Fig 1.
Presumed biosynthetic pathway of polyamines and deoxyhypusine/hypusine modifications on eukaryotic initiation factor 5A in E. histolytica.
Solid lines represent the steps catalyzed by the enzymes whose encoding genes are present in the E. histolytica genome, whereas dashed lines indicate those absent or not yet identified. Abbreviations: 5’-MTA, 5’-methylthioadenosine; MAT, S-adenosylmethionine synthetase; ODC, ornithine decarboxylase, AdoMetDC, S-adenosylmethionine decarboxylase; SpdS, spermidine synthase; SpmS, spermine synthase; eIF5A, eukaryotic initiation factor 5A; DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase.
Fig 2.
Multiple alignment of E. histolytica DHS and eIF5A protein sequences.
Conserved residues are marked by asterisks (*). Sequence alignment was performed using ClustalW. (A) Amino acid sequence alignment of E. histolytica DHS isoforms. Accession numbers of these sequences are as follows: EhDHS1 (XP_653614), EhDHS2 (XP_653426). The catalytic lysine residue is shown in red background, whereas NAD+ and spermidine binding sites are shown in green and yellow background, respectively. (B) Amino acid sequence alignment of E. histolytica eIF5A isoforms. Accession numbers of these sequences are as follows: EheIF5A1 (XP_657374); EheIF5A2 (XP_651531). The conserved lysine residues which are expected to be hypusinated are highlighted in red. Residues which are post-transnationally modified by acetylation and sulfation in human eIF5A, are shown in grey and cyan background, respectively.
Fig 3.
Characterization of wild type, mutant and co-expressed forms of EhDHS1 and EhDHS2 (A) Schematic representation of DHS1 and DHS2 mutant. Amino acid residues mutated are shown in grey. Amino acid positions are also shown. (B) Enzymatic activity of recombinant EhDHS1, EhDHS2, EhDHS1_K295F, and EhDHS2_F302K using EheIF5As substrates. The mean ± S.D. of three independent experiments performed in triplicate are shown. (C) Immunoblot analysis of co-expression of EhDHS1, which contains the amino-terminal His-tag, and EhDHS2, which contains the carboxyl-terminal S-tag, using anti-His and anti-S tag antibodies. (D) Specific activity of co-expressed recombinant EhDHS1 and EhDHS2 using EheIF5A1 and EheIF5A2 as substrates. The means ± standard deviations of three independent experiments performed in triplicate are shown.
Fig 4.
Determination of the size of the complex of EhDHS1 and EhDHS2.
(A) Purified Recombinant EhDHS1 and 2 were co-expressed, copurified, and were subjected to 4–16% BN-PAGE. After electrophoresis the gel was destained. Native page marker (Invitrogen) was used as a protein standard. (B) Immunoblot analysis following BN-PAGE of co-expressed N-terminal His-tagged EhDHS1 and C-terminal S-tagged EhDHS2, using anti-His and anti-S tag antibodies respectively.
Fig 5.
Effects of EhDHS2 gene silencing on gene expression of related genes and growth of E. histolytica trophozoites.
(A) Evaluation of gene expression by semi-quantitative RT-PCR analysis of EhDHS2 gene silenced transformant. The steady-state levels of transcripts of EhDHS1, EhDHS2, EheIF5A1, EheI5FA2 and EhRNA pol II genes were measured in trophozoites of G3 strain transfected with either empty vector (psAP2G) or the EhDHS2 gene silencing plasmid (psAP2G-DHS2). cDNA from the generated cell lines (psAP2G and DHS2gs strains) was subjected to 30 cycles of PCR using specific primers for the DHS2, DHS1, eIF5A1, eI5FA2 and RNA pol II genes. RNA polymerase II served as a control. PCR analysis of samples without reverse transcription was used to exclude the possibility of genomic DNA contamination. The densitometric quantification of the bands, shown in the right graph, was performed by Image J software, and the expression level of EhDHS1, EhDHS2, EheIF5A1, EheIF5A2, and EhRNA pol II was expressed in arbitrary units. (B) Growth kinetics of control (pSAP2G) and EhDHS2 gene silenced (DHS2gs) transformants. Approximately 60,000 amoebae in the logarithmic growth phase were inoculated into 6 mL fresh culture medium and amoebae were then counted every 24 hr. Data shown are the means ± standard deviations of five biological replicates. (C) Immunoblot analysis of control (pSAP2G) and EhDHS2 gene silenced (DHS2gs) transformants using hypusine antibody. Total cell lysate was electrophoresed on a 15% SDS-PAGE gel and subjected to an immunoblot assay with hypusine antibody and CS1 (loading control) antiserum. The intensity of the bands corresponding to hypusinated EheIF5A and CS1 was measured by densitometric quantification, analyzed by Image J software, and is shown in arbitrary units in the right graph.
Fig 6.
Cellular distribution of EhDHS and EheIF5A isoforms and post translational modification of EheIF5A1/2.
(A) Cellular fractionation of EhDHS1, EhDHS2, EheIF5A1, and EheIF5A2. Trophozoites of the transformants expressing HA-tagged EhDHS1, EhDHS2, EheIF5A1, and EheIF5A2 were fractionated as described in Materials and Methods, and subjected to immunoblot analysis using anti-HA monoclonal antibody, anti-hypusine, anti-CPBF1, and anti-CS1 polyclonal antisera. CPBF1 and CS1 served as control of organelle and cytosolic proteins, respectively. (B) PTMs of HA tagged EheIF5A. Approximately 30 μg of total lysates from the transformants expressing HA-tagged EheIF5A1 and EheIF5A2 were subjected to SDS-PAGE under reducing conditions and immunoblot analysis using anti-hypusine antibody.
Fig 7.
Silencing of EheIF5A2 caused growth retardation and up regulation of EheIF5A expression.
(A) Evaluation of gene expression by semi-quantitative RT-PCR analysis of EheIF5A2 gene silenced transformant. The steady-state levels of transcripts of EheIF5A1, EheIF5A2, and EhRNA pol II genes were measured in trophozoites of G3 strain transfected with either empty vector (psAP2G) or the EheIF5A2 gene silencing plasmid (psAP2G-eIF5A2). cDNA from the generated cell lines (psAP2G and EheIF5A2gs strains) was subjected to 25 cycles of PCR using specific primers for the eIF5A1, eI5FA2 and RNA pol II genes. RNA polymerase II served as a control. PCR analysis of samples without reverse transcription was used to exclude the possibility of genomic DNA contamination. (B) Growth kinetics of EheIF5A2 gene-silenced (EheIF5A2gs) and control (pSAP2G) transformants. Approximately 60,000 amoebae in the logarithmic growth phase were inoculated into 6 ml fresh culture medium and amoebae were then counted every 24 hr. Data shown are the means ± standard deviations of five biological replicates. (C) An immunoblot analysis of EheIF5A2gs and control (pSAP2G) transformants. Total cell lysates were electrophoresed on a 15% SDS-PAGE gel and subjected to an immunoblot assay with antibodies raised against EheIF5A1 and EheIF5A2.
Fig 8.
SUnSET analysis of active protein synthesis.
(A) SUnSET analysis of wild type strain. Trophozoites of E. histolytica clonal strain HM-1:IMSS cl 6 were incubated in normal growth medium with 100 μg/ml cycloheximide, 10 μg/ml puromycin, or both. Cell lysates were subjected to SDS-PAGE and immunoblot using antibody specific for puromycin. Note that trophozoites incorporated puromycin into proteins (P). The incorporation was blocked by cycloheximide treatment (C + P). These data validated the SUnSET analysis for assessing protein synthesis in E. histolytica. (B) SUnSET analysis of EheIF5A2gs strain. Trophozoites from EheIF5A2gs and control (pSAP2G) strains were subjected to SUnSET analysis as in (A). Equal protein loads were confirmed with CS1 antibody (lower panels). Representative data for at least 3 separate trials are shown.
Fig 9.
Steady state transcript levels of EieIF5A isoforms during excystation.
(A) Kinetics of excystation. Cysts were incubated at the indicated times, cysts and trophozoites were harvested and the excystation percentage was determined. The excystation efficiency was determined by counting trophozoites and cysts in a haemocytometer and experiments were performed in duplicate. Data shown are the means ± standard deviations of two independent experiments. (B) Semi-quantitative RT-PCR analysis of EieIF5A1, EieI5FA2, EieIF5A1 and EiActin genes during excystation. cDNA from 4 time points during excystation was subjected to 30 cycles of PCR using specific primers for eIF5A1, eI5FA2, eIF5A3, and actin genes. Actin gene served as a control. PCR from samples without reverse transcription served as controls to exclude the possibility of genomic DNA contamination. The densitometric quantification of the bands, shown in the right graph, was performed by Image J software, and the expression level of EieIF5A1, EieIF5A2, and EieIF5A3 and EiActin was expressed in arbitrary units.