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Infection and Immunity, March 2007, p. 1116-1128, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.00902-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Plasmodium falciparum and P. berghei Homologues of Macrophage Migration Inhibitory Factor{triangledown}

Kevin D. Augustijn,1 Robert Kleemann,2 Joanne Thompson,3 Teake Kooistra,2 Carina E. Crawford,3 Sarah E. Reece,3 Arnab Pain,4 Arjan H. G. Siebum,5 Chris J. Janse,1 and Andrew P Waters1*

Department of Parasitology, LUMC, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands,1 Vascular and Metabolic Diseases, TNO-Quality of Life, Zernikedreef 9, 2333 CK, Leiden, The Netherlands,2 Institute of Immunology and Infection Research, Kings Buildings, Ashworth Laboratories, West Mains Road, EH9 3JT Edinburgh, United Kingdom,3 The Wellcome Trust Sanger Institute, Hinxton, CB10 1SA Cambridge, Cambridge, United Kingdom,4 Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC, Leiden, The Netherlands5

Received 7 June 2006/ Returned for modification 16 August 2006/ Accepted 3 December 2006


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ABSTRACT
 
Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection.


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INTRODUCTION
 
Cytokines are the molecular messengers of the vertebrate immune system, coordinating the local and systemic immune responses to infective organisms. Macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered (4, 17) and is involved in both innate and adaptive immune responses. MIF counterregulates the anti-inflammatory effect of glucocorticoids (9) and is a key regulator of the proinflammatory response to endotoxins. For example, MIF-deficient mice survive a lethal dose of lipopolysaccharide (LPS) (6). MIF is released by immune cells in response to microbial products and proinflammatory cytokines, such as tumor necrosis factor alpha (10, 12). While mammalian MIF is normally found as a homomeric trimer, each subunit has two catalytic sites and two activities: a tautomerase activity, which requires an N-terminal proline residue (33), and an oxidoreductase activity (29), which is based on a thioredoxin-like motif. Although enzymatic activities are not normally found in cytokines, both of these activities have been linked to MIF's cytokine function (28, 38, 45). At the molecular level little is known about the exact mechanisms of MIF function; the biological substrates for the enzymatic activities of MIF, as well as its import and export pathways, are not fully understood. So far, a classical cytokine receptor for MIF has not been discovered, and the surface receptor (CD74) (32) and the intracellular factor Jun activation domain binding protein (Jab-1) (27) are the only functional MIF-binding partners that have been described.

Although the mode of action and the complete biological role of host-derived MIF remain to be established, it has been shown that MIF also has a critical role in determining the outcome of infections caused by parasites such as helminths (42), malaria parasites (34), and Leishmania (44). Interestingly, homologues of human MIF (huMIF) have been characterized in nematodes (39, 48, 49). Parasitic nematodes are long lived in their hosts and are able to modulate the immune response to evade killing by the immune system (for a review, see reference 27). Therefore, it has been suggested that the expression of MIF homologues plays a role in the immunobiology of and immune evasion by these nematodes. In support of this, workers have found evidence that nematode MIF has a role in activating macrophages and in recruitment of eosinophils (21). Genome sequencing of other human parasites has revealed that not only parasitic helminths but also protozoans, such as Plasmodium, contain genes encoding huMIF homologues (24). Plasmodium is the causative agent of malaria, which is responsible for over one million deaths annually and imposes a tremendous social and economic burden (7). The potential ability of Plasmodium to manipulate the host immune response though the secretion of cytokine homologues is clearly of interest.

In this paper we describe the first biochemical and genetic characterization of the Plasmodium homologues of MIF (PMIF) from two species, Plasmodium falciparum and Plasmodium berghei. We found that C-terminally His6-tagged PMIF exhibits biochemical and immunostimulatory features similar to those of huMIF and that it is expressed during the blood stages of parasite development in a mammalian host. Furthermore, gene deletion experiments showed that P. berghei MIF (PbMIF) is not required for completion of the parasite life cycle but significantly influences the number of reticulocytes in the circulation of mice during the early stages of infection. Furthermore, we found that PMIF is secreted from infected red blood cells and ruptured schizonts. Coupled with the lack of an essential intracellular function, therefore, our data indicate that PMIF most likely has a function in the interaction of the parasite with its host.


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MATERIALS AND METHODS
 
Plasmid construction. To obtain protein expression constructs, MIF reading frames were amplified from mixed blood stage P. falciparum (PlasmoDB entry PfMIF: PFL1420w) and P. berghei (GeneDB entry PbMIF: PB000372.03.0) cDNA by PCR using primers L1632 (5'-AAATTTCCGCATGCCGTGCTGTGAATTAATAAC-3') and L1554 (5'-ATTGGATCCACCAAATAGTGAGCCACTAAAAGC-3') (underlining indicates restriction sites) for P. berghei and primers L1667 (5'-AAAATTTCCGCATGCCTTGCTGTGAAGTAATAAC-3') and L1668 5'-ATTGGATCCGCCGAAAAGAGAACCACTGAAGGC-3' for P. falciparum, digested with SphI and BamHI, and ligated into C-terminally His6-tagged expression vector pQE-70 (QIAGEN). For genomic expression of green fluorescent protein (GFP)-tagged PbMIF, the MIF gene and promoter region (1,200-bp upstream sequence) were amplified from P. berghei genomic DNA using primers L1879 (5'-AAAGGTATTCCATGGAACCAAATAGTGAGCCACTAAAAGCA-3') and L1880 (5'-TAAATCAAAGCGGCCGCGAATCTTATCAACCATTTATACC-3'), digested with NcoI and NotI, and ligated into the C-terminally GFP-tagged integration construct pGFP-TAG (30) or pMYC-TAG, which was generated by transferring the c-myc tag from pSD141 (41), a kind gift from Oliver Billker (Imperial College, London, United Kingdon), into pGFP-TAG using primers L2300 (5'-CGGGGTACCGCGGCCGCTATCTAGACGAGGATCCATGGGGCCCGAACAAAAACTCATC-3') and L2301 (5'-GAGCAGATTGTACTGAGAGTGCACCATATGCGGTG-3'). To obtain a MIF knockout vector, an upstream region (positions –1200 to –300) and a downstream region (positions 785 to 1700) from the pbmif gene were amplified from P. berghei genomic DNA using primers L1466 (5'-TAAATCAAGGTACCGGCGAATCTTATCAACCATTTATACC-3') and L1467 (5'-TTGTATTTCATCGATCTCTTGTGATATTAATCCATACACGCC-3') and primers L1468 (5'-AAAGAGGCTGAATTCTATGTAAATTATTTTTCCTATGCCCTTAAC-3') and L1469 (5'-ATCGGATCCTTATGCGTATATATATTGAAGCATGGTG-3'), respectively. The PCR products were digested with Asp718 plus ClaI and EcoRI plus BamHI, respectively, and ligated into B3D (46). All constructs were sequenced to confirm MIF sequence identity.

Protein purification and refolding. P. falciparum MIF (PfMIF) and PbMIF were expressed in BL21(DE3)(pLysS) bacteria grown on Luria broth (Q-biogene) containing 30 µg/ml chloramphenicol and 0.05 µg/ml ampicillin. Bacteria were grown at 37°C to an optical density at 600 nm of 0.4 and were induced by addition of isopropyl-D-thiogalactoside to a final concentration of 1 mM. After 3 h, the cells were harvested and lysed in 50 mM Tris (pH 8.0), 500 mM NaCl, 10% (vol/vol) glycerol, 0.01% Nonidet P-40, 10 mM ß-mercaptoethanol in the presence of complete protease inhibitor cocktail (Roche) and 1 µg/ml lysozyme. Genomic DNA was fragmented by sonication, after which the lysate was cleared by centrifugation at 30,000 x g for 45 min at 4°C and loaded onto an Ni-nitrilotriacetic acid column (QIAGEN) equilibrated in 50 mM Tris (pH 8.0), 200 mM NaCl, 10% (vol/vol) glycerol, 20 mM imidazole, 10 mM ß-mercaptoethanol. After washing, the protein was eluted in a linear 20 to 400 mM imidazole gradient. Peak fractions were pooled, concentrated to 10 ml in an Amicon stirred ultrafiltration cell (Millipore) using a 10-kDa-cutoff filter, and loaded on a Superdex 75 gel filtration column (Amersham Pharmacia) equilibrated in 50 mM Tris (pH 8.0), 200 mM NaCl, 5% (vol/vol) glycerol, 5 mM ß-mercaptoethanol. These preparations and MIF preparations of refolded lyophilized recombinant MIF (3) were confirmed to be free of LPS (<5 ng of LPS/mg of protein) by the Limulus amoebocyte assay (Biowhittaker Inc., Walkersville, MD).

Oxidoreductase and tautomerase assays. Oxidoreductase and tautomerase assays were performed as described previously (29, 43), except that 1H nuclear magnetic resonance spectra during the tautomerase analysis were recorded with 1-min intervals using a Bruker DPX-300 with 3-(trimethylsilyl)tetradeuteropropionic acid sodium salt as the internal standard.

Antibody generation and Western blotting. Polyclonal antibodies against PbMIF were raised in New Zealand White rabbits by injecting 100 µg LPS-free PbMIF linked to keyhole limpet hemocyanin in complete Freund's adjuvant. After three boosts with 100 µg keyhole limpet hemocyanin-linked PbMIF in incomplete Freund's adjuvant, 5 ml of serum was collected and used at a 1/1,000 dilution as the primary antibody for Western blotting.

Immunofluorescence assays. Blood stages from overnight schizont cultures were fixed with paraformaldehyde, and the c-myc tag was visualized by incubation with anti c-myc monoclonal antibody (C3956; Sigma-Aldrich, The Netherlands), followed by staining with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G antibody. Parasite nuclei were stained using 4',6'-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, The Netherlands) according to the manufacturer's instructions. Fluorescence was visualized using fluorescence MDR microscopy (GFP and DAPI filter settings; Leica), and images were recorded using a DC500 digital camera.

AP-1 activation assay. The AP-1 assay was performed as described previously (26, 28). Briefly, 1.7 x 105 human embryonic kidney (HEK) cells per well were plated in 24-well cell culture plates. After 24 h, transfection with 50 ng pAP-1-luciferase reporter plasmid and 1 ng pRenilla control plasmid (dual-luciferase reporter assay system; Promega, Leiden, The Netherlands) per 1.7 x 105 HEK cells was performed using lipofectamine 2000 (Invitrogen) as the transfection reagent. Cells were allowed to recover from transfection for 18 h. Then transfected cells were preincubated with recombinant human MIF or PMIF or with control buffer for 1 h, followed by an 8-h coincubation with 3 nM phorbol myristate 13-acetate (PMA). Both the PMA concentration and the incubation time were optimized in pilot experiments to obtain the greatest induction of the AP-1 promoter without causing significant cell death. Control cells (basal) were not incubated with PMA. Cells were washed, and cell lysates were prepared using 100 µl passive lysis buffer (dual-luciferase reporter assay system; Promega) to quantify luciferase and renilla activities according to the protocol of the manufacturer.

Gene knockout and tagging in P. berghei. Transfection and selection of parasites with pbmif deleted or of tagged parasites were performed as previously described (18). Genomic integration of knockout and tagging vector DNA was confirmed by Southern blotting of pulse-field gel electrophoresis-separated chromosomes of the knockout or tagged parasites and probing with the 3' untranslated region of the PbDHFR gene. In addition, correct replacement of the PbMIF locus was verified by Southern blotting of AccI- and AccI/XbaI-digested wild-type and pbmif knockout (pbmif-ko) parasite genomic DNA and probing with the upstream pbmif knockout target region (positions –1200 to –300). The pbmif-ko lines and the line expressing c-myc-tagged PbMIF were cloned using limiting dilution, whereas the line expressing GFP-tagged PbMIF was not cloned using limiting dilution.

Virulence experiments. In two experiments, five hosts (mouse strains BALB/c and C57BL/6) were infected with HP-ANKA wild-type P. berghei parasites and five hosts were infected with pbmif-ko parasites (pbmif-ko1). In addition, in the second experiment, five hosts for both mouse strains were also infected with a second independently generated pbmif-ko parasite line (pbmif-ko2). All parasite lines were generated from the same stock (passage number). All mice (Haarlan, United Kingdom) were randomized into groups with respect to weight and red blood cell count, infected intraperitoneally with 1 x 105 ring stage parasites, and monitored daily from day 3 postinfection. Every day of sampling, mice were weighed, thin smears were prepared, and the red blood cell densities were calculated by flow cytometry (the mature red blood cells and reticulocytes in the size range from 3.3 to 10 µm in diameter in 2 µl of tail blood were counted according to the manufacturer's instructions; Coulter Electronics, United Kingdom). Mice were sampled until the level of parasitemia became very high (>50%) and/or substantial mortality occurred (day 12 postinfection for BALB/c hosts and day 7 postinfection for C57BL/6 hosts).

Analysis of virulence experiments. The proportion of parasitized red cells (parasitemia) and the proportion of red cells that were reticulocytes (reticulocytemia) were calculated daily from thin smears, and the results were transformed to obtain parasite and reticulocyte densities using red cell density counts. The following summary statistics for each infected host were calculated: cumulative densities for parasites and reticulocytes; red cell and mass loss; minimum red cell density and mass; and peak parasitemia and reticulocytemia. Proportion data were arcsin square root transformed to comply with the assumptions of parametric tests. For all experiments, general linear models (R Foundation for Statistical Computing, Austria) were used to compare infection parameters for pbmif-ko and wild-type infections, and Tukey tests were used to determine which groups differed when significant results were obtained.


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RESULTS
 
MIF is expressed constitutively by Plasmodium and secreted by blood stage forms. MIF homologues have been found previously in a number of parasitic nematodes (21, 48, 49) and have now also been found in apicomplexans, including Plasmodium (PlasmoDB entry PfMIF: PFL1420w; GeneDB entry PbMIF: PB000372.03.0). These parasite MIFs exhibit moderate levels of homology with mammalian MIFs and with each other (29% identity and 39% similarity between huMIF and PfMIF) (Fig. 1A). Nevertheless, a crystal structure determination of nematode MIF protein revealed that the overall structure of the divergent MIF proteins is highly conserved (45), and thus the sequence of parasite MIFs can be accurately threaded onto the human MIF structure. Conserved residues, shown in Fig. 1B in ball-and-stick form in the crystal structure of huMIF (33), mainly cluster around the tautomerase active site (residues 2, 32 to 34, and 64 to 66), indicating that the tautomerase activity is likely to be a shared property of mammalian and parasite MIFs. In contrast, the second cysteine in the mammalian MIF CXXC oxidoreductase motif does not seem to be as well conserved in the parasite MIFs (Fig. 1A).


Figure 1
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  		FIG. 1. Sequence alignment of mammalian and parasite MIFs. (A) Sequence alignment of human, mouse, P. berghei (Pb), and P. falciparum (Pf) MIF homologues, as well as two Brugia malayi MIF homologues (Bm-1 and -2). The arrow indicates the N-terminal catalytic proline associated with tautomerase activity, and the area enclosed in a box is the CXXC motif associated with oxidoreductase activity in the mammalian MIFs and the parasite variants. Asterisks indicate identical residues; colons and dots indicate residues with high and low levels of similarity, respectively. (B) Ribbon representation of the huMIF crystal structure (RCSB PDB entry: 1CA7 [33]). For clarity, only one monomer of the trimer is shown. Arrows indicate the positions of the tautomerase and oxidoreductase active sites. The first cysteine of the CXXC oxidoreductase motif (C57 in huMIF) that is conserved in the parasite MIFs is red. Conserved residues in the various MIFs mainly cluster around the tautomerase active site and are shown in ball-and-stick form.

To determine the expression profile of PbMIF, we analyzed P. berghei infection at various stages by Northern blotting. pbmif was transcribed throughout the asexual blood stages, and peak steady-state mRNA levels were observed at the late trophozoite and early schizont stages (Fig. 2A). Furthermore, we tracked PbMIF protein expression by examining generation of a knockin P. berghei strain expressing C-terminally GFP-tagged PbMIF under control of the endogenous promoter (Fig. 3B). Since the molecular weight of the GFP tag is about three times the molecular weight of the PbMIF monomer, the presence of GFP may affect the cellular distribution of PbMIF. Therefore, we did not draw any conclusion with regard to cellular localization of the fusion, other than presence or absence at a given stage of the life cycle. PbMIF-GFP was found to be expressed ubiquitously in the blood stages and in mosquito stage ookinetes and sporozoites (Fig. 2B to I).


Figure 2
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  		FIG. 2. Expression of PbMIF throughout the life cycle. (A) Time course Northern analysis of the PbMIF transcript in blood stages. Whole RNA extracted at various times from a synchronous P. berghei blood stage infection showed that there was production of PbMIF mRNA of the expected size (~0.7 kb) at 17 h postinfection (HPI) (trophozoite stage). Small quantities of transcript were also present in the sexual stages (Gct 1, enriched gametocytes; Gct 2, purified gametocytes) and the ookinete (Ook). An ethidium bromide-stained portion of the gel showing the positons of the 28S and 18S rRNAs was included as loading control. (B to E) Protein expression of the PbMIF-GFP fusion throughout the life cycle in a late ring/early trophozoite (B), mature trophozoite (C), schizont (D), and mature ookinete (some unfertilized female gametes are also visible) (E). Magnification, x100. (F and G) Protein expression of the PbMIF-GFP fusion in a day 18 oocyst. Magnification, x20 and x40, respectively. (H and I) Protein expression of the PbMIF-GFP fusion in salivary gland sporozoites. Magnification, x40 and x100, respectively. Bars, 10 µm.


Figure 3
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  		FIG. 3. Genetic manipulation of the pbmif locus. (A) pbmif-ko was made by double-crossover replacement using the 5' untranslated region (5'UTR) (positions –1200 to –300) and 3' untranslated region (3'UTR) (positions 785 to 1700) from the pbmif gene. Gene replacement with the TgDHFR/TS selection marker removed the XbaI site in the 3' untranslated region of PbMIF and introduced an additional AccI site. The sizes of restriction fragments are indicated between arrows, and the hatched box represents the region that was used as a probe in the Southern analysis whose results are shown in panel C. (B) PbMIF-GFP and c-myc fusions were made by a single-crossover knockin (insertion) event, which resulted in gene duplication of pbmif with one of the two copies tagged with GFP or c-myc, while the second copy remained wild type. (C) Southern blot analysis of AccI- and AccI/XbaI-digested genomic DNA from wild-type and knockout parasites. Using the 5' untranslated region integration target (hatched box in panel A) as the probe, the digestions should have yielded 2,447-, 2,100-, 2,616-, and 2,616-bp fragments, respectively.

Any function of PMIF in a parasite-host interaction or in immune evasion would require PMIF to be externalized by the parasite. However, like mammalian MIF, which is exported by nonclassical pathways likely involving an ABCA1 transporter (22), PMIF has no signal sequence. To analyze whether PMIF is released from parasite-infected erythrocytes, we developed a polyclonal antibody specific for PbMIF and immunoblotted supernatants and lysates of synchronous, cultured P. berghei blood stage parasites. This antibody showed extremely low cross-reactivity with recombinant huMIF during a long exposure (>5 min) of the blot (Fig. 4A, lanes 12 and 13), which is virtually identical to the results obtained with murine MIF (89% identity and 95% homology). Nevertheless, to prevent any confounding presence of murine MIF, we removed all leukocytes by passing the sample over a Plasmodipur filter (Euro-Diagnostica, Arnhem, The Netherlands). When grown in culture, P. berghei blood stage parasites arrest in the late schizont stage, since the infected erythrocytes do not rupture (47). This allows workers to discriminate between active secretion throughout asexual growth and release by the infected erythrocyte at the time of schizont rupture. Figure 4A shows that significant levels of PbMIF were present in the cytoplasm of the infected erythrocytes in both the trophozoite and schizont stages. A small quantity of PbMIF was also detected in the culture supernatant, but the amount varied between experiments (not shown), so the MIF was likely released by mechanical shearing of infected erythrocytes. Mechanical rupture of the parasitophorous vacuole of the schizonts, releasing the merozoites, resulted in release of an additional burst of PbMIF. Lysis of trophozoites or merozoites released additional MIF.


Figure 4
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  		FIG. 4. PbMIF is externalized by P. berghei and is released upon schizont rupture. (A) Western blot analysis using anti-PbMIF rabbit polyclonal antiserum. Lane 1 contained erythrocytes (ery.) infected with trophozoite (troph.) stage P. berghei that were isolated by heart puncture and lysed by osmotic shock. Parasite-derived MIF was pulled down from the lysis supernatant (sup.) (from the equivalent of 107 infected erythrocytes) using anti-PbMIF bound to protein G-Sepharose. Lane 2 contained control pulldown of the lysis supernatant using protein G-Sepharose alone. Lane 3 contained a trophozoite pellet (~2.0 x 105 parasites loaded). Lane 4 contained control pulldown of 100 ng of recombinant (rec.) C-terminally His6-tagged PbMIF using protein G-Sepharose alone. Lane 5 contained control pulldown of 100 ng of recombinant C-terminally His6-tagged PbMIF using anti-PbMIF loaded protein G-Sepharose. Lane 6 contained supernatant (10 µl of 100 ml) of an overnight schizont (schiz.) culture. Lane 7 contained supernatant (10 µl of 20 ml) of schizonts after disruption of the host erythrocyte membrane. Lane 8 contained phosphate-buffered saline wash supernatant (10 µl of 1 ml) of schizont pellet from lane 7. Lane 9 contained supernatant (10 µl of 1 ml) after mechanical rupturing of the schizonts into merozoites. Lane 10 contained phosphate-buffered saline wash supernatant (10 µl of 1 ml) of the merozoites from lane 9. Lane 11 contained solubilized merozoite pellet (~2.0 x 105 parasites loaded). Lane 12 contained 50 ng of recombinant C-terminally His6-tagged PbMIF. Lane 13 contained 100 ng of recombinant huMIF. (B) Immunofluorescent detection of c-myc-tagged PbMIF in blood stages. Thin smears from an overnight schizont culture expressing PbMIF-c-myc were stained using an anti-c-myc monoclonal antibody. (Panel 1) Intact schizont and trophozoite (arrowhead) show staining within the parasitophorous vacuole. (Panel 2) Intact schizont and ruptured schizont. (Panel 3) Wild-type P. berghei control stained with the anti-c-myc monoclonal antibody. The fluorescent image in panel 3 was taken with a 0.6-s exposure, while the fluorescent images in panels 1 and 2 were taken with a 0.3-s exposure. Bars, 10 µm. FITC, fluorescein isothiocyanate.

Rabbit polyclonal PbMIF antisera showed high nonspecific cross-reactivity in an immunofluorescence assay, precluding direct visualization of PbMIF in infected cells. Therefore, a knockin P. berghei strain expressing PbMIF with the much smaller (2.7-kDa) C-terminal c-myc tag (Fig. 3B) was generated, enabling direct detection of the fusion using monoclonal anti-c-myc antibodies. Figure 4B1 shows staining of the parasitophorous vacuole in trophozoites and schizonts, along with faint staining of the infected erythrocyte cytoplasm in the trophozoite. Schizont rupture resulted in a loss of fluorescence (Fig. 4B2), consistent with the results shown in Fig. 4A.

Expression and purification of recombinant PMIF-His6 and measurement of enzymatic activities. Although all MIFs are structurally conserved, the conservation of the residues associated with the two known enzymatic activities is greater for the tautomerase activity than for the oxidoreductase activity. Since the N-terminal proline is the catalytic residue in the tautomerase activity (33) and we wanted to compare the enzymatic properties of PMIF with those of huMIF, we located the His6 tag at the C terminus well away from any enzymatic site in our PbMIF, PfMIF, and huMIF expression constructs. The major peak of PMIF-His6 eluted at an apparent molecular mass of 30 kDa on a size exclusion column, while huMIF-His6 eluted at an apparent molecular mass of 22 kDa. Both values are consistent with a dimeric form in solution (Fig. 5A). We determined the tautomerase activity of the purified recombinant PMIF-His6 by monitoring the conversion of p-hydroxyphenylpyruvate from the enol form to the keto form by 1H nuclear magnetic resonance (43). PfMIF-His6 and PbMIF-His6 showed levels of tautomerase activity that were appreciably above the background level of the mock purification control, but the activity was fivefold lower that the activity of wild-type huMIF-His6, which was used as a control (Fig. 5B). We next tested whether PMIF-His6 exhibited oxidoreductase activity and quantified the NADPH-dependent reduction of 2-hydroxyethyldisulfide (HED). Figure 5C shows that the oxidoreductase activity of PfMIF-His6 was able to catalyze the reduction of HED at levels greater than the levels observed for the mock purification control. Surprisingly, given the lack of conservation, the oxidoreductase activity was also reduced only about fivefold compared to the activity of the huMIF-His6 wild-type control. In both assays, PbMIF-His6 exhibited less activity than PfMIF-His6 exhibited. Given the high level of conservation between these two MIFs, however, this was unlikely to be due to lower intrinsic activity but rather may have reflected small differences in refolding efficiency.


Figure 5
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  		FIG. 5. Recombinant PMIF elutes as a dimer in size exclusion chromatography and is active in tautomerase and oxidoreductase assays. (A) Chromatograms of C-terminally His6-tagged huMIF, which resulted in calculated molecular masses of 30 kDa for PbMIF and 22 kDa for huMIF. The reference compounds included bovine albumin (66 kDa; 153.93 ml), chicken ovalbumin (45 kDa; 170.93 ml), bovine carbonic anhydrase (30 kDa; 192.47 ml), and bovine {alpha}-lactalbumin (14.4 kDa; 217.39 ml). The inset shows a Coomassie blue-stained protein gel containing the peak fractions for PbMIF, which identified the void volume peak as PbMIF aggregates. The same results were obtained with PfMIF (data not shown). mAU, milli-absorbance units. (B and C) Recombinant MIF tautomerase activity with p-hydroxyphenylpyruvate (R = COOH and R' = C6H4-OH) (B) and oxidoreductase activity with 2-hydroxyethyldisulfide (R = CH2-OH) (C). Sample equations are shown for both conversions. The activities of PfMIF and PbMIF are expressed as percentages of the huMIF activity. In both sets of experiments, the PfMIF and PbMIF activities were greater than the activities of the mock purification control. Asterisks indicate that the P value is <0.05 (n ≥ 3).

Recombinant PMIF is as efficient as huMIF in inhibiting AP-1 stimulation. We investigated if the in vivo activity of PMIF-His6 mirrored the reduced enzymatic activity of PMIF-His6 compared to huMIF-His6 activity. huMIF can reduce AP-1 expression by physically interacting with the transcriptional coactivator Jab-1, an activity at least partially dependent on the oxidoreductase activity of huMIF (28). Therefore, we used an AP-1 reporter assay with HEK cells to examine the biological activity of recombinant PMIF-His6. Figure 6 shows that although both huMIF-His6 and PMIF-His6 showed a trend toward repression of the basal expression of AP-1, this effect became statistically relevant (P < 0.05; n ≥ 6) only after AP-1 expression was induced with PMA. The induction of the AP-1 promoter by PMA was quite subtle in HEK cells. Higher levels of induction could be obtained by increasing the PMA concentration and incubation time, but they were invariably associated with increased cell death. Nevertheless, the repression effect was on the same order of magnitude for both huMIF-His6 and PMIF-His6, which implies that PMIF is able to bind to and interfere with Jab-1 to the same extent as huMIF despite its reduced oxidoreductase activity.


Figure 6
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  		FIG. 6. Recombinant PMIF can inhibit AP-1 induction like huMIF. At the basal level of transcription, both huMIF and PMIF showed a nonsignificant trend toward inhibition of AP-1 transcription in HEK cells. However, upon stimulation with PMA, both huMIF and PMIF showed statistically relevant repression compared to buffer controls. Asterisks indicate that the P value is <0.05 (n ≥ 6).

PbMIF knockout parasites are viable throughout the life cycle and increase host reticulocyte density. To investigate the role of PbMIF during in vivo infection in the mouse host and mosquito vector, two independent pbmif-ko parasite lines were generated using double-crossover gene replacement. Correct integration of the selection marker and disruption of the pbmif locus were confirmed by pulse-field gel electrophoresis, PCR (not shown), and Southern blotting (Fig. 3). pbmif-ko parasites were viable in vivo, replicated asexually at the same rate as wild-type parasites, formed sexual stage parasites at the wild-type frequency, and were transmitted throughout the full life cycle (not shown). Since PbMIF may have a more subtle role in parasite-host interactions, we investigated pbmif-ko parasite virulence and infection dynamics in two inbred mouse strains, BALB/c and C57BL/6.

In C57BL/6 mice, P. berghei ANKA infection (intraperitoneal injection of 1 x 105 parasites) generally leads to cerebral complications, presenting as ataxia, shivering, lethargy, and death at days 8 to 9 after infection as the level of parasitemia reaches ~10% (16, 20). P. berghei ANKA infection in BALB/c mice may lead to similar cerebral complications in a small proportion of mice or, more commonly, to a syndrome termed severe malaria (19, 20), in which mice suffer from severe anemia, weight loss, and organ damage from day 8 after infection that are associated with a high level of parasitemia. Mice infected with wild-type or pbmif-ko parasites had symptoms consistent with these syndromes; infection of C57BL/6 mice resulted in death at day 8 postinfection, and infection of BALB/c mice resulted in high levels of parasitemia and severe anemia (Tables 1 and 2) in all cases.


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  		TABLE 1. Comparison of virulence parameters for infections with wild-type and pbmif-ko parasitesa


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  		TABLE 2. Comparison of virulence parameters for infections with wild-type and two independently generated pbmif-ko parasitesa

In both experiments, with two independently generated pbmif-ko lines and two mouse strains, hosts infected with the pbmif-ko lines had significantly higher reticulocyte densities and peak levels of reticulocytemia than hosts infected with wild-type parasites (Fig. 7; Tables 1 and 2). In contrast, none of the other virulence parameters revealed consistently significant differences between infections with pbmif-ko parasites and infections with wild-type parasites. In both experiments, infections with the first pbmif-ko line (pbmif-ko1) did not differ significantly from infections with wild-type parasites in terms of cumulative parasite density, peak parasitemia, minimum mass, mass loss, minimum red cell density, or red cell loss. When infections initiated with the second pbmif-ko line (pbmif-ko2) were compared to infections with wild-type parasites, there were no significant differences in peak parasitemia, minimum mass, mass loss, or red cell loss. However, compared to infections with wild-type parasites, for infections with pbmif-ko2 parasites the cumulative parasite densities were significantly higher, but only in BALB/c mice, and the minimum red cell densities were significantly lower, but only in C57BL/6 mice. Therefore, only reticulocyte parameters differed consistently and significantly between infections with both pbmif-ko lines and wild-type infections in BALB/c and C57BL/6 mice (Fig. 7; Tables 1 and 2).


Figure 7
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  		FIG. 7. Parasite and reticulocyte densities in pbmif-ko-infected mice. The values are means and standard errors for asexual stage and reticulocyte density during infections in experiment 2, obtained by using BALB/c mice (A.1 and A.2) and C57BL/6 mice (B.1 and B.2). For each host strain, five infections were initiated with either wild-type parasites (WT) or one of two independently generated pbmif-ko lines (KO1 and KO2). In both host strains the densities of circulating reticulocytes were significantly lower in infections initiated with wild-type parasites than in infections initiated with pbmif-ko parasites (A.1 and B.1). In contrast, the densities of asexual parasites did not differ significantly in infections initiated with wild-type parasites and infections initiated with pbmif-ko parasites (A.2 and B.2).


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DISCUSSION
 
It is now clear that the cytokine MIF is an evolutionarily ancient protein with a widespread distribution. MIF homologues in parasitic nematodes have been functionally characterized (21, 49). Furthermore, genome-sequencing projects have shown that MIF homologues are present in the protozoan parasites Toxoplasma gondii and Leishmania major, but genes encoding MIF are absent from the published genomes of other protozoans, including the related apicomplexan parasites Cryptosporidium parvum, Theileria parvum, and Theileria annulata, and free-living eukaryotes, including Dictyostelium discoideum and the ciliate Tetrahymena thermophila. Thus, at present the expression of MIF homologues in unicellular eukaryotes is consistently associated with a group of parasitic protozoans that engage in specialized interactions with host cells in blood. Here we describe our initial functional characterization of the Plasmodium MIF homologue. The use of the term "homologue" instead of "orthologue" in the case of the parasite-derived MIF is justified, since while we show that there is conservation in function, we cannot at this time rule out additional properties of parasite-derived MIF that are not shared with host MIF.

While PbMIF transcription appears to peak at the trophozoite stage in asexual blood stage parasites, PbMIF is expressed in all parasite forms examined throughout the life cycle and is distributed in the cytoplasm of both the parasite and the infected erythrocyte. These results correlate with P. falciparum transcriptome data (5, 32) (PlasmoDB entry PFL1420w). Furthermore, proteome surveys have indicated that the protein is also present in all P. falciparum and P. berghei life cycle stages analyzed (23, 25). The apparent discrepancy between the presence of the messenger and the presence of the protein might be explained by differences in protein and mRNA stability and turnover. In blood stage parasites, release of PMIF is most likely episodic, coinciding with rupture of the infected erythrocytes and release of merozoites. Recombinant His6-tagged PMIF and huMIF elute at apparent molecular masses of 30 and 22 kDa in size exclusion chromatography, and these sizes are both consistent with a dimeric form in solution. Until recently, the oligomerization state of huMIF had been under debate, with reports showing monomeric, dimeric, and trimeric forms based on cross-linking experiments and dimeric forms based on size exclusion chromatography analyses. A trimeric form in solution was firmly established in sedimentation equilibrium studies (reference 40 and references therein). Since PMIF-His6 appeared to behave like huMIF-His6 in size exclusion chromatography and huMIF-His6 appeared to behave like untagged huMIF in previous work (37), the His6 tag does not appear to affect oligomerization. However, a more in-depth study is required to determine the exact oligomerization status of PMIF in solution.

There have been conflicting reports about the relevance of the C terminus of huMIF with regard to enzymatic activity (2, 36). In our assays, a C-terminal His6 tag did not appear to influence enzymatic activity. Recombinant PMIF-His6 showed tautomerase and oxidoreductase activities, although in both assays the activities were ~20% of the recombinant huMIF-His6 activities. It is surprising that PMIF-His6 retains oxidoreductase activity in the absence of the second cysteine of the CXXC motif and with the low level of conservation of surrounding sequences. However, given the low level of conservation, we cannot directly compare the PMIF and its oxidoreductase activity to the human C60S MIF mutant (which exhibited only background activity compared to wild-type MIF in the HED assay) and its activity. For example, the cysteine in PMIF that would correspond to C57 in mammalian MIF is shifted by one register in the alignment (Fig. 1A). Rather, the remaining oxidoreductase activity in PMIF can be seen as an intermediate between the activity of human C57S MIF (which exhibits about 60% of the wild-type MIF activity in the HED assay) and the activity of C60S MIF (29). However, we cannot exclude the possibility that the N-terminal cysteine residues may contribute to this effect by transferring protons from reduced glutathione to HED. It is also possible that the structural requirements for oxidoreductase activity are fulfilled in the PMIF multimer.

Despite the relatively low enzymatic activity and low sequence similarity compared to huMIF, we found that recombinant PMIF-His6 and the huMIF-His6 control are equally efficient in reducing AP-1 activation in HEK cells. The exact structural requirements for Jab-1 binding and the inhibition of Jab-1-mediated activation of AP-1 are currently unknown. Previous studies have implied that in huMIF, a structural rearrangement involving the oxidoreductase activity (involving C60) is required for this activity (8, 28, 38). However, peptides spanning the CXXC motif with either the wild-type or C57S/C60S double point mutant sequence can compete for huMIF binding to Jab-1 (27). In the absence of a strong oxidoreductase activity and the crucial C60 residue in PMIF, therefore, structural differences may account for PMIF's ability to inhibit Jab-1 function. Alternatively, differences in protein uptake or stability in HEK cells between PMIF and huMIF might explain this effect.

PMIF is not essential for any phase of the Plasmodium life cycle. Furthermore deletion of PbMIF did not consistently influence standard virulence parameters, such as red blood cell loss, weight loss, and parasite growth, in the early stages of a P. berghei infection. However, infections in two host strains revealed a clear and significant increase in reticulocyte production in the early stages of pbmif-ko infection such that the pbmif-ko-infected hosts had 1.5- to 4.5-fold more circulating reticulocytes than wild-type-infected hosts. Accurately characterizing the phenotypic effects of MIF expression is difficult using these combinations of P. berghei and inbred mice due to the rapid and lethal progression of the infections. Such studies may be more informative if they are carried out with Plasmodium chabaudi, which causes a chronic infection in inbred mouse strains. These studies should rely on the P. chabaudi genetic transformation technology that has been developed recently (S. Reece and J. Thompson, unpublished data).

None of the studies on the host MIF response to Plasmodium infection published thus far have taken the presence of parasite-encoded MIF into account (1, 13, 14, 15, 34, 35). Intriguingly, two of these studies clearly showed that MIF has a role as a host-derived factor inhibiting erythropoiesis in the context of P. chabaudi infection in BALB/c mice (34, 35). Our results showing increased reticulocyte numbers in pbmif-ko infections match well with these studies and indicate that parasite-encoded MIF might work in concert with host MIF to suppress erythropoiesis in the context of a Plasmodium infection. Furthermore, McDevitt et al. observed a mild increase in host survival when infection was carried out with MIF knockout mice. Therefore, it should be of interest to monitor the course of infection of the pbmif-ko parasite in a MIF knockout mouse strain. At this time it is unclear how this inhibitory effect on reticulocyte numbers might benefit the parasite; since P. berghei is a reticulocyte-preferring parasite, PbMIF appears to act to reduce the preferred host blood cell pool for invasion. However, suppression of erythropoiesis may lead to maintenance of a lower level of parasitemia, which might contribute to a longer-lasting infection. Thus, it is clear that further research on the behavior of the pmif-ko parasites in the context of chronic and also genetically diverse infections is required to understand the action of PMIF.

Since MIF is a central regulator of the inflammatory response in vertebrates (for a review, see reference 11), release of a parasite homologue is likely to influence this response. It seems counterintuitive that parasites produce a protein that could both initiate a potentially lethal inflammatory immune response and demonstrably reduce the population of red cells preferred for asexual proliferation. However, huMIF has been shown to be able to act upon both pro- and anti-inflammatory pathways, depending on the context and concentration (28; for a review, see reference 11). This raises the intriguing possibility that parasites produce MIF homologues to subversively switch the host immune response from a proinflammatory setting to an anti-inflammatory setting, for instance by expressing an excess amount of parasite MIF that competes with host MIF for effector binding sites and so desensitizes host signaling for the proinflammatory response. Preliminary data indeed show that PMIF binds to CD74, which has been identified as a MIF surface receptor (31), with higher affinity than huMIF binds (unpublished observation). We are currently generating a transgenic P. berghei parasite which overexpresses PbMIF in the hope that further characterization of the role of PMIF in an in vivo setting and further biochemical studies with recombinant PMIF will provide additional insights into the role of this cytokine homologue in relation to the host response. Alternatively, parasites may purposefully induce an inflammatory response, using the host immune system as a means to regulate the population of competing parasites within hosts. Further characterization of host responses to PMIF after short-term exposure, as well as long-term exposure, is required to distinguish between these two possibilities. Finally, since parasite MIF homologues are not limited to Plasmodium, the findings presented in this study should have implications for the study of host-parasite dynamics in other parasites.


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ACKNOWLEDGMENTS
 
We thank Jai Ramesar for technical assistance with the P. berghei transfections and Andrew Read, Rick Maizels, and Judith Allen for critical reading of the manuscript and useful discussions.

This work was supported by grant 072171 from The Wellcome Trust and by grants 812.05.002 and 816.02.001 from The Netherlands Organization for Scientific Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Parasitology, LUMC, Albinusdreef 2, Room P4-35, 2333 ZA Leiden, The Netherlands. Phone: 31-71 5265069. Fax: 31-71 5266907. E-mail: waters{at}lumc.nl. Back

{triangledown} Published ahead of print on 11 December 2006. Back

Editor: J. F. Urban, Jr.


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Infection and Immunity, March 2007, p. 1116-1128, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.00902-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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