| Malaria remains a major cause of morbidity and mortality in the tropical countries.Plasmodium falciparum is responsible for the deadliest form of human malaria causing1–3million deaths a year. The pathogenicity is related to its ability to evade the human immunesystem by modifying infected host red blood cells to bind to different host vascularadhesins and to undergo antigenic variation. The cytoadhesion of infected red blood cellsleads to many of the severe manifestations of the disease, including cerebral malaria andpregnancy associated malaria. The major antigenic ligands found to be responsible for thecytoadhesive properties of the iRBC are P. falciparum erythrocyte membrane protein1(PfEMP1), encoded by the var multigene family. There are about60var genes found in thesequenced genome of P. falciparum clone3D7. The var genes can be further classifed bypromoter sequence into fve distinct types (UpsA, UpsB, UpsC, UpsD and UpsE), whichare strongly associated with specifc chromosomal locations and orientations oftranscription. Mass research demonstrate that the type of var gene expressed is related tothe outcome of malaria infection.In order to protect the limited var gene repertoire, each time only one gene isexpressed while the others are silenced, known as mutually exclusive expression.Multi-layers of regulations are involved in the mutually exclusive manner, includingnuclear repositioning, histone modifcations, and interaction between the var5’promoterand var intron promoter. PfEMP1stimulate the acquired immune responses, therefore theswitching of expression between different var genes is important for the parasites to avoidthe antibody response that the host generates against PfEMP1. The mechanism of antigenicvariation in P. falciparum are far from being fully understood. Nevertheless, In vitrostudies of malaria parasites have shown that variant switching is non-random and partlygene specific. For example, the switching rates are suggested to be intrinsic and constantfor the same var gene but different for individual var genes. The ability to switch to certainvariant types may depend on the antigenic switching history of the parasite.Most of the recent advances in var gene expression and switching have beengenerated through experiments with laboratory adapted parasite lines but few in the fieldisolates. The main obstacle are the hypervariable var gene sequences and there is littlegenomic overlap of var genes between different P. falciparum isolates. However, theparasite may lose some characteristics because of the long-term culture without the immune pressure. It is important to make sure whether the inferences made fromlaboratory strains are same in the field isolates or there may be new discoveries.In this paper the P. falciparum field isolate FCYN0906was studied. CloneFCYN0906-5H was derived from the field isolate and50var genes were identified bysolexa sequencing and assembling. Then50var gene specific primer sets were designed todetect the dynamic transcriptional levels of individual var genes, and further to analyze theswitching patterns of var genes in the field isolates. The main steps and results are asfollows:1. Parasite Culture and the Generation of clonesThe P. falciparum field isolate FCYN0906was collected from the border of Chinaand Myanmar. It was thawed and cultivated followed the standard protocol. Six cloneswere generated from the field isolate by limiting dilution. Clone4C,4H,5H,6G had thesame genotype according to the DNA fragment length of8microsatellite alleles (ARA2，TA1，TA60，TA81，TA87，TA109，PfPK2，Poly), while clone4A and6E were different.So clone4C,4H,5H,6G were used for the following study.2. Solexa sequencing, Bioinformatic analysis and PCR amplification to assemblethe var genes of clone FCYN0906-5HThe genomic DNA of clone FCYN0906-5H was prepared for the solexa sequencing.We got10,259,515paired end reads in all,93.15%of them could map to the genome of3D7isolate and the average coverage was90X. However, var gene sequences werediversity among different isolates and the reads couldn’t map to the variable regions of thevar genes from3D7isolate. So the reads were assembled into contigs in order to find thevar genes of the field isolate. There were8,281contigs (>500bp) in all with N50=1684bp.After blasting with the PfEMP1protein sequences in NCBI database,217contigsbelonged to var genes, of which66contigs were predicted to contain partial or entireDBL domains. Nineteen primers were designed based on the hypervariable regions in theDBL sequence to complete the DBL domains combined with primers DBL AF/BRthrough PCR, sequencing. Finally,50sequences with DBL domains were affirmed torepresent the50var genes of the field isolate individually since each sequence had a DBLdomain,42of them had NTS domain.The50var genes above mentioned were subgrouped according to the upstreamsequences blasted with the5’ regions of60var genes in3D7isolate. The primersupsB-5’UTR and upsC-5’UTR previous described were also helpful to identify3upsB subtype (var149,var25,var46) and4upsC subtype (var131,var163,var170,var51) var genesrespectively. The phylogenetic analysis of50DBL showed that although upsB and upsCsubgroups were overlapping, the upsA subgroup could be distinguished from others. Inparticular, var4and var84had no upstream sequences but were assumed to be upsAsubtype since they were also within the upsA subgroup. Finally we got11upsA,18upsB,7upsC,1upsD,1upsE subtype var genes and the rest12var genes belonged to either upsBor upsC subgroup.3. The design of50var gene specific primer setsA total of50var gene specific primer sets were designed depending on thehypervariable regions of the DBL. All primer pairs were tested on genomic DNA of cloneFCYN0906-5H firstly. PCR products were sequenced and compared with the var genesequences in MEGA to make sure the specificity of the primers. The amplifcationeffciencies of the primers were tested on10-fold dilutions of genomic DNA. Quantitativeamplifcation was performed and all primers had amplification efficiencies(E) between1.96and2. Specifcity of amplifcation was ascertained by melting-curve analysis of eachPCR product. So we confirmed that the50primers were effective to detect thetranscription levels of the var genes. We also showed that the four clones (4C,4H,5H,6G)had the same sequences amplifed by the50primers, which ascertained that the four cloneshad the same genotype.4.The allelic exclusion of var genes in the field isolatesThe var genes of the3D7isolate were mutually exclusive expressed to protect thelimited antigens from the host immune system. In order to prove whether the var genes ofthe field isolates had the same expression manner, the four clones (FCYN0906-4C,4H,5H,6G) were thawed and cultivated again. Synchronic ring-stage parasites (10-15h) wereharvested. RNA was isolated and treated with Dnase I to remove the potentialcontamination of genomic DNA. Then cDNA was synthesized and the transcriptionallevels of all the var genes in the four clones were detected respectively. The result showedthat all the four clones had different dominant var genes, of which the proportion of totalsignal was61%,69%,44%and57%respectively at the latest timepoint. Therefore,although there were minor transcripts, the allelic exclusion of var genes was quite obviousin the field isolate as in laboratory-adapted isolate3D7.5. Various var gene switching patterns in the field isolatesIt is obvious that most available data about the expression and switching of var genes are based on analyses of parasite strains that are cultured for long times in vitro. In thisstudy,50var gene specific primer sets were designed, which made it possible to analyzethe switching patterns of var genes in the field isolates. We divided each clone cultureabove mentioned into two biological replicates to see whether the almost identicalreplicates may display the same switching pattern. For the next50generations, ring-stageparasites were harvested every10generations and transcription levels of the var geneswere measured by realtime PCR with50gene specifc primers. Then the clones werethawed and the experiment was repeated with the same time course, so there were fourreplicates for each clone in all.The results showed different switching pathways of the four clones as a whole. Eachclone favored some special var genes that were not expressed dominantly in other clones,like var147, var98, var143in clone4C and var47, var163, var136in clone6G. Accordingto initial transcription profiles, the main difference of the four clones was the expression ofdistinct dominant var genes, leading to different and favored switching pathways.It was unexpected to find various switching directions even in the four replicates ofeach clone. First, our results indicated that, for a given var gene, the switching on/off ratewas not always the same, it could be activated and silenced at high speed or just with slowprocess. For example, var98switched on at high speed in clone4C-B (2.7%), while atlower speed in clone4C-D (0.3%). It offered more switching patterns since the dominantvar gene can either persist for a long time, switch back, be replaced by an alternatedominant var gene slowly or immediately. That was an important factor causing variousswitching directions of the replicates. Then the next dominant transcripts were shown to bediverse and chosen randomly, which was another cause leading to different pathways. Forthe clone4C, var167switched off, replaced by var143in4C-C while var21in4C-D.We also found similar changes of the transcription profiles of the two replicatesderived from the same culture especially within the first three time-points, which suggestedthe conserved program in the switching pattern. The switching pathway was even the samein6G-C and6G-D. In all the four clones, var21was more active and seemed to act as arole of transition between the two dominant transcripts.So our results showed that both the conserved and random parts exist in the switchingprocess. The initial dominant var genes and the variable switching on/off rates were alsoinvolved in the different switching patterns.The whole transcriptional levels of the upsA group var genes were low, the occasional up-regulation was random and unrepeatable since there was no selective pressure in vitro.Summary:In this study, we attempt to obtain the var gene repertoire of the clone5H generatedfrom the plasmodium falciparum field isolate FCYN0906by illumina solexa sequencing.Although the diversity of the var gene sequences, we still assemble50partial var genesequences of the field isolate from the contigs based on the DBL domains through PCR,sequencing and bioinformatic analysis. A total of50var gene specific primers are designedrelying on the hypervariable regions in the DBL sequences.This is the first time that the transcriptional levels of the entire var gene repertoire arequantitative and comparable in the field isolate. We find that the var genes are mutuallyexclusive expressed in the field isolate as well. The switching rates of the var genes fromthe field isolate are not constant, which is opposed to the previous views. The switchingpatterns are more abundant and consist of both conserved and random parts, which shouldnot be separated in the various switching pathways.These separated parts should be taken together with the previous findings from3D7isolate to reflect the complete switching process as described in our results, which is morecomplex than we imagine:1) the var genes are mutually exclusive expressed in the cloneof the field isolate;2) the var genes on/off rates are variable and the dominant var gene caneither persist for a long time, switch back, be replaced by an alternate dominant var geneslowly or immediately;3) the transcriptional variations follow a conserved switchingprogram when the initial dominant var gene maintains or reduces gradually;4) theexpression of the dominant var genes falls accompanied by the switching on of other vargenes favored;5) it seems random to choose another dominant transcript from the vargenes switched on;6) most of upsA group var genes are silenced with occasional andunrepeatable up-regulation in vitro. |
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