Proteomic Comparison of Entamoeba histolytica and Entamoeba dispar and the Role of E. histolytica Alcohol Dehydrogenase 3 in Virulence

The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1∶IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1∶IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1∶IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba

Lipids induce expression of serum-responsive transmembrane kinase EhTMKB1-9 in an early branching eukaryote Entamoeba histolytica

Mechanisms underlying the initiation of proliferative response are known only for a few organisms, and are not understood for the medically important organisms including Entamoeba histolytica. The trans membrane kinase EhTMKB1-9 of E. histolytica is one of the early indicators of proliferation and its' expression is regulated by serum, one of the components necessary for cellular proliferation in vitro. In this study we show that bovine serum albumin (BSA) can induce EhTMKB1-9 expression in place of serum, and that both follow the same mechanism. Both serum and BSA use the same promoter element and the activation process is initiated through a PI3 kinase-mediated pathway. We further show that BSA activates EhTMKB1-9 due to the lipids associated with it and that unsaturated fatty acids are responsible for activation. These results suggest that lipid molecules are ligand(s) for initiation of a signaling system that stimulates EhTMKB1-9 expression

Entrepreneurial sons, patriarchy and the Colonels' experiment in Thessaly, rural Greece

Existing studies within the field of institutional entrepreneurship explore how entrepreneurs influence change in economic institutions. This paper turns the attention of scholarly inquiry on the antecedents of deinstitutionalization and more specifically, the influence of entrepreneurship in shaping social institutions such as patriarchy. The paper draws from the findings of ethnographic work in two Greek lowland village communities during the military Dictatorship (1967–1974). Paradoxically this era associated with the spread of mechanization, cheap credit, revaluation of labour and clear means-ends relations, signalled entrepreneurial sons’ individuated dissent and activism who were now able to question the Patriarch’s authority, recognize opportunities and act as unintentional agents of deinstitutionalization. A ‘different’ model of institutional change is presented here, where politics intersects with entrepreneurs, in changing social institutions. This model discusses the external drivers of institutional atrophy and how handling dissensus (and its varieties over historical time) is instrumental in enabling institutional entrepreneurship

Tissue Invasion by Entamoeba histolytica: Evidence of Genetic Selection and/or DNA Reorganization Events in Organ Tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver

Global analysis of gene expression in response to L-Cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica</it>, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. <it>E. histolytica </it>completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in <it>E. histolytica</it>, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism.</p> <p>Results</p> <p>In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion.</p> <p>Conclusions</p> <p>To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol.</p

Small RNAs with 5′-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica

Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5′-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5′-polyphosphate siRNAs extends to single-celled eukaryotic organisms

The effect of the timing of exposure to Campylobacter jejuni on the gut microbiome and inflammatory responses of broiler chickens

Background Campylobacters are an unwelcome member of the poultry gut microbiota in terms of food safety. The objective of this study was to compare the microbiota, inflammatory responses, and zootechnical parameters of broiler chickens not exposed to Campylobacter jejuni with those exposed either early at 6 days old or at the age commercial broiler chicken flocks are frequently observed to become colonized at 20 days old. Results Birds infected with Campylobacter at 20 days became cecal colonized within 2 days of exposure, whereas birds infected at 6 days of age did not show complete colonization of the sample cohort until 9 days post-infection. All birds sampled thereafter were colonized until the end of the study at 35 days (mean 6.1 log10 CFU per g of cecal contents). The cecal microbiota of birds infected with Campylobacter were significantly different to age-matched non-infected controls at 2 days post-infection but generally the composition of the cecal microbiota were more affected by bird age as the time post infection increased. The effects of Campylobacter colonization on the cecal microbiota were associated with reductions in the relative abundance of OTUs within the taxonomic family Lactobacillaceae and the Clostridium cluster XIVa. Specific members of the Lachnospiraceae and Ruminococcaceae families exhibit transient shifts in microbial community populations dependent upon the age at which the birds become colonized by C. jejuni. Analysis of ileal and cecal chemokine/cytokine gene expression revealed increases in IL-6, IL-17A and Il-17F consistent with a Th17 response but the persistence of the response was dependent on the stage/time of C. jejuni colonization that coincide with significant reductions in the abundance of Clostridium cluster XIVa. Conclusions This study combines microbiome data, cytokine/chemokine gene expression with intestinal villus and crypt measurements to compare chickens colonized early or late in the rearing cycle to provide insights into the process and outcomes of Campylobacter colonization. Early colonization results in a transient growth rate reduction and pro-inflammatory response but persistent modification of the cecal microbiota. Late colonization produces pro-inflammatory responses with changes in the cecal microbiota that will endure in market ready chickens

Amebiasis in HIV-1-Infected Japanese Men: Clinical Features and Response to Therapy

Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection

Progesterone after previous preterm birth for prevention of neonatal respiratory distress syndrome (PROGRESS): a randomised controlled trial

Background: Neonatal respiratory distress syndrome, as a consequence of preterm birth, is a major cause of early mortality and morbidity during infancy and childhood. Survivors of preterm birth continue to remain at considerable risk of both chronic lung disease and long-term neurological handicap. Progesterone is involved in the maintenance of uterine quiescence through modulation of the calcium-calmodulin-myosin-light-chain-kinase system in smooth muscle cells. The withdrawal of progesterone, either actual or functional is thought to be an antecedent to the onset of labour. While there have been recent reports of progesterone supplementation for women at risk of preterm birth which show promise in this intervention, there is currently insufficient data on clinically important outcomes for both women and infants to enable informed clinical decision-making. The aims of this randomised, double blind, placebo controlled trial are to assess whether the use of vaginal progesterone pessaries in women with a history of previous spontaneous preterm birth will reduce the risk and severity of respiratory distress syndrome, so improving their infant's health, without increasing maternal risks. Methods Design: Multicentred randomised, double blind, placebo-controlled trial. Inclusion Criteria: pregnant women with a live fetus, and a history of prior preterm birth at less than 37 weeks gestation and greater than 20 weeks gestation in the immediately preceding pregnancy, where onset of labour occurred spontaneously, or in association with cervical incompetence, or following preterm prelabour ruptured membranes. Trial Entry & Randomisation: After obtaining written informed consent, eligible women will be randomised between 18 and 23+6 weeks gestation using a central telephone randomisation service. The randomisation schedule prepared by non clinical research staff will use balanced variable blocks, with stratification according to plurality of the pregnancy and centre where planned to give birth. Eligible women will be randomised to either vaginal progesterone or vaginal placebo. Study Medication & Treatment Schedules: Treatment packs will appear identical. Woman, caregivers and research staff will be blinded to treatment allocation. Primary Study Outcome: Neonatal Respiratory Distress Syndrome (defined by incidence and severity). Sample Size: of 984 women to show a 40% reduction in respiratory distress syndrome from 15% to 9% (p = 0.05, 80% power). Discussion: This is a protocol for a randomised trial.Jodie M. Dodd, Caroline A. Crowther, Andrew J. McPhee, Vicki Flenady, and Jeffrey S. Robinso