and MPO promoted an increase in amoeba viability in the mice. The mouse ALAs showed a more profound presence of NETs and MPO than did the hamster ALAs. We concluded that both effector mechanisms were essential for the amoebic damage and could prevent the formation of ALAs in the resistant model.

Functional characterization and subcellular distribution of two recombinant cytosolic HSP70 isoforms from Entamoeba histolytica under normal and stress conditions.

Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.

Low Serum Interleukin-6 Is a Differential Marker of Obesity-Related Metabolic Dysfunction in Women and Men.

There is scant information regarding the role of interleukin (IL)-6 in obesity-related metabolic dysfunction in humans. Thus, we studied the serum levels of IL-6 in normal weight, overweight, and obese subjects, and examined associations of IL-6 with hyperglycemia, insulin resistance, dyslipidemia, and systemic inflammation. One hundred three women and men were included in the study. Anthropometric parameters, blood glucose, insulin, total cholesterol, and triglycerides were measured. Serum levels of tumor necrosis factor-alpha (TNF-alpha), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance (ANOVA) showed a 2.5-fold significant decrease in serum IL-6 in overweight and obese individuals when compared with normal weight controls. Serum IL-6 exhibited significant inverse correlations with body mass index (r = -0.39/P < 0.0001), waist circumference (r = -0.42/P < 0.001), blood glucose (r = -0.40/P < 0.0001), triglycerides (r = -0.34/P < 0.0001), and TNF-alpha (r = -0.48/P < 0.0001), whereas a strongly positive correlation was found with IL-10 (r = 0.77/P < 0.0001). Multiple linear regression analysis revealed that behavior of IL-6 was mainly influenced by IL-10 (beta = 0.28/P = 1.95 × 10-6), TNF-alpha (beta = -0.67/P = 0.0017), and body fat percentage (beta = -5.95/P = 7.67 × 10-5) in women. In contrast, IL-10 (beta = 0.37/P = 1.34 × 10-9), TNF-alpha (beta = -0.85/P = 0.0005), and triglycerides (beta = 1.07/P = 0.0007) were major influencing factors of IL-6 in men. This study demonstrates that IL-6 is a marker of metabolic dysfunction that is differentially regulated in obese women and men. [Figure: see text].

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Mechanisms of natural resistance of Balb/c mice to experimental liver amoebiasis.

Entamoeba histolytica is the parasite responsible for human amoebiasis. The analysis of the natural resistance mechanisms of some rodents to amoebic liver abscess (ALA) may reveal alternative pathogenicity mechanisms to those previously discovered in the experimental model of ALA in hamsters. In this work the natural resistance of BALB/c mice to ALA was explored by performing: (i) in vivo chemotaxis analysis with a specifically designed chamber; (ii) in vitro amoebic survival in fresh and decomplemented serum; (iii) histological temporal course analysis of ALA development in mice with different treatments (hypocomplementemic, hyperimmune and treated with iNOS and NADPH oxidase inhibitors) and (iv) mouse liver amoebic infection by both in situ implantation of ALA from hamsters and inoculation of parasites into the peritoneal cavity. The results show that E. histolytica clearance from the mouse liver is related to a low chemotactic activity of complement, which results in poor inflammatory response and parasite inability to cause tissue damage. Also, the absence of amoebic tropism for the mouse liver is correlated with resistance to experimental liver amoebiasis.

Control and regulation of the pyrophosphate-dependent glucose metabolism in Entamoeba histolytica.

Entamoeba histolytica has neither Krebs cycle nor oxidative phosphorylation activities; therefore, glycolysis is the main pathway for ATP supply and provision of carbon skeleton precursors for the synthesis of macromolecules. Glucose is metabolized through fermentative glycolysis, producing ethanol as its main end-product as well as some acetate. Amoebal glycolysis markedly differs from the typical Embden-Meyerhof-Parnas pathway present in human cells: (i) by the use of inorganic pyrophosphate, instead of ATP, as the high-energy phospho group donor; (ii) with one exception, the pathway enzymes can catalyze reversible reactions under physiological conditions; (iii) there is no allosteric regulation and sigmoidal kinetic behavior of key enzymes; and (iv) the presence of some glycolytic and fermentation enzymes similar to those of anaerobic bacteria. These peculiarities bring about alternative mechanisms of control and regulation of the PPi-dependent fermentative glycolysis in the parasite in comparison to the ATP-dependent and allosterically regulated glycolysis in many other eukaryotic cells. In this review, the current knowledge of the carbohydrate metabolism enzymes in E. histolytica is analyzed. Thermodynamics and stoichiometric analyses indicate 2 to 3.5 ATP yield per glucose metabolized, instead of the often presumed 5 ATP/glucose ratio. PPi derived from anabolism seems insufficient for PPi-glycolysis; hence, alternative ways of PPi supply are also discussed. Furthermore, the underlying mechanisms of control and regulation of the E. histolytica carbohydrate metabolism, analyzed by applying integral and systemic approaches such as Metabolic Control Analysis and kinetic modeling, contribute to unveiling alternative and promising drug targets.

Characterization of proteolytic activities of Giardia lamblia with the ability to cleave His-tagged N-terminal sequences.

Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites.

Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins.

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

Complement is a rat natural resistance factor to amoebic liver infection.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica This illness is prevalent in poor countries causing 100,000 deaths worldwide. Knowledge of the natural resistance mechanisms of rats to amoebic liver abscess (ALA) development may help to discover new pathogenic factors and to design novel therapeutic strategies against amoebiasis. In this work, histologic analyses suggested that the complement system may play a central role in rat natural resistance to ALA. E. histolytica trophozoites disappeared from rat liver within 6 h post-infection with minimal or no inflammatory infiltrate. In vitro findings indicate that rat complement was lethal for the parasite. Furthermore, hamsters became resistant to ALA by intravenous administration of fresh rat serum before infection. The amoebicidal potency of rat complement was 10 times higher than hamster complement and was not related to their respective CH50 levels. The alternative pathway of complement plays a central role in its toxicity to E. histolytica since trypan blue, which is a C3b receptor inhibitor, blocks its amoebicidal activity. These results suggest that amoebic membrane affinity, high for C3b and/or low for Factor H, in comparison with the hamster ones, may result in higher deposition of membrane complex attack on parasite surface and death.

First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target.

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

Serum Levels of Interleukin-13 Increase in Subjects with Insulin Resistance but Do Not Correlate with Markers of Low-Grade Systemic Inflammation.

Experimental evidence in mice suggests a role for interleukin- (IL-) 13 in insulin resistance and low-grade systemic inflammation. However, IL-13 serum levels have not been assessed in subjects with insulin resistance, and associations of IL-13 with parameters of low-grade systemic inflammation are still unknown. Our main goal was to examine the systemic levels of IL-13 in patients with insulin resistance, while also studying the relationship of IL-13 with anthropometric, metabolic, and low-grade systemic inflammatory markers. Ninety-two participants were included in the study and divided into insulin-resistant patients and noninsulin-resistant controls. Blood levels of IL-13, glucose, insulin, triglycerides, cholesterol, tumor necrosis factor-alpha (TNF-α), IL-10, proinflammatory (Mon-CD11c+CD206-), and anti-inflammatory (Mon-CD11c-CD206+) monocytes, as well as anthropometric parameters, were measured in all volunteers. Insulin-resistant patients showed 2.5-fold higher serum levels of IL-13 than controls (P < 0.0001) and significantly increased values of TNF-α and Mon-CD11c+CD206-, with concomitant reductions in IL-10 and Mon-CD11c-CD206+. Increased IL-13 was extraordinarily well associated with hyperglycemia (r = 0.7362) and hypertriglyceridemia (r = 0.7632) but unexpectedly exhibited no significant correlations with TNF-α (r = 0.2907), IL-10 (r = -0.3882), Mon-CD11c+CD206- (r = 0.2745) or Mon-CD11c-CD206+ (r = -0.3237). This study demonstrates that IL-13 serum levels are elevated in patients with insulin resistance without showing correlation with parameters of low-grade systemic inflammation.

Parallel ELISAs using crude soluble antigen and excretory-secretory antigen for improved serodiagnosis of amoebic liver abscess.

Crude soluble antigen (CSA) produced from Entamoeba histolytica trophozoite is conventionally used for serodiagnosis of invasive amoebiasis. However, high background seropositivities by CSA-assay in endemic areas complicate the interpretation of positive result in clinical settings. Instead, incorporating a second assay which indicates active or recent infection into the routine amoebic serology could possibly complement the limitations of CSA-assay. Hence, the present study aimed to evaluate the diagnostic efficacies of indirect ELISAs using CSA and excretory-secretory antigen (ESA) for serodiagnosis of amoebic liver abscess (ALA). Reference standard for diagnosis of ALA at Hospital Universiti Sains Malaysia is based on clinical presentation, radiological imaging and positive indirect haemagglutination assay (titer ≥256). Five groups of human serum samples collected from the hospital included Group I - ALA diagnosed by the reference standard and pus aspirate analysis using real-time PCR (n=10), Group II - ALA diagnosed by the reference standard only (n=41), Group III - healthy control (n=45), Group IV - other diseases control (n=51) and Group V - other infectious diseases control (n=31). For serodiagnosis of ALA serum samples (Group I and II), CSA-ELISA showed sensitivities of 100% for both groups, while ESA-ELISA showed sensitivities of 100% and 88%, respectively. For serodiagnosis of non-ALA serum samples (Group III, IV and V), CSA-ELISA showed specificities of 91%, 75% and 100%, respectively; while ESA-ELISA showed specificities of 96%, 98% and 100%, respectively. Indirect ELISAs using CSA and ESA have shown distinct strength for serodiagnosis of ALA, in terms of sensitivity and specificity, respectively. In conclusion, parallel analysis by both assays improved the overall efficacies of amoebic serology as compared to either single assay.

Roles of acetyl-CoA synthetase (ADP-forming) and acetate kinase (PPi-forming) in ATP and PPi supply in Entamoeba histolytica.

BACKGROUND: Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here. METHODS: The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae. RESULTS: Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels. CONCLUSIONS: These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis. SIGNIFICANCE: The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.

The oxygen reduction pathway and heat shock stress response are both required for Entamoeba histolytica pathogenicity.

Several species belonging to the genus Entamoeba can colonize the mouth or the human gut; however, only Entamoeba histolytica is pathogenic to the host, causing the disease amoebiasis. This illness is responsible for one hundred thousand human deaths per year worldwide, affecting mainly underdeveloped countries. Throughout its entire life cycle and invasion of human tissues, the parasite is constantly subjected to stress conditions. Under in vitro culture, this microaerophilic parasite can tolerate up to 5 % oxygen concentrations; however, during tissue invasion the parasite has to cope with the higher oxygen content found in well-perfused tissues (4-14 %) and with reactive oxygen and nitrogen species derived from both host and parasite. In this work, the role of the amoebic oxygen reduction pathway (ORP) and heat shock response (HSP) are analyzed in relation to E. histolytica pathogenicity. The data suggest that in contrast with non-pathogenic E. dispar, the higher level of ORP and HSPs displayed by E. histolytica enables its survival in tissues by diminishing and detoxifying intracellular oxidants and repairing damaged proteins to allow metabolic fluxes, replication and immune evasion.

Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica.

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.

In vivo identification of the steps that control energy metabolism and survival of Entamoeba histolytica.

The steps that control the Entamoeba histolytica glycolytic flux were here identified by elasticity analysis, an experimental approach of metabolic control analysis. The concentrations of glycolytic metabolites were gradually varied in live trophozoites by (a) feeding with different glucose concentrations and (b) inhibiting the final pathway steps; in parallel, the changes in the pathway flux were determined. From the metabolite concentration-flux relationship, the elasticity coefficients of individual or groups of pathway reactions were determined and used to calculate their respective degrees of control on the glycolytic flux (flux control coefficients). The results indicated that the pathway flux was mainly controlled (72-86%) by the glucose transport/hexokinase/glycogen degradation group of reactions and by bifunctional aldehyde-alcohol dehydrogenase (ADHE; 18%). Further, inhibition of the first pathway reactions with 2-deoxyglucose (2DOG) decreased the glycolytic flux and ATP content by 75% and 50%, respectively. Cell viability was also decreased by 2DOG (25%) and more potently (50%) by 2DOG plus the ADHE inhibitor tetraethylthiuram disulfide (disulfiram). Biosate as an alternative carbon (amino acid) source was unable to replace glucose for ATP supply, which indicated that glucose was the main nutrient for amoebal ATP synthesis and survival. These results indicated that glycolysis in the parasite is mainly controlled by the initial pathway reactions and that their inhibition can decrease the parasite energy load and survival.