Entwicklung einer Screeningmethode in Mikrotiterplatten für Produkt-Vektor-Stamm-Kombinationen unter Berücksichtigung von drei Proteinen unterschiedlichen Molekulargewichts

In dieser Arbeit soll eine Screeningmethode in Mikrotiterplatten entwickelt werden, mit der die Möglichkeit besteht, viele verschiedene Produkt-Vektor-Stamm-Kombinationen sehr rationell zu testen. Dafür werden die drei Proteine unterschiedlichen Molekulargewichtes in jeweils drei Vektoren kloniert und in je sechs Stämme transformiert. Anschließend soll pro Produkt ein “Best-Off-Candidate“ identifiziert werden. Dieses Screening in Mikrotiterplatten stellt im Vergleich zu Versuchen im Schüttelkolben einen viel geringeren Arbeitsaufwand dar und ist daher deutlich lukrativer für ein kommerziell arbeitendes Unternehmen

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.AUTHOR SUMMARY: The pathogen Entamoeba histolytica can live asymptomatically in the human gut, or it can disrupt the intestinal barrier and induce life-threatening abscesses in different organs, most often in the liver. The molecular framework that enables this invasive, highly pathogenic phenotype is still not well understood. In order to identify factors that are positively or negatively correlated for invasion and destruction of the liver, we used a unique tool, E. histolytica clones that differ dramatically in their pathogenicity, while sharing almost identical genetic background. Based on comprehensive transcriptome studies of these clones, we identified a set of candidate genes that are potentially involved in pathogenicity. Using ectopic overexpression of the most promising candidates, either in pathogenic or in non-pathogenic Entamoeba clones, we identified genes where high expression reduced pathogenicity and only one gene that increased pathogenicity to a certain extend. Taken together, the current study identifies novel pathogenicity factors of E. histolytica and highlights the observation that various different genes contribute to pathogenicity

Synthetic analogs of an Entamoeba histolytica glycolipid designed to combat intracellular Leishmania infection

Intracellular pathogens belonging to the genus Leishmania have developed effective strategies that enable them to survive within host immune cells. Immunostimulatory compounds that counteract such immunological escape mechanisms represent promising treatment options for diseases. Here, we demonstrate that a lipopeptidephosphoglycan (LPPG) isolated from the membrane of a protozoan parasite, Entamoeba histolytica (Eh), shows considerable immunostimulatory effects targeted against Leishmania (L.) major, a representative species responsible for cutaneous leishmaniasis (CL). Treatment led to a marked reduction in the number of intracellular Leishmania parasites in vitro, and ameliorated CL in a mouse model. We next designed and synthesized analogs of the phosphatidylinositol anchors harbored by EhLPPG; two of these analogs reproduced the anti-leishmanial activity of the native compound by inducing production of pro-inflammatory cytokines. The use of such compounds, either alone or as a supportive option, might improve the currently unsatisfactory treatment of CL and other diseases caused by pathogen-manipulated immune responses

Phenotypical characterisation of clones A1^np, B2^p and B8^np.

<p>Phenotypical characterisation of clones A1^np, B2^p and B8^np.</p

Analysis of ALA formation after infecting gerbils with various <i>E</i>. <i>histolytica</i> transfectants.

<p>B2^p (A), A1^np (B) and B8^np transfectants (C) overexpressing various genes differentially expressed in clones A1^np and B2^p were used to intrahepatically infect mice. Seven days post infection, ALA formation was determined [area of ALA (mm²)]. Significance (<i>p</i>-values) was established by the Mann-Whitney <i>U</i> test. <i>p</i>-values were calculated relative to control (pNC transfectants of clone B2^p (A), A1^np (B) or B8^np (c), respectively). *<i>p</i> < 0.05, **<i>p</i> < 0.005, ***<i>p</i> < 0.001, **** <i>p</i> < 0.0001, ns: not significant. For detailed information see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005853#ppat.1005853.s001" target="_blank">S1 Table</a>.</p

Determination of cysteine peptidase activity.

<p>(A) CP activities of cell line HM-1:IMSS-A, clones A1–A12, cell line HM-1:IMSS-B and clones B1–B12 were measured at least four times in duplicate. (B) Substrate gel electrophoresis of clones A1–A12 and clones B1–B12. Cell lysates were separated on SDS-PAGE co-polymerised with gelatine. To visualise CP activity of proteins, gels were stained with Coomassie blue and the images were inverted. Standards are indicated on the left (kDa). Significance (<i>p-</i>values) was established using an unpaired t test. <i>p</i>-values were calculated relative to control (HM-1:IMSS-A or HM-1:IMSS-B, respectively). *<i>p</i> < 0.05, **<i>p</i> < 0.005, ***<i>p</i> < 0.001, ns: not significant. For detailed information see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005853#ppat.1005853.s001" target="_blank">S1 Table</a>.</p

Analysis of ALA formation after infecting gerbils with various sub-clones derived from clone B8^np and clone B2^p.

<p>Clones B8^np and B2^p were sub-cloned by a limited dilution method, resulting in five sub-clones each (B8_1–5 and B2_1–5, respectively). Gerbils were infected with 1 × 10⁶ trophozoites of the various sub-clones. Seven days post infection, liver abscess formation was analysed, and area of ALA (mm²) was determined. Significance (<i>p</i>-values) was established by the Mann-Whitney <i>U</i> test. <i>p</i>-values were calculated relative to control (clone B8^np or clone B2^p, respectively). For detailed information see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005853#ppat.1005853.s001" target="_blank">S1 Table</a>.</p

Analysis of the time course of ALA formation after infecting gerbils (A) and mice (B) with clones A1^np, B2^p or B8^np.

<p>Gerbils were infected with 1 × 10⁶ trophozoites, and mice were infected with 2.5 × 10⁵ trophozoites. Abscess sizes were determined on days 3, 5, 7 and 10 post infection using magnetic resonance imaging (MRI). Abscesses are shown in red circles. Significance was established using an unpaired t test. <i>p</i>-values were calculated relative to control (clone B2^p). *<i>p</i> < 0.05, **<i>p</i> < 0.005, ***<i>p</i> < 0.001, <i>****p</i> < 0.0001. For detailed information see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005853#ppat.1005853.s001" target="_blank">S1 Table</a>.</p

Genes differentially more highly expressed in clone A1^np than in clone B2^p (threshold ≥ 3.0, padj ≤ 0.05).

<p>Genes differentially more highly expressed in clone A1^np than in clone B2^p (threshold ≥ 3.0, padj ≤ 0.05).</p

mRNASeq and quantitative real-time PCR data for genes whose expression was at least ≥ 3-fold higher in clone A1^np than in clone B2^p.

<p>mRNASeq and quantitative real-time PCR data for genes whose expression was at least ≥ 3-fold higher in clone A1^np than in clone B2^p.</p