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Background: In its effort to survive the human immune system, Plasmodium falciparum uses several parasite-derived antigens most of which are expressed at the surface of the parasitized red blood cells (pRBCs). Recently SURFINs, a new family of antigens encoded by the surf multi-gene family, has been reported. One member of the family, SURFIN4.2, was found present both at the pRBC-surface and at the merozoite apex. Methods: The presence of a second SURFIN member, SURFIN4.1 (PFD0100c, PFD0105c) is reported here. Bioinformatic tools were used to study the structure of the surf4.1 gene. To investigate the expression of surf genes PCR and real-time quantitative PCR (Rt-QPCR) were employed and Northern and Western blots were used to confirm the size of the surf4.1 gene and the SURFIN4.1 protein respectively. Localization of SURFIN4.1 was determined using immunofluorescence assays. Results: The surf4.1 gene was found present in one copy by Rt-QPCR in some parasites (3D7AH1, 3D7S8, 7G8) whereas six copies of the gene were identified in FCR3 and FCR3S1.2. surf4.1 was found transcribed in the late asexual stages of the parasite beginning ≈32 hours post invasion and throughout the schizont stages with the level of transcription peaking at late schizogony. The levels of transcript correlated with the number of gene copies in FCR3 and 3D7S8. surf4.1 was found to encode a polypeptide of ≈Mw 258 kDa (SURFIN4.1) present within the parasitophorous vacuole (PV), around free merozoites as merozoite-associated material, but not at the pRBC-surface. Despite multiple surf4.1 gene copies in some parasites this was not reflected in the levels of SURFIN4.1 polypeptide. Conclusion: SURFIN4.1 is a member of the SURFINs, present in the PV and on the released merozoite. The results suggest different SURFINs to be expressed at different locations in the parasite and at distinct time-points during the intra-erythrocytic cycle. Published: 1 July 2008 Malaria Journal 2008, 7:116 doi:10.1186/1475-2875-7-116 Received: 12 November 2007 Accepted: 1 July 2008 This article is available from: http://www.malariajournal.com/content/7/1/116 © 2008 Mphande et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Malaria Journal 2008, 7:116 http://www.malariajournal.com/content/7/1/116


Background
The erythrocytic life cycle of Plasmodium falciparum is characterized by complex interactions of parasite derived surface exposed antigens with host receptors and immune defence proteins. At the surface of the the parasitized red blood cells (pRBCs), parasite antigens provide adhesive properties and at the same time allow for limited parasite resistance to the host immune response by virtue of great sequence variability. In case of the well-characterized surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a multi-adhesive variable ligand of 200-400 kDa, this is achieved by clonal exchange, "switching" of the mutually expressed PfEMP1 encoding var gene. A unique set of ≈60 var genes is present in each parasite genome [1][2][3]. With the release of merozoites from the host cell, a number of additional parasite proteins are exposed to the immune system, which, in response to the host immune pressure, show polymorphisms or are differentially expressed [4].
The merozoite, which is the invasive form of P. falciparum, invades erythrocytes in a cascade of events in which multiple receptor-ligand interactions facilitate parasite attachment, reorientation, junction formation and entry [5][6][7][8][9]. The machinery employed in this process is complex and involves proteins at the merozoite surface and in specialized secretory organelles at the merozoite apex [10]. Triggered by the initial parasite attachment to the host cell, micronemes and/or rhoptries release their contents. The Duffy binding-like (DBL) family (EBA-175 and its paralogues EBA-140 (BAEBL) and EBA-181 (JESEBL) [11,12]) in micronemes and the reticulocyte binding like (RBL) family (PfRh1, PfRh2a, PfRh2b, PfRh3 and PfRh4 [13][14][15][16]) in the neck of rhoptries have been shown to be relevant for merozoite infectivity [4]. In P. falciparum, several copies of Pf Rh1 gene were reported in FCR3, W2mef, and FCB1 (13, 3 and 4 respectively) when compared to 3D7. Thus, apart from the different protein antigens expressed on the surface of the merozoite, the complexity of the malaria parasite is increasing by the introduction of gene duplications and deletions [17][18][19].
SURFINs are a small family of large (200-300 kDa) proteins, which are composed of domains structurally related to PvSTP1 of Plasmodium vivax but also other exported and surface exposed parasite proteins [20], including PfEMP1, the P. vivax VIR family and Plasmodium knowlesi variant surface antigen (SICAvar). In accordance with this structural relationship, SURFIN 4.2 was found co-transported with PfEMP1 to the pRBC-surface. The protein is also observed on the merozoite as merozoite-associated material (MAM) at the apical end of the released merozoites. In the 3D7 parasite SURFINs are encoded by 10 surface-associated interspersed (surf) genes, which are located close or within the subtelomeric region of five chromosomes. Fre-quent recombination events in this area are responsible for the considerable variability of proteins encoded in this region, also observed in SURFINs. N-terminally, SURFINs contain a cysteine rich domain (CRD) defined by six conserved cysteine residues, five of which are found positionally conserved in PvSTP1. The CRD precedes a region of high size and sequence variability, followed by a putative transmembrane (TM) region. In contrast to the variable N-terminal region, the C-terminal domain of SURFINs is relatively conserved and characterized by tryptophane rich domains (WRD) of approximately 150 amino acid residues, separated by less conserved sequences [20]. The WRDs establish a structural link to several Plasmodium surface proteins, i.e. PfEMP1, PkSICAvar and also the giant erythrocyte membrane protein 332 (Pf332), of which recent data also suggests surface exposure [21].
The objectives of the study were to analyze the surf 4.1 gene structure to determine whether it is a pseudo gene, as previously annotated, or a complete functional gene. The transcription pattern of the surf 4.1 gene and localization of SURFIN 4.1 on the protein level were also investigated.

Analysis of surf gene presence and sequence heterogeneity in various parasite lines
Based on the 3D7 sequences of the annotated SURFINs, two sets of forward and reverse primers (Primer set 1 and Primer set 2), were designed for each surf gene. Some of the primers were designed to cross introns while others were within the open reading frames (ORFs) (Additional Files 1 and 2). Genomic DNA was extracted from trophozoite stages of 3D7S8, NF54, 7G8, DD2, FCR3, FCR3S1.2, TM284, TM180, R29, UAM25, UKS03, and UKS05 using the DNeasy Blood and Tissue kit according to the manufacturer's instructions (Qiagen, USA). Using two sets of independently designed surf gene specific primers, DNA was PCR amplified from each parasite. The following were the PCR conditions 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C 60 sec.

transcript analysis
Highly synchronized 3D7S8 and FCR3 parasites were harvested post invasion (p.i.) at early ring (10 h), late ring (20 h), early trophozoite (30 h), and schizont (40 h) parasite stages. The parasites were resuspended in pre-warmed TRIzol (Invitrogen, USA) (37°C) and incubated at 37°C for 5 minutes. Total RNA was extracted using the manufacturer's instructions (Invitrogen, USA) and DNAse treated (Ambion, USA). The RNA (100 ng) was reverse transcribed (RT+) using MuLV reverse transcriptase enzyme and random hexamers (Applied Biosystems, USA) according to manufacturer's instructions. For each RT+ experiment, one RT-reaction (with reverse transcriptase omitted) was performed to ensure that there was no DNA contamination in the RNA. surf 4.1 is currently present as PFD0100c in GeneDB, and PlasmboDB v5.4 but it was previously indicated as a truncated gene in the PlasmoDB v4.3 malaria database and was then suggestively comprised two distinct genes PFD0105c and PFD0100c. Primers (surfC 4.1 F/R and Sintra 4.1 F/R) were consequently designed on opposite sides of the intergenic region of the two previously annotated genes (yielding amplification products of 1160 bp and 450 bp respectively, see Additional File 2). cDNA from ring stages, trophozoite stages and schizont stages was amplified using surfC 4.1 F and surfC 4.1 R. The RT-PCR cycling conditions were as follows: 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C 60 sec. The cDNA from the different parasite stages was also amplified using primers specific to the surf 4.1 gene sections, these included 5CS-1F/R, 5CS-2F/R, surfC 4.1 F/R, S 4.1 F/R, 1C-S1F/R, 1C-S2F/R and 1C-S3F/R (Additional File 2).

Identification of the PFD0105c intron location
PCR was conducted using primers surfC 4.1 F/R (Additional File 2) that were designed to cross the intergenic region and at the same time also read through the intron of the first part of the surf 4.1 gene (PFD0105c). The primers were used to amplify cDNA and gDNA from FCR3S1.2, 3D7S8, and NF54. The cDNA and gDNA PCR products were cloned into TOPOII vector (Invitrogen USA) and prepared for sequencing using the BigDye v3.1 kit (Applied Biosystems, USA) according to manufacturer's instructions.

Sera and specific antibodies
A fragment of surf 4.1 (from the non-cytoplasmic part of the protein), SURFIN 4.1 -C1 was PCR amplified from 3D7S8 using primers 5'-atcgagctcTTGGATAATACAGGT-GAT-3' and 5'-gatctcgagTTCCTTATGATGTTTTGGT-3'. The amplified DNA was cloned into TOPOII vector and sequenced. The SURFIN 4.1 -C1 fragment was subsequently cloned into the PQE Trisystem -His strep 2 expression vector (Qiagen, USA) which adds a stretch of 6 histidine residues to the C terminus and 8 streptavidin residues at the N-terminus. The fusion protein was purified on His-Trap columns (Amersham Biotec, Sweden) charged with Nickel ions (1 M, NiCl 2 , SIGMA, USA) according to manufacturer's instructions. The purified fusion protein was used to immunize rabbits (500 μg/injection) and rats (100 μg/injection) in Freund's complete adjuvant (SIGMA, USA) and boosted three times at one-month intervals with the same protein concentration in Freund's incomplete adjuvant. The animals were bled 10 days after the last boost.

Real-time quantitative PCR analysis of surf 4.1 copy numbers and transcription levels
Copy numbers of surf 4.1 were determined for 7G8, 3D7AH1, FCR3 and their daughter clones 3D7S8 and FCR3S1.2. Genomic DNA (gDNA) was prepared using Easy-DNA™ Kit (Invitrogen) as previously described [17]. In addition, transcriptional levels were analysed for 3D7S8 and FCR3. Parasites were kept tightly synchronized using 5 % (w/v) sorbitol, and were harvested at 4 h post invasion intervals for two consecutive parasite generations. RNA from each time point was isolated using the RNeasy ® Mini Kit according to the manufacturer's instructions and contaminating gDNA was removed using the RNase-Free DNase Set (Qiagen). Total RNA was reversely transcribed with SuperScript III RNase H reverse transcriptase (Invitrogen), with random hexamers and oligo(dT) [12][13][14][15][16][17][18] (300 ng/ml and 25 ng/ml respectively; Invitrogen) at 50°C for two hours. For each cDNA synthesis reaction, a control reaction without reverse transcriptase (RT-) was performed with identical amounts of template. For real-time quantitative PCR (Rt-QPCR) determination of copy numbers as well as to monitor relative transcription of surf 4.1 to the endogenous control seryl-tRNA synthetase, the primers surf 4.1 were employed: 5'-TTTGAAGCTCCTGGTCAAGGA-3', surf 4.1 -3': TTGTTT-GTGCAAGTGTTTTGAAAG, PF07_0073-5': TATCATCT-CAACAGGTAT CTACATCTCCTA and PF07_0073-3': TTTGAGAGTTACATGTGGTATCATCTTTT. These primers as well as the complete Rt-QPCR amplified product were designed to conserved regions in the parasite lines studied. Amplification efficiencies of the primer pairs were tested on dilution series of both gDNA and cDNA from all parasites, and were proven sufficiently close (ranging from 94.4 to 99.2%) to obviate the need for a correction factor. Amplification reactions for both relative copy numbers (gDNA) and relative transcription (cDNA) were done in quadruplicate in 20 μl, containing Power SYBR Green master mix (Applied Biosystems), 300 nM of each forward and reverse primer and 2 ng of template. Forty five cycles (95°C for 15 sec and 60°C for 1 min) were performed in an ABI sequence detector 7500 (Applied Biosystems). Relative copy numbers were analysed according to the ΔΔCt method and plotted using SigmaPlot 9.0 (Systat Software Inc.

Indirect immunofluorescence on air-dried monolayers and localization of SURFIN 4.1
For immunofluorescence assays (IFA), monolayers of pRBCs were prepared as previously described [24]. Monolayers were incubated with rabbit anti-SURFIN 4.1 -C1 antibody (1:400) or pre-immune serum for 30 min at room temperature and subsequently with a secondary anti-rabbit Alexa 488 (1:100; Jackson, USA) for 30 min. The cells were then incubated with propidium iodide (1:100) for nuclear staining for 30 min. Preparations were washed three times with PBS between each antibody preparation.
In the co-localization assay with EBA175, monolayers were incubated with rabbit anti- HOESCHT and/or DAPI (Jackson, USA) were used for nuclear staining. All incubations were carried out at room temperature in a humid chamber. Slides were analysed with a Nikon Optiphot 2 UV microscope.

Agglutination assays
To analyse the ability of the immune anti-SURFIN 4.1 -C1serum to agglutinate pRBCs, synchronized parasite cultures of 3D7S8 and FCR3 were incubated with serum as described by Barragan et al [25]. The samples were then analysed under the microscope for presence of clumps or agglutinates. Acridine orange was used for staining of the parasite.

Results
The data presented here shows a revised gene structure of surf 4.1 , which combines two previously identified open reading frames (PFD0100c and PFD0105c) (plasmodb.org v4.3 and Figure 1A). This was obtained by bioinformatic analysis and confirmed by PCR, which shows that surf 4.1 is one complete gene with three exons separated by two introns ( Figure 1B). In PlasmoDB, surf 4    others were amplified using two primer sets. These two separate primer sets were used to account for any sequence differences and to ensure that as many surf genes were amplified from the different parasites. Genomic DNA sequences from 3D7, HB3, D10, DD2 and 7G8 from surf4.1 (1-2320 bp.) showed sequence polymorphisms within the parasite isolates (Additional File 4).

surf 4.1 exists in different copy numbers in different parasite strains
Six copies of surf 4.1 gene were observed in FCR3 and its daughter clone FCR3S1.2 and one copy in each of 3D7AH1, 3D7S8, and 7G8 (Figure 2A and Additional File 5). Achieved copy numbers confirm previous findings where microarray and fluorescent in situ hybridizations were used for quantification [18].

surf genes are differentially transcribed dependent on developmental stage and strain
To understand the transcription patterns of the surf genes in the 3D7S8 parasite clone, RT-PCR was performed with primers specifically targeting individual sequences of all 10 surf genes (Additional File 1). Different surf genes were found expressed at different times during the erythrocytic cycle (  Figure 2B and Additional File 6). An obvious difference in transcript levels was observed between these two parasite lines, corresponding to the difference in copy numbers of the surf 4.1 gene.
The transcription pattern of surf 4.1 was confirmed with Northern blot experiments using specific probes. surf 4.1 mRNA of approximately 9 kB was detected in schizonts > 40 h post infection ( Figure 1C).

Identification of SURFIN 4.1 by immunoblotting
Immunoblot analysis was performed with SDS-lysates obtained from synchronized in vitro cultures of 3D7S8 and FCR3. Using polyclonal rabbit-anti-SURFIN 4.1 -C1 antibodies, a band of approximately 250 kDa was detected in schizont stage parasites (36-40 h), which corresponds to the predicted SURFIN 4.1 protein mass of 258 kDa ( Figure 1D). Similar intensities were observed in both FCR3 and 3D7S8. Achieved data is consistent with the RT-PCR analysis suggesting PFD0100c and PFD0105c to form a single open reading frame.

SURFIN 4.1 is localized to the parasitophorous vacuole (PV)
To study the localization of SURFIN 4.1 , IFA was carried out on 3D7S8 and FCR3 air-dried monolayers using purified rabbit-anti-SURFIN 4.1 -C1 IgG. SURFIN 4.1 was found expressed during the mature stages of the parasite (30 h and onwards, Figure 3). There was no recognition of The genes that are expressed are indicated by "+" while those that are not expressed are indicated by "-".  (Figure 3). When a SURFIN 4.2 antibody was used on the same parasite stages, the same pattern of staining was achieved for both trophozoite and early schizont stages. However in the ruptured schizont, SURFIN 4.2 antibody showed a distinct staining of the merozoite apex while the pattern observed with SURFIN 4.1 antibody was not apical but rather spread around the merozoite (Figure 3).
Co-localization experiments were carried out between SURFIN 4.1 and the micronemal protein EBA175, and SURFIN 4.2 . In the intact schizont, SURFIN 4.1 appears to co-localize in part with EBA175, but the proteins differentiate in the ruptured schizont. SURFIN 4.1 is spread around the pre-released merozoites, while EBA175 localizes at the apical end of the merozoite ( Figure 4A). SURFIN 4.1 is also co-localized with SURFIN 4.2 in the intact schizont but again the two proteins differentiate in the ruptured (segmented) schizont as SURFIN 4.1 is spread around the prereleased merozoites while SURFIN 4.2 is also seen on the merozoites but with apical staining (Figure 4A-B).

SURFIN 4.1 seems not to be exposed on the infected erythrocyte surface
Agglutination assay on pRBCs and RBC binding assay on uninfected RBCs with rabbit-anti-SURFIN 4.1 -C1 whole serum showed no agglutinates in either 3D7S8 or FCR3 (30 h and 40 h cultures).

Discussion
SURFINs of P. falciparum, encoded by the surf multi gene family, compose a new family of surface proteins. Not much is known about the protein family and its function [20]. This paper reports on the identification of SURFIN 4.1 a member of the SURFINs present in the parasitophorous vacuole (PV) and in part at the level of the merozoite. SURFIN 4.1 is encoded by the surf 4.1 gene, which, as is SURFIN 4.2 (the only member of the family thoroughly studied), located on chromosome 4 of P. falciparum.
In order to investigate the presence of surf genes in both laboratory strains and clinical isolates of P. falciparum, PCR was carried out on all the 10 surf genes using two different primer sets for each gene. Two independent primer sets were used in order to reduce bias in gene amplification, which may arise with the use of only one primer set due to sequence variation (Additional File 3). The outcome showed that all the genes were amplified in either laboratory adapted or clinical isolates, but not every gene was amplified in every parasite (Additional File 3).
In the same manner the surf 4.1 gene was also found amplified in all parasite lines, but for isolate R29 (Additional  Figure 1A) [20]. Differences in length of the first intron were observed between FCR3 and 3D7S8 parasite strains with FCR3 having a longer intron. It is not established whether the difference in intron length and or presence of SNPs could contribute to the surf 4.1 gene maintaining a complete open reading frame in certain P. falciparum strains. The gene encodes a 258 kDa protein, SURFIN 4.1 , which is of a slightly smaller molecular weight than the previously characterised SURFIN 4.2 (286.4 kDa) ( Figure 1D).
With the knowledge that surf 4.1 exists as one complete gene, copy number polymorphisms were analysed in different parasite isolates. FCR3 and its daughter clone FCR3S1.2 (cloned by micromanipulation) were found to have six copies of the gene, in concordance with copy number estimates performed previously using microarrays [18]. The increase in surf 4.1 gene copy numbers correlated with a five-fold increase in RNA transcription observed in FCR3 compared to the 3D7S8 parasite strain. However, the increase in copy number of the gene was not reflected on level of protein, rather that the protein was present a similar levels in the two parasites. This might be due to sequence differences in the protein in FCR3, resulting in poorer recognition by the antibodies raised using a protein construct from 3D7S8 And another possibility may be that there is a certain level of translational repression as has been previously described in P. falciparum [26].
The temporal transcriptional profile of surf 4.1 was analysed in the 3D7S8 and FCR3 parasite strains. surf 4.1 is transcribed during the late stages from ≈32 h post invasion peaking at the late schizont stages (44-48 hrs). The difference in transcription patterns between surf 4.1 (late trophs and schizonts) and surf 4.2 (transcribed from early rings to the schizont stages) may suggest differences in function of the proteins encoded by the respective genes. The transcription profile of surf 4.1 correlated well with the protein expression, observed in both FCR3 and 3D7S8 parasite lysates (Figure 3). No detectable protein for SURFIN 4.1 was observed in early parasite stages.
In the trophozoite stage, SURFIN 4.2 is present in the PV as is SURFIN 4.1 , which appears during the late trophozoite or early schizont stages and is observed in segmented sch-Localization of SURFIN 4.1 in respect to other merozoite associated proteins by immunofluorescence staining Figure 4 Localization of SURFIN 4.1 in respect to other merozoite associated proteins by immunofluorescence staining. A). Co-localization study between rat anti-SURFIN 4.1 and rabbit anti-EBA175 was carried out on 3D7S8 air dried monolayers. EBA175 is a micronemal protein hence localizes at the merozoite apex. The parasite nucleus was stained in blue using Hoescht. In the intact schizont SURFIN 4.1 and EBA175 partially co-localize as shown in the merge of the two photos. B). Co-localization between SURFIN 4.1 (green) and SURFIN 4.2 (red) is observed in the intact schizont as indicated by the yellow colour in the merged photos. In the ruptured schizont on the other hand, SURFIN 4.1 (green) is spread around the merozoites (blue) while SURFIN 4.2 (red) is observed as a distinct dot on the merozoite (blue). The merge of the two colocalization patterns shows that SURFIN 4 izonts carrying newly formed merozoites. SURFIN 4.1 is also observed as amorphous material (MAM) around the merozoites, which could suggest that it plays a role in the merozoite invasion process. It is interesting to note that SURFIN 4.1 partially co-localises with both SURFIN 4.2 and EBA175 in the intact schizont but the proteins differentiate in the segmented schizonts. Both SURFIN 4.2 and EBA175 are observed at the apical end of merozoites in the segmented schizont while SURFIN 4.1 is spread around the merozoite (Figure 4A-B). SURFIN 4.1 seems not to be exposed on the pRBC surface as shown by failure of the anti-SURFIN 4.1 -C1 serum to agglutinate IE in FCR3 and 3D7S8. This observation is in line with the IFA on air-dried monolayer results, which indicates that SURFIN 4.1 remains within the PV and is not present in the pRBC cytoplasm or at the surface of IE. Immunofluorescence assay on live cells showed no reactivity with SURFIN 4.1 -C1 serum. Still both SURFINs have PEXEL-like motifs: SURFIN 4.1 has a PEXEL-like motif (RNVFE) located at aa 25-29 while in SURFIN 4.2 the PEXEL motif is RKIFE though at the same aa position. The motifs differ but whether this difference could affect the differential transport of SURFINs to the IE is to be elucidated.
The function of SURFIN 4.1 is not yet known, but other members of the SURFINs have been associated with putative functions such as SURFIN 4.2 , which might be involved in erythrocyte invasion. PFA0650w (surf 1.2 ) has been suggested to be involved in surface adhesion [27] and PFD0100c (part of surf 4.1 gene) has been shown to share Myb-protein domains and GO annotations with PFI1480w and PFL0815w, which have putative functions regarding transcriptional regulation [28].
Antigenic variation has been reported in some multi-gene families including the var gene family [29][30][31]. The surf multi-gene family shows differential expression of its genes ( Table 1). Some of the genes are expressed simultaneously during the entire blood stage parasite cycle; some are expressed throughout the life cycle while one third is expressed during the later stages of the cycle. It is not yet established whether differential expression of the surf genes could suggest different functions.

Conclusion
In summary, surf 4.1 is one complete, and not truncated, gene in P. falciparum parasites, with gene-copy number polymorphisms existing amongst different clones and strains. The