Journal: PLoS Pathogens

Article Title: AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica

doi: 10.1371/journal.ppat.1006882

Figure Lengend Snippet: (A) Verification by PCR of the lost genomic region, containing the EHI_176590 gene, in KU27. (B) Immuno-detection of EHI_176590 in KU27 and KU50. Total cell lysates, from the indicated strains, were analyzed by immunoblot analysis with anti-EHI_176590 antibody. The arrow depicts the EHI_176590 protein, which is present in KU50, but missing in KU27; asterisk indicates the protein band detected by non-specific binding or cross reaction. (C) Immuno-detection of EHI_176590-HA. Total lysates from EHI_176590-HA-expressing and mock transformants were analyzed by immunoblot analysis with an anti-HA antibody (left panel) or anti-EHI_176590 antibody (right panel). Black arrowheads indicate full-length EHI_176590-HA, and gray arrowheads indicate minor truncated forms of EHI_17690-HA. The arrow indicates intrinsic EHI_176590. Asterisks depict protein bands detected by non-specific binding or cross reaction. (D) Low magnification confocal immunofluorescence images of the EHI_176590-HA-expressing and mock-transfected transformants. Trophozoites were fixed and reacted with anti-Igl and anti-HA antibodies. Merged fluorescent (IGL/HA) and differential interference contrast (DIC) images are also shown. (E) Increased adherence to HRBCs by EHI_176590 overexpression. EHI_176590-HA-expressing (filled bars) and mock (open bars) transformants were co-cultured with HRBCs on ice for 30 min, and the number of adherent HRBCs per ameba were counted. The total number of trophozoites was set to 100% and the percentages of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of four biological replicates (2292 and 1762 trophozoites were counted for the EHI_176590-HA-expressing and mock transformants, respectively). **p-value <0.01, ***p-value <0.001 (F) Cell motility of the EHI_176590-HA-expressing and mock-transfected transformants. Time-lapse images of CellTracker Green-loaded EHI_176590-HA-expressing and mock (“HA”) transformants were analyzed by ICY software to measure cell motility. The average speed and standard deviation of the motility of EHI_176590-HA and mock vector transformants (HA) are shown; 135 and 106 trophozoites, respectively, were monitored in four independent experiments.

Article Snippet: Anti-HA rabbit polyclonal antibody was purchased from MBL.

Techniques: Western Blot, Binding Assay, Expressing, Immunofluorescence, Transfection, Over Expression, Cell Culture, Software, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica

doi: 10.1371/journal.ppat.1006882

Figure Lengend Snippet: (A) Immunofluorescence image, obtained by confocal microscopy, showing localization of EHI_176590. EHI_176590-HA-expressing amoebae were fixed and reacted with anti-HA antibody. (B) Magnified image of the inset in (A). Arrows indicate protrusions. (C) Immunofluorescence image, obtained by confocal microscopy, showing localization of EHI_176590-HA and the intermediate subunit of galactose/N-acetylgalactosamine-specific lectin (Igl). EHI_176590-HA-expressing amoebae were fixed and stained with anti-HA and anti-Igl antibodies. Arrow indicates protrusion. Islet showing signal-enhanced image to highlight the protrusion. (D) Effect of EHI_176590-HA expression on the percentage of amoebae that possess protrusions. EHI_176590-HA-expressing and mock transformants (HA) were immunostained as in (C) and the number of cells with protrusions, longer than 0.7 μm, was counted and are presented as percentages. The averages of three independent experiments, in which 100–200 amoebae were counted, are shown. (E) Effect of EHI_176590-HA expression on the number of protrusions per amoeba. EHI_176590-HA-expressing and mock transformants (HA) were immunostained as in (C), and the number of protrusions, per amoeba, was counted. The averages of three independent experiments, in which 100–200 amoebae were counted, are shown. * indicates p<0.01.

Article Snippet: Anti-HA rabbit polyclonal antibody was purchased from MBL.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Staining

Journal: PLoS Pathogens

Article Title: AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica

doi: 10.1371/journal.ppat.1006882

Figure Lengend Snippet: EHI_176590-HA expression in KU27 (A) Immuno-detection of EHI_176590-HA ectopically expressed in strain KU27. Total cell lysates, from mock-transfected and EHI_176590-HA-expressing strain KU27 trophozoites, were analyzed by immunoblot analysis with anti-EHI_176590 (left panel) and anti-HA antibody (right panel). The arrow indicates EHI_176590-HA, and asterisks indicate protein bands detected by non-specific immunoreactivity. (B) Low-magnification confocal immunofluorescence images of EHI_176590-HA-expressing and mock-transfected KU27. Trophozoites were fixed and reacted with anti-Igl and anti-HA antibodies. Merged fluorescent (IGL/HA) and DIC images are also shown. (C) Adhesion of the parental, EHI_176590-HA-expressing, and mock-transfected KU27 to HRBCs. Parental (open bars), EHI_176590-HA-expressing (black bars), and mock-transfected (gray bars) KU27 strains were co-cultured with HRBCs on ice for 30 min, and adherent HRBCs per ameba were counted. The total number of trophozoites was set to 100%, and the percentage of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of four biological replicates.

Article Snippet: Anti-HA rabbit polyclonal antibody was purchased from MBL.

Techniques: Expressing, Transfection, Western Blot, Immunofluorescence, Cell Culture

Journal: PLoS Pathogens

Article Title: AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica

doi: 10.1371/journal.ppat.1006882

Figure Lengend Snippet: (A) Confirmation of EHI_176590 gene silencing by immunoblot analysis. Total lysates, from EHI_176590 gene-silenced and mock transformants, were subjected to immunoblot analysis using anti-EHI_176590 (upper panel) and control anti-CS1 antibodies (lower panel). (B) Effect of EHI_176590 gene silencing on liver abscess formation. Approximately 1×10 6 trophozoites of EHI_176590 gene-silenced and mock-transfected strains (created from animal-passaged virulent Cl6) were injected into the livers of hamsters. Injected animals were sacrificed 6 days post-infection, and the liver and abscesses were dissected and weighed separately. Averaged percentages of the weight of abscesses per liver are shown. *p<0.004. (C) Adhesion of EHI_176590 gene-silenced and mock-transfected strains to HRBCs. Mock-transfected (black bars) and EHI_176590 gene-silenced (white bars) strains were co-cultured with HRBCs on ice for 30 min, and adherent HRBCs per ameba were counted. The percentage of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of three biological replicates. (D) Confirmation of EHI_176590-HA expression by immunoblot analysis. Total lysates from EHI_176590-HA and mock transformants were analyzed by immunoblot with anti-HA, anti-EHI_176590, and anti-CS1 antibodies (upper, middle, and lower panels, respectively). (E) Effect of EHI_176590-HA overexpression on liver abscess formation. Approximately 3×10 6 trophozoites, of EHI_176590-HA and mock-transfected transformants, were injected into the liver of hamsters, and liver abscess formation was evaluated as in (B). **p<0.003. (F) Adhesion of EHI_176590-HA and mock-transfected strains to HRBCs. Trophozoites of mock-transfected (black bars) and EHI_176590-HA transfected (white bars) strains were co-cultured with HRBCs, and adhesion was evaluated as in (C).

Article Snippet: Anti-HA rabbit polyclonal antibody was purchased from MBL.

Techniques: Western Blot, Control, Transfection, Injection, Infection, Cell Culture, Expressing, Over Expression