Cryptosporidium parvum has an active hypusine biosynthesis pathway

Abstract

The protozoan parasite Cryptosporidium parvum causes severe enteric infection and diarrheal disease with substantial morbidity and mortality in untreated AIDS patients and children in developing or resource-limited countries. No fully effective treatment is available. Hypusination of eIF5A is an important post-translational modification essential for cell proliferation. This modification occurs in a two step process catalyzed by deoxyhypusine synthase (DHS) followed by deoxyhypusine hydroxylase. An ORF of 1086bp was identified in the C. parvum (Cp) genome which encodes for a putative polypeptide of 362 amino acids. The recombinant CpDHS protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. Sequence analysis and structural modeling of CpDHS were performed to probe differences with respect to the DHS of other species. Unlike Leishmania, Trypanosomes and Entamoeba, Cryptosporidium contains only a single gene for DHS. Phylogenetic analysis shows that CpDHS is more closely related to apicomplexan DHS than kinetoplastid DHS. Important residues that are essential for the functioning of the enzyme including NAD(+) binding residues, spermidine binding residues and the active site lysine are conserved between CpDHS and human DHS. N(1)-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of DHS caused an effective inhibition of infection and growth of C. parvum in HCT-8 cells.

Keywords: Cryptosporidium parvum; Deoxyhypsuine synthase; Hypusine pathway; Protozoan parasite.

Fig. 1

Phylogenetic analysis. Sequence based phytogeny of DHS sequences from C. parvum lowa II(CGD2_3930); C. muris (CMU_003030); E. multilocularis (EmW_000780100); S. japonicum (Sjp_0017580); S. mansoni (Smp_065120); E. hellem (EHEL_090880); N. ceranae (NCER_100851); G. Assemblage B (GL50581_338; GL50803_15535;GLP15_78;); T. gondii (TGGT1_070160); N. canium (NCLIV_050000); T. vaginalis (TVAG_359990); E. invadens (EIN_107550; EIN_017320); E. dispar (EDL161760; EDL198820); E. histolytica (EHI_098350; EHI_006030); T. brucei (Tb927.1.870; Tbg972.1.280; Tb427.10.2750; Tb927.10.2750; Tbg972.10.3430; Tb427.01.870); L. mexicana (LmxM.20.0250; LmxM.33.0330); T. cruzi (Tc00.1047053504119.29; Tc00.1047053506195.300); T. congolense (TcIL3000.1.360); P. yoelli (PY01546); P. berghei (PBANKA.103000); P. chabaudi (PCHAS_103080); P. knowlesi (PKH_133500); P. falciparum (PF14_0125); P. vivax (PVX_085825); T. parvum (TP02_0058); T. annulata (TA13570); B. bovis (BBOV_III010890); E. intestinalis (Ein09_0870); L. tarentolae (LtaP20.0250; LtaP34.0370); L. major(LmjF.20.0250; LmjF.34.0330); L. braziliensis (LbrM.20.4450; LbrM.20.0300); L.infantum (LinJ.20.0270; LinJ.34.0350); DHYS_PYRCJ (A3MVC9); DHYS_METS5 (A4YHK6); DHYS_IGNH4 (A8AA61); DHYS_HALS3 (B0R5L2); DHYS_SULIA (C3N5B4); DHYS_METTH (026230); DHYS1_ARCFU (028088); DHYS_PYRHO (050105); DHYS_HUMAN (P49366); DHYS_DEBHA (Q6BJH5); DHYS_CANGA (Q6FRN2); DHYS_PICTO (Q6KZL5); DHYS_SOLLC (Q9AXR0); DHYS_ENCCU (Q8SQN2); DHYS1_METAC (Q8TS38); DHYS_METKA (Q8TXD7); DHYS2_METMA (Q8Q051); DHYS_AERPE (Q9YE72); L. donovani (LdBPK_340350; LdBPK_200270) were derived using neighbor joining method. Bootstrap values are shown at the nodes.

Fig. 2

Structural comparison of the human DHS structure with C. parvum DHS Model and L. donovani DHS Model. Structural comparison of the human DHS structure (PDB: 1 DHS; Violet) with C. parvum DHS Model (cgd2_3930; Olive green) and L. donovani DHS Model (LdBPK_340350; Bright Orange). NAD⁺ binding residues are shown as red sticks. Catalytic lysine is shown as pink spheres. Residues at the tetramer interface in the human DHS structure (PDB:1RQD) and their equivalent residues in the C. parvum DHS and L. donovani DHS models are shown as white spheres. The loop insertions (Insertion 1 & Insertion 2) are shown in dotted lines in the L. donovani model. The 14 residue loop insertion (Insertion 3) in the L. donovani model and the equivalent interface residues in the C. parvum model and the human structure are shown in white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Purification and biochemical characterization of recombinant CpDHS. (A) Purification of His-tagged CpDHS protein on Ni ²⁺Nitrolotriacetic acid affinity resin. Lane 1: Molecular weight marker; lane 2, flow through; lanes 3 and 4 eluted fractions showing purified protein. (B) Determination of oligomerization state of CpDHS by chemical crosslinking. Lane 1, Molecular weight marker; Lane 2, human His-DHS; lane 3, human His-DHS incubated with 20 mM glutaraldehyde; lane 4, His-CpDHS; lane 5–8, His-CpDHS incubated with 20 mM glutaraldehyde for 2,5,15 and 30 min respectively; lane 9, human His-DHS treated with 20% SDS and 20 mM glutaraldehyde. (C) Comparison of trichloroacetic acid precipitable counts obtained from enzyme assay mixture using different amount of recombinant CpDHS. Results are mean ± S.D. of triplicate samples. (D) Dependence of CpDHS reaction on eIF5A concentration. Varying concentrations of eIF5A (µM) with 1 mM NAD⁺, 2.5 µM (4 µCi) of [1,8-³H] spermidine and 1 µg of CpDHS were used. The K_m and V_max value were 0.9163 µM and 1.473 pmol/h, respectively. (E) Dependence of CpDHS reaction on spermidine concentration. Varying concentrations of spermidine (µM) with 1 mM NAD, 5µM eIF5Aand 1 µg of CpDHS were used. The K_m and V_max value were 12.29µM and 4.86 pmol/h, respectively. (F) Radiolabeling of eIF5A by in vitro CpDHS and human DHS reaction. Radiolabeling of eIF5A was confirmed by SDS-PAGE of the DHS reaction mixture. Fluorogram showing the position of eIF5A. Lane 1: recombinant CpDHS plus eIF5A, Lane 2: recombinant human DHS plus eIF5A.

Fig. 4

Kinetics and stoichiometry of NADH bound to CpDHS. The final component of the reaction mixture was added in the fluorescence cell at r= 0 and emission at the fixed wavelength, 441 nm (excitation at 340 nm), was followed over the indicated time course. (A) NADH fluorescence with addition of 100 µM spermidine and 1 mM NAD⁺. (B) NADH fluorescence with addition of 1 mM NAD⁺ followed by 100 µM spermidine and its inhibition by 100 µM GC7. (C) Quantification of the AU of the NADH (1 µM) formed. 1.1 µM NAD⁺ and 100 µM spermidine were incubated with increasing the amount of DHS for 25 min. The fit of the experimental points to a hyperbola gave a maximum value of 31.92 (indicated by arrow). (D) DHS (1.3 µM) was incubated with 100 µM spermidine and increasing concentrations of NAD⁺.The arbitrary unit (A.U.) after the reaction was divided by 31.92 to give the equivalent NADH concentration in µM.

Fig. 5

Inhibition of CpDHS by various guanyldiamines. The reactions were carried out as described under methods using 1 µg of recombinant CpDHS in 50 µl of reaction volume with indicated concentrations of various guanyldiamine. Results are mean ± SD of triplicate samples. * p < 0.05; ** p < 0.01; *** p < 0.005 and ns indicates not significant (p>0.05).

Fig. 6

Inhibition of C. parvum infection and growth by GC7. (A) Inhibition of C. parvum oocyst production by DHS inhibitors C. parvum (3 × 10⁴) oocysts were inoculated to a confluent layer of HCT-8 cells and incubated for 2 h after which time the media was replaced with fresh medium containing stated amount of test compound and incubated for 24 h; oocytes were harvested and counted as described under the Methods. C. parvum oocyst production in the presence of 0.02 µM (), 0.04 µM (), or 0.021 µM () GC7; 0.45 µM (), 0.90 µM (), or 4.5 µM () GC7G; 24µM diaminooctane (DAO); 36 µM diaminononane (DAN); 10 µM agmatine (AGM). Results are expressed as + SD of triplicate experiments compared to control oocysts lacking added compounds. Paromomycin (150 µM) caused 58% inhibition of C. parvum oocysts production. (B) Inhibition of C. parvum infection and growth by GC7. C. parvum oocyst production after 24 h incubation with HCT-8 cells. Oocysts () or sporozoites () preincubated for 1.5 h with HCT-8 cells prior to addition of GC7; Oocysts () or sporozoites () preincubated with GC7 for 30 min prior to infecting HCT-8 cells.