File S1 - Gene Expression Profiling in Entamoeba histolytica Identifies Key Components in Iron Uptake and Metabolism

Supporting information. Figure S1, Growth of Entamoeba histolytica trophozoites under normal iron conditions (▴) and in iron-deficient medium (▪). Trophozoites were counted in a Neubauer chamber every 24 hrs for 5 days. The cell count corresponds to the mean of three observations. Figure S2, A Venn diagram of genes differentially expressed in three conditions. Upper panel: upregulated genes. Lower panel: downregulated genes. A total of 224 transcripts were significantly modulated in one or more of the three conditions. In iron deficiency, 9 transcripts were upregulated and 11 were downregulated. In iron deficiency + Hb, 107 transcripts were upregulated and 50 were downregulated. Under low-iron conditions, 34 transcripts were upregulated and 46 were downregulated. Figure S3, Alignments of selected E. histolytica genes. A) amoebic ComEC orthologs (EHI_169340 and EHI_156240) and ComEC from Bacillus licheniformis (WP_003183688.1, 20% similarity), B) amebic P-glycoprotein-5 (EHI_125030) and PvdE from Pseudomonas aeruginosa (YP_002440490.1, 33% similarity), C) amoebic MFT (EHI_173950) and MFS1 exporter from Azotobacter vineldii (YP_002797373.1, 33% similarity), D) amoebic MFT (EHI_173950) and human FLVCR1 (NP_054772.1), E) amoebic hypothetical protein (EHI_009840) and heme-degrading monooxygenase (isdG) from Staphylococcus aureus (NP_645835.1, 38% similarity), F) amoebic hypothetical protein (EHI_095090) and ferrochelatase from Nitrosomonas sp. (YP_004294842.1, 27% similarity), and G) amoebic hypothetical protein (EHI_138420) and uroporphyrin-III C-methyltransferase from Actinobacillus pleuropneumoniae (WP_005597783.1, 32% similarity). Residues with 100% and 80% homology are highlight in gray and black, respectively. The comparisons were performed using the CLUSTALW alignment tool from the WebExPASy Molecular Biology Server (http://ca.expasy.org). Table S1, Quantification of iron in the TYI-S-33 and TYI-S-33ΔFe medium. Footnote: The ferrozine method described in the Material and Methods section was used to quantify iron in the TYI-S-33 medium, incomplete TYI-S-33ΔFe medium (no supplementation with AFC, vitamins and serum) and TYI-S-33ΔFe complete (no supplementation with AFC but supplemented with vitamins and serum) medium. Serum accounts for 55.5 µM iron, i.e. the difference between complete and incomplete media. Peptone accounts for 39.7 µM iron, i.e. the level determined in the incomplete TYI-S-33ΔFe medium. AFC accounts for 78 µM iron, i.e. the difference between the complete TYI-S-33 and complete TYI-S-33ΔFe media. AFC: ammonium ferric citrate; Hb: hemoglobin. Values correspond to the mean of experiments performed in triplicate. Table S2, Differentially expressed genes in iron deficiency. Footnote: FC: fold-change; BY: the false discovery rate according to Benjamini and Yekutieli multiple testing; rawp: the unadjusted P-value. Table S3, Differentially expressed genes in iron deficiency with Hb supplementation. Footnote: FC: fold-change; BY: the false discovery rate according to Benjamini and Yekutieli multiple testing; rawp: the unadjusted P-value. Table S4, Differentially expressed genes under low-iron conditions. Footnote: FC: fold-change; BY: the false discovery rate according to Benjamini and Yekutieli multiple testing; rawp: the unadjusted P-value. Table S5, Fold-changes for genes differentially expressed in normal medium + Hb for 2 hours. Footnote: The AmoebaDB ID and GenBank ID numbers refer to the gene's accession number in AmoebaDB and NCBI GenBank, respectively; “Normal iron + Hb for 2 h” refers to the fold-change in expression in TYI-S-33 medium supplemented with Hb for 2 hours, compared with the normal iron condition (as detected by quantitative real-time PCRs). Table S6, Fold-changes for genes differentially expressed in iron deficiency for 24 hours. Footnote: The AmoebaDB ID and GenBank ID numbers refer to the gene's accession number in AmoebaDB and NCBI GenBank, respectively; “Normal iron to iron deficiency for 24 h” refers to the fold-change in expression in TYI-S-33 medium without AFC supplementation for 24 hours, when compared with the normal iron condition (as detected by quantitative real-time PCRs). The trophozoites incubated in iron-deficient medium for 24 hours were recovered and incubated for an additional 24 hours in normal iron medium (“iron deficiency 24 h to normal iron 24 h”). Gene expression was detected using quantitative real-time PCRs. Table S7, List of primers used for real time-PCRs. Footnote: Position: the relative nucleotide position of the primer's 5′ end, where 0 refers to the first nucleotide of the start codon; Sequence: sequence of the primer from 5′ to 3′. Note that the reverse primer's sequence is reversed and complemented. (DOC)