35
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background

          Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules.

          Principal Findings

          Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite.

          Conclusions

          Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

          Related collections

          Most cited references36

          • Record: found
          • Abstract: found
          • Article: not found

          Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

          Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Amoebiasis.

            Amoebiasis is the second leading cause of death from parasitic disease worldwide. The causative protozoan parasite, Entamoeba histolytica, is a potent pathogen. Secreting proteinases that dissolve host tissues, killing host cells on contact, and engulfing red blood cells, E histolytica trophozoites invade the intestinal mucosa, causing amoebic colitis. In some cases amoebas breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses consisting of a few E histolytica trophozoites surrounding dead and dying hepatocytes and liquefied cellular debris. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. Evidence that what we thought was a single species based on morphology is, in fact, two genetically distinct species--now termed Entamoeba histolytica (the pathogen) and Entamoeba dispar (a commensal)--has turned conventional wisdom about the epidemiology and diagnosis of amoebiasis upside down. New models of disease have linked E histolytica induction of intestinal inflammation and hepatocyte programmed cell death to the pathogenesis of amoebic colitis and amoebic liver abscess.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              The genome of the protist parasite Entamoeba histolytica.

              Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2009
                23 December 2009
                : 4
                : 12
                : e8424
                Affiliations
                [1 ]Institut Pasteur, Unité Biologie Cellulaire du Parasitisme, Paris, France
                [2 ]INSERM U786, Paris, France
                INSERM U567, Institut Cochin, France
                Author notes

                Conceived and designed the experiments: CFS JSR DP CW NG. Performed the experiments: CFS JSR DP CW. Analyzed the data: CFS JSR DP CW NG. Contributed reagents/materials/analysis tools: NG. Wrote the paper: CFS JSR DP NG.

                [¤]

                Current address: Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, España

                Article
                09-PONE-RA-13167R1
                10.1371/journal.pone.0008424
                2793006
                20037645
                9809773d-dbcb-464a-ad92-9c4bb612ef18
                Solis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 25 September 2009
                : 24 November 2009
                Page count
                Pages: 11
                Categories
                Research Article
                Infectious Diseases/Gastrointestinal Infections
                Infectious Diseases/Neglected Tropical Diseases
                Infectious Diseases/Protozoal Infections

                Uncategorized
                Uncategorized

                Comments

                Comment on this article