Sulfur mobilization for Fe-S cluster assembly by the essential SUF pathway in the Plasmodium falciparum apicoplast and its inhibition

Abstract

The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS.

FIG 1

Schematic representation of the steps involved in Fe-S cluster assembly on target apoproteins by the SUF pathway.

FIG 2

Expression of recombinant PfSufS and PfSufE and their detection in the parasite. (A) Coomassie-stained SDS-PAGE of purified recombinant PfSufS expressed as a 6×His-tagged protein in E. coli (i) and detection of the protein in P. falciparum lysate (ii) by Western blotting with preimmune serum (Pre-I) and anti-SufS serum (I). (B) Purified recombinant PfSufE seen in a Coomassie-stained SDS-PA gel (i) and detection of the protein in parasite lysate by Western blotting with anti-PfSufE Ab (I) (ii).

FIG 3

Immunofluorescence localization of PfSufS and PfSufE. (A) Immunofluorescence assay of P. falciparum 3D7 cells with anti-PfSufS Ab with anti-PfHU Ab as an apicoplast marker (i) or Mitotracker Red (ii) indicates targeting of PfSufS to the apicoplast. (B) Colocalization of PfSufE and apicoplast-targeted GFP observed in the P. falciparum D10 ACP_leader-GFP cell line with anti-PfSufE and anti-GFP Abs (i). No overlap of PfSufE signal was observed with Mitotracker Red in P. falciparum 3D7 cells (ii), indicating apicoplast localization of PfSufE. DIC, differential interference contrast; DAPI, 4′,6-diamidino-2-phenylindole.

FIG 4

PfSufS is a PLP cofactor-dependent desulfurase. (A) UV-VIS absorption spectrum of PfSufS with and without treatment with NaBH₄. The inset shows the effect of NaBH₄ on the absorption spectrum of E. coli SufS. The 420-nm peak of PfSufS-bound PLP shifts to a 335-nm peak of reduced PLP after treatment with NaBH₄. (B) Enhancement of cysteine desulfurase activity of PfSufS with increasing concentrations of PfSufE. All of the reaction mixtures except the control (20 μM PfSufE alone) contained 5 μM PfSufS. (C) Effect of the PfSufS Cys₄₉₇Ala mutation on sulfide release in the presence of PfSufE.

FIG 5

PfSufS and PfSufE complex in vitro and in vivo. (A) Coimmunoprecipitation of recombinant PfSufS and PfSufE with PfSufS Abs cross-linked to protein A Sepharose beads, followed by Western blotting with anti-His Ab. Arrows indicate ∼53-kDa PfSufS and ∼20-kDa PfSufE (lane 2). No signal was observed in beads cross-linked with preimmune serum (lane 1) or in the presence of PfSufE alone (lane 3). Lane 5 is purified PfSufE used as size marker. (B) Coimmunoprecipitation of PfSufE with PfSufS from parasite lysates with beads cross-linked to anti-PfSufS Ab. Coprecipitated PfSufS (i) and PfSufE (ii) were detected in Western blot assays with anti-PfSufS and anti-PfSufE Abs, respectively. (C) Parasite proteins were cross-linked in vivo by DSP and then treated with increasing concentrations of DTT to break the complex(s). SDS-PA gels of the samples were probed with anti-PfSufE Ab (i) or anti-PfSufS Ab (ii) in Western blot assays to detect the complex and released proteins.

FIG 6

Structural model of PfSufS-PfSufE interaction. (A) Protein docking with the GRAMMX server identified a complex with proximal positioning of the critical cysteine residues of the interacting proteins. A large insertion in PfSufS cannot be modeled and appears as a loop. (B) Enlarged portion of panel A that shows the active site and the PfSufS-PfSufE interface. The PLP cofactor is in black.

FIG 7

DCS inhibits PfSufS activity. (A) UV-VIS absorption spectrum of PfSufS in the presence of DCS showing a reduction in the 420-nm bound PLP peak accompanied by the appearance of a peak at 320 nm representing a PLP-DCS adduct. (B) Effect of increasing concentrations of DCS on cysteine desulfurase activity of PfSufS. The IC₅₀ of DCS was determined to be 29.2 ± 2.9 μM.