Table 1.
Oligonucleotides used in this study.
Figure 1.
The effect of GS on the resistance of E.histolytica to HS and oxidative stress.
The viability of trophozoites grown in glucose-free Diamond's TYI-S-33 media for 12 hours (A), subjected to heat shock at 42°C (B) and to oxidative stress (2.5 mM H2O2) (C) was measured and compared to control trophozoites grown in complete Diamond's TYI-S-33 medium. The number of trophozoites at the beginning of each experiment was taken as 100%. Data are expressed as the mean and standard deviation of three independent experiments that were repeated twice with a P-value <0.05.
Figure 2.
The effect of GS on virulence.
The hemolytic activity (A), and the cytopathic activity (B), adhesion (C) and mobility (D) of E.histolytica trophozoites were examined. Control cells were grown in complete TYI medium, while the glucose-starved trophozoites were grown in glucose-free TYI for 12 hours. The respective value of the control for each activity was taken as 100%. Data are expressed as the mean and standard deviation of three independent experiments that were repeated twice with a P-value <0.05.
Figure 3.
Quantitative changes in protein expression following glucose starvation.
(A) Total lysates of control and glucose-starved E.histolytica trophozoites used for stable isotope dimethyl labeling of peptides for quantitative proteomics analysis. The pies represent proteins whose amounts were increased (upper panel) or decreased (lower panel) following glucose starvation. The proteins were annotated according to their biological role as classified in the Pfam databases. Percentage number is given for each protein family. (B) Transcript amounts for Ap-A, CP-A5, URE-3BP and HSP70 in control and glucose-starved (GS) E.histolytica trophozoites were analyzed by Northern blot analysis. HSP70 whose expression is only slightly reduced in glucose-starved trophozoites (Table S1) was used as a control for loaded RNA. (C) Western blot analysis of Ap-A and KRiP1 expression in glucose-starved trophozoites. Cytoplasmic and nuclear protein fractions of E.histolytica were separated on 12% SDS-PAGE, transferred to a nitrocellulose membrane and incubated respectively with the KRiP1, Ap-A, or actin antibody. (D) Western blot analysis of LgL1 amounts in control and glucose-starved (GS) trophozoites. Membrane protein fractions were separated on 12% SDS-PAGE, transferred to nitrocellulose membrane and incubated with LgL1 antibody. Ponceau stain was used to control for protein loading.
Table 2.
Quantitative proteomic analysis of proteins with increased abundance following glucose starvation.
Table 3.
Quantitative proteomics analysis of proteins with decreased abundance following glucose starvation.
Figure 4.
Antisense down-regulation of KRiP1 expression.
(A) Determination of KRiP1 expression in sense KRiP1 and antisense KRiP1 transfectants by Western blot analysis. Nuclear protein fractions of E.histolytica sense KRiP1 and antisense KRiP1 transfectants grown in the presence of 40 µg per ml G418 were separated on 12% SDS-PAGE and analyzed by Western blot using a KRiP1 antibody, and an actin antibody. Identical results were obtained for three independent experiments. (B) Cytopathic activity of sense KRiP1 and antisense KRiP1 transfectants grown in complete TYI-S-33 medium. The cytopathic activity of the sense KRiP1 transfectant was taken as 100%. Data are expressed as the mean and standard deviation of three independent experiments that were repeated twice with a P-value <0.05. (C) Amoebic liver abscess formation by E. histolytica transfected with sense- and antisense-KRiP1 expression plasmids. Hamsters were infected by intraportal vein injection with trophozoites (1×106) expressing a KRiP1-sense (upper row) or -antisense (lower row) plasmid. A total of 6 animals, in two independent experiments, were infected with each amoeba type. Liver abscess formation was analysed at 7 days post-infection. Images show representative fields from 5 µm sections of formaldehyde-fixed tissue, stained with Harris' haematoxylin, periodic acid-Schiff reagent to visualize the hamster cells. Trophozoites were revealed by monoclonal antibody CD6 directed against E. histolytica Gal/GalNAc lectin heavy subunit and HRPO-coupled anti-mouse antibody. Scale bar, 60 µm for upper left panel and 40 µm for the three other panels.
Figure 5.
Cytopathic activity under GS of strains with reduced expression of Ap-A, CP-A5, Lgl1, and KRiP1.
Cytopathic activity of E.histolytica Ap-A mutant (G3), and CP-A5 mutant (RB8), and LgL1 mutant (RB9), and E.histolytica sense KRiP1 and antisense KRiP1transfectants that were cultivated in control and glucose-free TYI-S-33 medium. The cytopathic activity of each strain before glucose starvation (GS) was taken as 100%. Data are expressed as the mean and standard deviation of three independent experiments that were repeated twice with a P-value <0.05 (with assays/measurements repeated twice.