Clone And Identification Of The Promoter Of Toxoplasma Gondii LDH2 Gene

Toxoplasmosis,caused by an intracellular protozoan parasite named Toxoplasma gondii(T.gondii) which has the ability to infect a variety of warm-blooded vertebrates including human beings,is widespread throughout the world.In healthy humans,infection is often asymptomatic in immuno-competent individuals and progresses from a rapidly replicating tachyzoite stage to a dormant bradyzoite stage in response to the immune system,which is the most important stage in toxoplasmosis.The bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.In patients with immuno-deficiencies such as AIDS or other malignancies,bradyzoites that differentiate into tachyzoites after release from the cysts can give rise to a recurrent infection that can be fatal.At present,no effective treatment for chronic toxoplasmosis is available,because bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.While within a human host,the opportunistic pathogen T.gondii relies heavily on glycolysis for its energy needs no matter whether in rapidly replicating tachyzoite stage or in dormant bradyzoite stage.Lactate dehydrogenase(LDH),the terminal enzyme in anaerobic glycolysis,is necessary for NAD⁺ regeneration.This enzyme plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,whereas glycolysis is the only energy source in bradyzoites.LDH is considered as a promising target for developing drugs for chronic toxoplasmosis because its unique structural,physical,chemical and kinetic properties differ from those of human host cells.What's more,study of the differentiation of LDH helps a lot to understand the genetic regulatory signals that control differentiation between tachyzoites and bradyzoites.There are two isozymes of LDH expressed in T.gondii.The mRNA of LDH2 is detected in the bradyzoite stage only.The transcript of LDH1,on the other hand, is found in both bradyzoites and tachyzoites.But the LDH1 gene product is only expressed in tachyzoites,whereas that of LDH2 is expressed in only bradyzoites. The genes encoding the isozymes show 64%nucleotide sequence identity,and their gene products share 71%amino acid sequence identity.It is speculated that LDH1 is replaced by LDH2 during development from tachyzoites to bradyzoites. But it is unknown that whether the differentiation is a consequence of post-transcriptional induction or it happens during the course of transcription.Since LDH plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,for bradyzoites in chronic toxoplasmosis,the pathogenecity of T.gondii depends very much on the type and activity of LDH.However,the regulatory mechanism of LDH differentiation in tachyzoites and bradyzoites is unknown at present.In this study,we extensively studied the promoter of LDH2 gene in T.gondii bradyzoites.PART ONE Amplification of 5'-flanking Sequence of LDH2 Gene in T.gondii BradyzoitesObjective:The aim of this part is to clone the 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites.Methods and results:In order to study the promoter and regulation of LDH2 gene,we amplified 5'-flanking sequence of T.gondii LDH2 gene using Thermal Asymmetric Interlaced PCR(TAIL-PCR).Then inserted it into pMD18-T vector and sent it for sequencing. Conclusion:We got the right 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites after checking the already-known information about LDH2.PART TWO Analysis of T.gondii LDH2 Gene 5'-Flanking Sequence Using Bio-Informatic ToolsObjective:To examine T.gondii LDH2 gene candidate promoter and transcriptional start site(TSS) by online computer-based bio-informatics tools.Methods and results:1.Methprimer obtains 11 CpG islands by screening in the whole LDH2 gene sequence.Among these islands,there are 5 islands within about 1Kb fragment upstream the start code ATG.This indicates that the code sequence of LDH2 gene connects with CpG islands.2.Promoterscan result shows a candidate promoter located in the fragment from 320bp to 509bp of input sequence,-540bp to -351bp of LDH2 gene.It gives a score of 53.25,whereas the threshold is 50.00.3.McPromoter finds two candidate promoters,one is around 100bp of the input sequence,and another is near 600bp.The scores of them are 0.02 and 0.03, much lower than the threshold.While the candidate near 600bp has fragment overlapped with the candidate promoters of both NNPP and Pomoterscan.4.Promoter2.0 finds that there is a highly likely promoter fragment near 900bp of the input sequence.5.NNPP gives only one candidate promoter,which is from 327bp to 376bp of the input sequence.The score of this candidate is 0.98,much more than the threshold 0.8.This fragment is partially overlapped with the result of Promoterscan.But the advantage of this software is that it can give shorter fragment than the other tools used in this study.6.The other software used in this study did not find any fragments with promoter similar activity,or TSS.Conclusion:Although the results obtained from each software varies from each other,we put them together,and still can speculate that the candidate promoter should locate at the fragment from 300bp to 700bp of the sequence inputted.PART THREE Identification of the Activity of the Candidate Promoter of T.gondii LDH2 GeneObjective:The aim of this section is to identify the activity of the candidate promoter which we got in the second section by bio-informatics tools using dual luciferase reporter assay system.Methods and results:At present,the exact mechanism of the regulation of T.gondii LDH2 gene has not been clearly elucidated and it will help us to understand the differentiation between tachyzoites and bradyzoites.Firstly,we inserted the candidate promoter into the upstream of the Firefly luciferase of pTL₃-Basic by Kpnâ… /Xhoâ… to generate pTL₃-Lp,after PCR amplifying the fragment between 300bp to 700bp of the input sequence using primers by PCR with Kpnâ… and Xhoâ… restriction sites.In the same way,we generated pTL₃-993 the luciferase reporter construct of 5'-flanking sequence of T.gondii LDH2 gene.pRL-Tg vector which has Renilla luciferases drived byβ-tubulin gene promoter used as an internal control to normalize transfection efficiencies.The constructs,negative control pTL₃-Basic(without any promoter or enhancer) and positive control pTL₃-Control(withβ-tubulin gene promoter upstream the Firefly luciferase ORF) were objectively transfected into T.gondii RH strain tachyzoites by electrotransformation.Ninety-six hours after transfection,tachyzoites and bradyzoites cells were harvested and assayed for the luciferase activity.The pTL₃-Lp gives 0.054 and 0.053 in luciferase activity in both tachyzoites and bradyzoites,similar with the negative control pTL₃-Basic(0.052 and 0.051). Whereas pTL₃-Control gives 1.753 in tachyzoites and 1.802 in bradyzoites, pTL₃-993 gives 0.052 and 2.572.Conclusion:The candidate promoter has no function on the transcription of the downstream Firefly luciferase gene.PART FOUR Identification of the core promoter of T.gondii LDH2 geneObjective:In this section,we went back to traditional method to study the functional promoter within a not-very-known fragment.Methods and results:We did not get any functional elements in the third section based on computer searching.In order to search the core promoter of T. gondii LDH2 gene,we generated 9 luciferase reporter constructs by 5'-deletion mutagenesis,and identified the activity using dual luciferase reporter assay system. To search the TSS of LDH2 gene,primer extension was performed according to the lab protocols.The identification of the core promoter(which is the minimal fragment to control the transcription) is very important to study the regulatory mechanism of T.gondii LDH2 gene.1.In tachyzoites,all the 9 luciferase reporter constructs did not give any activities.This proves that mRNA of LDH2 is not detected in the tradyzoite stage at some aspects.2.The constructs with 393-fragment located at the upstream of the luciferase reporter gene gives 50 fold of the activity of the negative control pTL₃-Basic, about 1.5 folds of the positive control pTL₃-Control.The luciferase activities of the constructs without this fragment are similar to the negative control.3.Primer extension experiment shows that T.gondii LDH2 gene TSS is just 134bp upstream of the start code ATG.4.All these results show that the core promoter of T.gondii LDH2 gene is located at -259～1 of LDH2 gene.5.LDH2 gene expression is tightly regulated at the transcriptional level.And LDH2 replaces LDH1 during the development from tachyzoites to bradyzoites.It is therefore a regulation in transcription initiation stage instead of the post-transcription stage.Conclusion:we successfully located the core promoter of T.gondii LDH2 gene at the place of-259bp～+1bp of LDH2 gene.PART FIVE Identification of the Functional Cis-element of T.gondii LDH2 gene by EMSAObjective:In this section,we checked whether there are some protein factors in the nuclear extracts that can bind the core promoter fragment of T.gondii LDH2 gene.Methods and results:Whether a promoter is founctional or not depends on whether there are some transcriptional factors that can bind the promoter or not.So it is important to study the binding sites and transcriptional factors that bind to these sites.The results show that it does have some proteins in tachyzoites and bradyzoites nuclear extracts which can bind to the core promoter,but we have not identified these proteins factors yet.What's more,the results indicate that the protein that binds the promoter in tachyzoites nuclear extracts,might be different form that in bradyzoites.This also explains that why LDH2 only expresses in bradyzoites.Conclusion:There are some Cis-elements existed in the core promoter region of LDH2 gene of T.gondii.Some proteins could specifically bind to these Cis-elementshas.What we found provides a new insight into the transcriptional regulation of T.gondii LDH2 gene.