The apicomplexan Cryptosporidium parvum possesses a single mitochondrial-type ferredoxin and ferredoxin:NADP+ reductase system

Abstract

We have successfully expressed recombinant mitochondrial-type ferredoxin (mtFd) and ferredoxin:NADP(+) reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time for an apicomplexan. Both C. parvum mtFd (CpmtFd) and FNR (CpmtFNR) were obtained and purified as holo-proteins, in which the correct assembly of [2Fe-2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/vis and electron paramagnetic resonance. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c-coupled assay that followed a typical Michaelis-Menten kinetics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals and could be explored as potential drug targets in Cryptosporidium and other apicomplexans.

Figure 1

The electron transfer system conducted by ferredoxin (Fd) and FAD-containing FNR. The use of cytochrome c (cyt c) and an artificial electron acceptor in the assay is also illustrated.

Figure 2

Strategy of subcloning of CpmtFd and CpmtFNR into pMAL-c2X-derived MBPHT-Cherry2 and pET21a expression vectors, respectively. The N-terminal sequences for CpmtFd and CpmtFNR excluded from expression are highlighted in red. Arrows indicate the cleavage sites by TEV protease and factor Xa, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Structures of mtFd-II–type CpmtFd (A) and mtFNR-type CpmtFNR (B) and sequence comparison of their conserved domains and motifs with representative species. In CpmtFd, the conserved C-terminal VDGxxPxPH motif characteristic to Type II mitochondrial-typetferredoxin (mtFd-II) is boxed in gray. The four Cys residues involved in the binding of [2Fe–2S] cluster is marked by stars. In CpmtFNR, six conserved motifs are identified, including four motifs contain highly conserved residues (marked by stars) that are previously known to be involved in FAD-pyrophosphate binding and NADP-pyrophosphate binding. The N-terminal sequence without a defined upstream start Met residue but predicted as a secretory signal (if translated) is question marked. In both proteins, regions expressed as recombinant proteins are shown. A sequence log display for those regions can be found in Supporting Information Figure S1.

Figure 4

Phylogenetic reconstructions reveal apicomplexan mtFd-II and mtFNR are unique groups of proteins that are highly divergent from their counterparts in humans and animals. A: Phylogenetic tree inferred from mtFd-II proteins with 38 taxa and 112 amino acid (aa) positions by Bayesian inference (BI) and ML bootstrapping methods. B: Phylogenetic tree inferred from mtFNR proteins with 60 taxa and 245 aa positions. Both BI and ML analyses used a WAG amino acid substitution model with the consideration of fraction of invariance (F_inv) and 12-rate of gamma distribution [Γ₍₁₂₎]. PP values in BI analysis were summarized after 25% first trees (resulted from 106 generation of runs) were discarded. ML bootstrap proportion (BP) support values were summarized from 100 replicated sequences under a majority ruling law. In both trees, only nodes supported by ≥50% PP or BP values are labeled. Nodes highlighted with solid circles (or solid diamonds) are supported by PP at 1.0 and BP values ≥ 95.0% (or 90.0–94.9%), respectively.

Figure 5

Expression profiles of CpmtFd and CpmtFNR genes in parasite oocysts and intracellular developmental stages as determined by real-time quantitative RT-PCR. Fold changes are plotted in relative to that in the oocyst stage.

Figure 6

SDS-PAGE fractionation of recombinant CpmtFd and CpmtFNR proteins in which the MBP or His tags were removed. Tobacco etch virus (TEV) protease was also loaded for comparison.

Figure 7

A: UV/vis spectrum of recombinant CpmtFd displayed multiple peaks of absorbance at 460, 416, 343, and 278 nm, which are characteristic to the [2Fe–2S]-containing proteins such as that for Escherichia coli ferredoxin (superimposed here based on Ref. 33). B: Electron paramagnetic resonance (EPR) spectrum of CpmtFd in reduced form at 4 K.