Fig 2: The host eIF5A protein is sequestered at the parasite surface and released upon MC2646 treatment.a The MC2646-click compound (MC4404) is localized in the vicinity of the parasite nuclei in the schizont. Microscopic images of infected TBL3 cells showing immunofluorescence of the MC2646 click compound (MC4404, Azide-Alexa Fluor 594, red) and DAPI-stained nuclei, with or without (Ctrl) MC2646 treatment. Leica microscope x100 magnification; the scale bar corresponds to 5 µm. b A control TCP-click compound did not localize to the parasite surface in the presence of absence of the MC2636 treatment. Microscopic images of infected TBL3 cells showing immunofluorescence of the Tranylcypromine click compound (Azide-Alexa Fluor 594, red) and DAPI-stained nuclei, with or without (Ctrl) MC2646 treatment. Leica microscope x100 magnification; the scale bar corresponds to 5 µm. c MC2646 treatment released eIF5A from the parasite schizont. We performed immunofluorescence analysis to localize eIF5A (Alexa Fluor 488, green) adjacent to parasite nuclei (DAPI), distinct from MC2646 click MC4404 (Azide-Alexa Fluor 594, red). The eIF5A was released from the schizont upon MC2646 treatment. Leica microscope x100 magnification; the scale bar corresponds to 5 µm. d Reconstructed 3D microscopy images of infected TBL3 cells stained with mab1C12 to mark the surface of the parasite macroschizont structure (green) and a specific anti-eIF5A antibody (red) in cells treated or not with MC2646 compound. The parasite and host nuclei are marked by DAPI staining (blue). The host eIF5A protein is recruited to the parasite surface in TBL3 cells and released upon MC2646 drug treatment. Leica microscope x100 magnification; the scale bar corresponds to 5 µm. e Inhibition of eIF5A hypusination. Infected TBL3 cells were treated with MC2646 with or without increasing concentration of the DHPS inhibitor GC7 and analysed by Western blot to monitor eIF5A, hypusinated eIF5A, TFEB, ATG3, and p62 proteins. Decreased eIF5A hypusination led to reduced TFEB and Atg3 levels and p62 accumulation. f Inhibition of eIF5A hypusination rescued parasite survival. Infected TBL3 cells were subjected to the above conditions (Fig. 4c) and monitored for parasite load by counting parasite nuclei per cell. At least 50 host cells were counted per condition (n = 3). Statistical significance One way-Anova, Dunnett’s multiple comparison test, **p < 0.014; ****p < 0.0001. g Inhibition of eIF5A hypusination reversed autophagic flux. Infected TBL3 cells were subjected to the above conditions (Fig. 3c) and monitored for LC3B puncta by immunofluorescence. At least 50 host cells were counted per condition. Results are significant under Kruskal-Wallis followed by a Dunn’s multiple comparison test ****p < 0.0001. h Effect of eIF5A depletion. Tac12 infected macrophages expressing stable sh_eIF5A (compared to control sh_Ctrl) were analysed by Western blot upon MC2646 treatment. eIF5A knockdown resulted in reduced levels of TFEB, ATG3, and p62. At least 50 host cells were counted per condition. Results are significant under Kruskal-Wallis followed by a Dunn’s multiple comparison test ****p < 0.0001. i eIF5A depletion rescued parasite survival. Tac12 cells expressing sh_eIF5A showed restored parasite load (parasite nuclei per host cell) upon MC2646 treatment. At least 50 host cells were counted per condition. Results are significant under Kruskal-Wallis followed by a Dunn’s multiple comparison test ****p < 0.0001. j eIF5A depletion blocked autophagosome formation. Immunofluorescence analysis of Tac12 cells expressing sh_eIF5A showed reduced LC3B puncta in the presence of MC2646 drug. At least 50 host cells were counted per condition. Results are significant under Kruskal-Wallis followed by a Dunn’s multiple comparison test ****p < 0.0001. In all experiments, cells were incubated with 50 ng/ml Buparvaquone or 4 µM MC2646 for 24 h and/or BafA1 (50 nM for 3 h). Results are representative of 3 independent experiments. Statistical analysis Dunnett’s multiple comparison test. **p < 0.01, ****p < 0.0001. The boxplots in graphs indicate the 25% (bottom), 50% (center) and 75% quartiles (top). Whiskers represent the minimum (bottom) and the maximum (top).

Fig 3: eIF5A hypusination in P. falciparum is inhibited by DFMO and GC7.(A) Schematic of polyamine biosynthesis and hypusination pathways in P. falciparum. (B) Western blot analysis of eIF5A and hypusinated eIF5A using lysates from parasites grown with or without DFMO (IC50 concentration, 0.5 mM) for 0, 12, 24, 36, 48, and 72 hours. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a loading control. (C and D) Relative levels of eIF5A and hypusinated eIF5A in DFMO-treated and untreated parasites. (E) Western blot analysis of eIF5A and hypusinated eIF5A in parasites grown with or without DFMO (IC50 concentration, 0.5 mM) or DFMO plus 9.2 μM PUT (+PUT), 9.2 μM SPD (+SPD), or 9.2 μM SPM (+SPM) for 72 hours. GAPDH was used as a loading control. (F and G) Relative levels of eIF5A and hypusinated eIF5A under the aforementioned different media conditions. (H) Western blot analysis of eIF5A and hypusinated eIF5A in parasites grown with or without GC7 (IC50 concentration, 15 μM) for 0, 12, 24, 36, 48, and 72 hours. (I and J) Relative levels of eIF5A and hypusinated eIF5A in untreated and GC7-treated parasites. (K) Western blot analysis of eIF5A and hypusinated eIF5A in parasites grown with or without GC7 (IC50 concentration, 15 μM) or GC7 plus 9.2 μM PUT (+PUT), 9.2 μM SPD (+SPD), or 9.2 μM SPM (+SPM) for 72 hours. (L and M) Relative levels of eIF5A and hypusinated eIF5A under these different conditions. Data are presented as means ± SD of three independent experiments. Statistical significance of differences was calculated using Welch’s t test. *P < 0.05 and **P < 0.01; ns, nonsignificant P value.

Fig 4: PUT depletion results in altered eIF5A hypusination (BdeIF5AHyp) in B. duncani.(A) Schematic representation of the eIF5A hypusination pathway in B. duncani. (B) In vitro efficacy of GC7 (100 μM) against B. duncani in DMEM/F12 and DMEM/F12 supplemented with 9.2 μM of either PUT, SPD, or SPM. (C) Western blot analysis using anti-eIF5A and anti-hypusine antibodies using lysates from parasites grown in the absence or presence of GC7 (100 μM) for 0, 12, 24, 36, and 48 hours. Anti–Bdhsp70-2 antiserum was used as a loading control. (D) Relative levels of BdeIF5A in lysates from untreated or GC7-treated parasites. (E) Relative levels of hypusinated eIF5A (BdeIF5AHyp) in parasite lysates from untreated or GC7-treated parasites. (F) Immunoblot analyses using anti-eIF5A and anti-hypusine antibodies using lysates from parasites cultured in the absence of PUT for 0, 12, 24, 36, and 48 hours. Anti–Bdhsp70-2 antiserum was used as a loading control. (G and H) Relative levels of BdeIF5A (G) and BdeIF5AHyp (H) in lysates from parasites maintained in PUT-depleted medium at different time points. (I) Western blot analysis using anti-eIF5A and anti-hypusine antibodies using lysates from parasites cultured in PUT-depleted (−PUT) medium, PUT containing medium (+PUT), and −PUT media supplemented with either SPD (+SPD; 9.2 μM) or SPM (+SPM; 9.2 μM) for 48 hours. Anti-Bdhsp70–2 antiserum was used as a loading control. (J and K) Relative levels of eIF5A (J) and BdeIF5AHyp (K) in lysates from parasites cultured in the aforementioned media. All data (A to K) presented as means ± SD of three independent experiments. Statistical significance of differences was calculated using Welch’s t test. *significant P < 0.05, **significant P < 0.001, ****P < 0.0001; ns, no significant difference.

Fig 5: Model of the modes of PUT acquisition, polyamine biosynthesis, and eIF5A hypusination used by B. duncani and P. falciparum.In B. duncani, PUT is salvaged from the host, through a putative PGT, and used for the synthesis of SPD and SPM. In P. falciparum, PUT production relies on the conversion of arginine to ORN by the parasite arginase and subsequent conversion of ORN to PUT by ODC. Alternatively, P. falciparum can also salvage PUT from host via PGT. In both parasites, PUT is processed further to form SPD and SPM, and SPD is used as a substrate in the hypusination pathway and results in the addition of hypusine to eIF5A. The hypusinated eIF5A is critical for protein translation. SPS, SPM synthase; SMO, SPM oxidase; ODC, ORN decarboxylase; SPDS, SPD synthase; DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase.