An FtsH Protease Is Recruited to the Mitochondrion of <i>Plasmodium falciparum</i>

<div><p>The two organelles, apicoplast and mitochondrion, of the malaria parasite <i>Plasmodium falciparum</i> have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (<i>Pf</i>FtsH1) that exhibits ATP- and Zn²⁺-dependent protease activity. <i>Pf</i>FtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged <i>Pf</i>FtsH1, and immunofluorescence assay with anti-<i>Pf</i>FtsH1 Ab demonstrated that the protein localises to <i>P. falciparum</i> mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in <i>Pf</i>FtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of <i>Pf</i>FtsH1 in <i>Escherichia coli</i> converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the <i>Plasmodium</i> factor on the bacterial homolog, indicative of functional conservation with <i>Ec</i>FtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in <i>P. falciparum</i>.</p> </div

Localization of <i>Pf</i>FtsH1 to the mitochondrion is confirmed by immunofluorescence with anti-FtsH1 Ab.

<p>Confocal immunofluorescence microscopy of <i>P. falciparum</i> 3D7 infected erythrocytes using Mitotracker Red and anti-FtsH1 Ab shows localization of <i>Pf</i>FtsH1 in the parasite mitochondrion.</p

ATP- and Zn²⁺-dependent protease activity of <i>Pf</i>FtsH1.

<p>(A) <i>Pf</i>FtsH1 cleaves α-casein in a time-dependent manner in the presence of zinc and ATP. Addition of EDTA inhibits protease activity of <i>Pf</i>FtsH1 indicating the requirement of Zn²⁺ for <i>Pf</i>FtsH1-catalysed proteolysis. (B) <i>Pf</i>FtsH1 binds ATP as indicated by quenching of intrinsic fluorescence from the single tryptophan residue of the recombinant protein upon incubation with ATP. (C) ATP hydrolysis, and not just binding of the nucleotide to <i>Pf</i>FtsH1, is required for proteolytic activity. Proteolysis of α-casein was measured in the presence of ATP or its non-hydrolysable analog AMPPNP, and control sets lacking nucleotide or the enzyme. Proteolysis was observed only in the presence of ATP.</p

Localisation of <i>Pf</i>FtsH1 in a <i>P. falciparum</i> 3D7 transfectant line carrying C-terminal 3xHA-tagged <i>Pf</i>FtsH1.

<p>(A) Western with anti-HA mAb recognises an intact ~105 kDa (FtsH + HA tag) product and a ~38 kDa band likely to represent the cleaved ~35 kDa C-terminal region fused with HA. (B) Immunofluorescence localization of <i>Pf</i>FtsH1-HA using the anti-HA mAb and antibody against the apicoplast marker ACP. No overlap of <i>Pf</i>FtsH1-HA signal was observed with the apicoplast marker. (C) <i>Pf</i>FtsH1 co-localizes with the mitochondrial signal in trophozoites (upper panel) and appears as punctuate signals lining the organelle defined by the mitochondrial stain Mitotracker Red in schizonts (lower panel). (D) Confocal microscopy <i>Pf</i>FtsH1-HA expressing parasites showing co-localisation of <i>Pf</i>FtsH1 with the mitochondrion which is stained with Mitotracker Red.</p

Defective cytokinesis observed in a fraction of <i>E. coli</i> cells expressing <i>Pf</i>FtsH_int.

<p><i>E. coli</i> cells transformed with pGEX + RIG or pGEX-<i>Pf</i>FtsH_int +RIG were grown for 3 h at 20°C after induction, fixed and stained with DAPI.</p

Recombinant expression of <i>Pf</i>FtsH1 in <i>E. coli</i> and its detection in the parasite lysate.

<p>(A) Line-drawing showing <i>Pf</i>FtsH1 stretches expressed in <i>E. coli</i> and the probable protein cleavage site. (B) Purified GST-<i>Pf</i>FtsH_int visualised in a coomassie-stained SDS-PA gel (left panel) and western blot analysis of purified protein using anti-GST Ab (right panel). (C) Purified His-<i>Pf</i>FtsH1 ATPase + protease domain on a coomassie-stained SDS-PA gel (left panel) and western blot of the protein with anti-His Ab (right panel). (D) <i>P. falciparum</i> lysate probed with anti-<i>Pf</i>FtsH1 Ab (I) detects a ~101 kDa band and a major ~66 kDa band. A minor band is also seen at ~72 kDa. No signal is detected with pre-immune serum (Pre-I).</p

<i>Pf</i>FtsH1 is a membrane-associated protein.

<p><i>P. falciparum</i> D10 ACP_leader-GFP parasites were <i>s</i>equentially treated with Tris, sodium carbonate and Triton X-100 to investigate membrane association. Unlike apicoplast lumenal GFP, all <i>Pf</i>FtsH1 was Tris-insoluble and only partially solubilised by carbonate buffer, indicating membrane association. Almost all <i>Pf</i>FtsH1 was solubilised by Triton X-100. S, soluble fraction; IS, insoluble fraction.</p

Phylogenetic tree of FtsH proteins from Bacteria, diatoms, yeast, alga, protozoa, plants and humans.

<p>Phylogenetic analysis using the maximum likelihood method indicates that the apicomplexan FtsH sequences form three distinct clades. Two of these (including the <i>Pf</i>FtsH analysed in this study, indicated by *) are closely allied with proteases known to face into the mitochondrial intermembrane space (i-AAA proteases), a third is allied with kinetoplastida proteases and proteases known to face into the mitochondrial matrix (m-AAA proteases). Athal, <i>Arabidopsis thaliana</i>; bbov, <i>Babesia </i><i>bovis</i>; cmer, <i>Cyanidioschyzon</i><i>merolae</i>; cpar, <i>Cryptosporidium parvum</i>; ecol, <i>Escherichia coli</i>; hnep, <i>Hyphomonas</i><i>neptunium</i>; hsap, <i>Homo sapiens</i>; lmaj, <i>Leishmania major</i>; mmus, <i>Mus musculus</i>; ncan, <i>Neospora</i><i>caninum</i>; nitr, <i>Nitrosomonas</i> sp. AL212; ntab, <i>Nicotiana tabacum</i>; pfal, <i>Plasmodium falciparum</i>; pviv, <i>P. vivax</i>; rglu, <i>Rhodotorula</i><i>glutinis</i>; scer, <i>Saccharomyces cerevisiae</i>; syne, <i>Synechocystis</i> sp. PCC 6803; tequ, <i>Taylorellaequigenitalis</i>; tgon, <i>Toxoplasma gondii</i>; ther, <i>Thermodesulfobacterium</i> sp. OPB45; tmar, <i>Thermotoga maritima</i>; tpse, <i>Thalassiosira pseudonana</i>; tviv, <i>Trypanosoma</i><i>vivax</i>; vcho, <i>Vibrio cholerae</i>. Sequences used in the alignment are available in File S2.</p

Processing and oligomeric assembly of <i>Pf</i>FtsH1 in the parasite.

<p>(A) Schematic of <i>Pf</i>FtsH1 processing inferred from results shown below and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074408#pone-0074408-g002" target="_blank">Figure 2D and Figure 3A</a>. * identifies <i>Pf</i>FtsH fragments detected by anti-HA mAb; # denotes fragments recognized by the anti-FtsH Ab generated against the ATPase+protease domain. (B) Pulse-chase of <i>P. falciparum</i>-infected erythrocytes followed by immunoprecipitation with anti-<i>Pf</i>FtsH antibody. Parasites at the early trophozoite stage were labeled with ³⁵S methionine and cysteine for 90 min and harvested immediately after the pulse (0 h, lane 1) and after 1 h, 2.5 h and 5 h of chase (lanes 2-4). Lane 5 is immunoprecipitation at 0 h with pre-immune serum. A seven-day exposure of X-ray film was required to detect the signals above. (C) Parasite proteins were cross-linked in vivo by DSP followed by treatment with increasing concentrations of DTT to break the complex(s). <i>Pf</i>FtsH1 was detected by western blot analysis using anti-<i>Pf</i>FtsH1 Ab. (D) <i>Pf</i>FtsH1 exists in oligomeric complexes in the parasite as detected by BN-PAGE followed by western blotting with anti-<i>Pf</i>FtsH1 Ab.</p

Linkage between regulatory region SNPs and correlation of haplotypes with plasma TNF levels

<p><b>Copyright information:</b></p><p>Taken from "Polymorphisms of -enhancer and gene for FcγRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population"</p><p>http://www.malariajournal.com/content/7/1/13</p><p>Malaria Journal 2008;7():13-13.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2245971.</p><p></p> A, rLD plot of four SNPs (-1031, -863, -308, -238). Value in each cell is the percent D' between SNP pairs. B, Correlation of promoter haplotype (-1031C, -863A, -857C, -308G, -238G) with elevated TNF levels compared to the major haplotype TCCGG. Mean plasma TNF levels were compared using Mann-Whitney test (z = -3.73, P = 0.0002). C, Plasma TNF level in patients and controls. Pairwise comparison of means by non-parametric Mann-Whitney test: severe versus control, z = 4.41, P < 0.0001; non-severe versus control, z = 0.67, P = 0.25; severe versus non-severe, z = 3.13, P = 0.0009