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First published online 11 December 2007
doi: 10.1242/jcs.014050

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Conservation of the pro-apoptotic nuclease activity of endonuclease G in unicellular trypanosomatid parasites

Laboratory of Bacterial, Parasitic and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda MD 20892, USA

View larger version (86K): [in this window] [in a new window]   Fig. 1. CLUSTAL W alignment of Leishmania major and Trypanosoma brucei putative endoG with mammalian and nematode homologs. Only the partial amino acid sequences encompassing the predicted nuclease domains are shown (arrow marks). Numbers indicate starting and end amino acids used for alignment. The shaded area represents identical residues, and dashes represent gaps. The DRGH-motif, containing the catalytically important amino acid residues, is underlined. The amino acid residues important for activity such as histidine (*), the cofactor binding asparagine () and glutamic acid (*) are indicated. The percentage identities of full-length L. major endoG with those of T. brucei, C. elegans (NUCG_CAEEL), human (NUCG_HUMAN) and mouse (NUCG_MOUSE) endoG are shown in the parentheses.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Nuclease activity of recombinant T. brucei endoG protein (-rTbEG). (A) Polyclonal antibodies raised against full-length T. brucei endoG protein reacted with procyclic T. brucei lysates (L) and the TbEG recombinant protein (R). Pre-immune serum was used as a control (NRS). (B) A nuclease cleavage assay was performed with wild-type (rTbEG) or mutant TbEG (H220A and E261A, rTbEGm) proteins using genomic DNA (gDNA). Treatment with aurintricarboxylic acid (ATA) inhibited DNA degradation, confirming the specificity of TbEG for nucleic acid substrates. (C) Nuclease assay as in B, using total RNA as a substrate. (D) TUNEL assay of p-formaldehyde-fixed T. brucei procyclic cells incubated with rTbEG (light black line) or rTbEGm protein (dark black line) analyzed by flow cytometry. Untreated T. brucei cells (gray shaded peak) served as a control.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Nuclease activity of Leishmania endoG. (A) Western blot showing the reactivity of antibodies against HA with lysates of promastigotes (Pro) and axenic amastigotes (AxAm) of transfectants overexpressing the Leishmania endoG-HA protein. (B) Immunoprecipitated material (equal volumes) with antibodies against HA from KS, EG and EGm axenic amastigote cell lysates were resolved by SDS-PAGE and the blot was probed with biotinylated antibodies against rTbEG. (C) The immunoprecipitated material shown in B was incubated with 1 µg of plasmid DNA, and the cleavage products were resolved on a Bioanalyzer. The numbers on the x-axis represent the size of the DNA fragments in base pairs (bp). The peaks below 10380 bp in the LdEG and rTbEG panels represent cleavage products. rTbEG was used as a positive control, and KS controls (parasites transfected with the pKSNEO plasmid alone) are shown in A, B and C.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Immunofluorescence staining of wild-type T. brucei, Leishmania and Leishmania-transfectant cells. (A) T. brucei procyclic cells and (B) Leishmania promastigotes were stained with antibodies against TbEG (-rTbEG) as primary, and FITC-conjugated anti-rabbit IgG as secondary, antibodies. To visualize mitochondria, cells were labeled with Mitotracker Red (Mito Red). Pre-immune serum (NRS) was used in control staining experiments. (C) Axenic amastigotes overexpressing endoG (LdEG) were labeled with antibodies against the HA epitope (-HA). Mitochondria were labeled with Mitotracker Red. LdKS transfectants were used as a control. Bars, 5 µm (A); 2.5 µm (B,C).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effects of overexpression of endoG on Leishmania growth and sensitivity to oxidant stress. (A) The growth of L. donovani axenic amastigote (LdAxAm) cultures – wild-type (WT), plasmid control (KS) or wild-type (EG) or mutant endoG transfectants (EGm) – was monitored. The percentage of viable cells was determined by propidium iodide (PI) staining followed by FACS analysis. Only the PI-negative cell populations are plotted. The results are the mean of three independent experiments. (B) LdAxAm transfectants (KS, EG and EGm) in the early log phase of growth were subjected to TUNEL labeling and observed by fluorescence microscopy. Nucleic acids were stained with DAPI. Bar, 2.5 µm. (C) TUNEL-positive cells in wild-type and transfectants, as in B, were quantified by FACS analysis over 5 days in culture. The results are the mean of three independent experiments. (D) Leishmania amastigote parasite sensitivity to undergoing cell death in response to treatment with H2O2 was measured by FACS analysis of TUNEL-labeled wild-type (WT), KS-, EG- and EGm-expressing cells. The area shaded gray indicates the TUNEL-positive cells without H2O2 treatment, and the histogram demarcated by a black line overlay indicates TUNEL-positive cells after treatment with 2 mM H2O2 for 1 hour. The numbers represent the percentage TUNEL-positive cells. (E) Data from three independent experiments, as in D. Asterisk (*) indicates significant difference (P<0.05). Error bars indicate the standard deviation.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Effect of overexpression of endoG on differentiation and survival of Leishmania in macrophages. (A) Human macrophages differentiated from monocytes were infected with purified metacyclic parasites from L. major transfectant cultures (KS, EG, EGm) for six hours (10:1 parasite-to-macrophage ratio), stained with Diff-quick reagents, and the numbers of amastigotes in these cultures were determined over a period of 5 days by microscopic observation. The data are expressed as the number of amastigotes per 100 macrophages. Error bars indicate the standard deviation. (B) DAPI and TUNEL staining of infected macrophages at the 6 hour time point. (C) Macrophages infected with either wild-type L. major (WT) or transfectant parasites (KS, EG, EGm) were stimulated with LPS and human IFN-. DAPI and TUNEL stainings after 48 hours of stimulation are shown. DAPI and TUNEL stainings of non-stimulated macrophages infected with wild-type parasites are shown as controls (top panels).

View larger version (32K): [in this window] [in a new window]   Fig. 7. H2O2-triggered release of endoG from mitochondria. (A) L. donovani axenic amastigote transfectants (LdKS, LdEG) treated with or without H2O2 under the conditions used to trigger cell death were incubated in a buffer containing digitonin, and fractions representing cytosol (C) and mitochondria (M) were analyzed by SDS-PAGE and western blotting using antibodies against HA (-HA), T. brucei endoG (-rTbEG) and Leishmania cytochrome c (-LdCyt C). The blots were re-probed with antibodies against T. brucei Hsp70 (-TbHsp70) and Leishmania adenine phosphoribosyltransferase (-LdAPRT). (B) Western blot showing the cross-reactivity of the antibody against TbHsp70 with Leishmania lysates.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effect of knockdown of endoG in T. brucei. (A) Growth of transfected procyclic T. brucei either induced (RNAi+) or non-induced (RNAi–) with tetracycline. Northern blot from induced (+) or non-induced (–) cells collected at day 6 (gray arrowhead) and probed with a [32P]-labeled T. brucei endoG-specific probe (endoG; inset). Ribosomal RNA bands (rRNA) stained with ethidium bromide and the results of re-probing the blot with a [32P]-labeled -tubulin probe are shown (inset). (B) EndoG-depleted (RNAi+) cells and un-induced T. brucei controls (RNAi–) were treated with H2O2, labeled with TUNEL mix and analyzed by flow cytometry. The area shaded gray indicates the TUNEL-positive cells without H2O2 treatment, and the histogram demarcated by a black line overlay indicates TUNEL-positive cells after treatment with 2 mM H2O2 for 1 hour. (C) TUNEL-positive cells from RNAi-induced and non-induced controls were quantified by FACS analysis, and the data represented here were obtained from three independent experiments. (*) indicates significance (P<0.05). Error bars indicate the standard deviation.