I also certify that it has not previously been submitted in whole or in part to this university or any other university or institution for the award of a degree or diploma. It is hereby stated that the thesis entitled "Reverse Genetics Reveals the Critical Role of Sporozoite-Specific Genes SSPELD and SCOT-3 in Plasmodium berghei Liver Stage Development" is a record of bona fide work by Mr. The dissertation has not previously been submitted in part or in full to this or any other university or institution for the award of a degree or diploma.
I acknowledge the financial support of DBT, CSIR, ICMR, UGC, PURSE, DBT‐CREEB and DST‐FIST to the Department and School of Life Sciences.
Introduction
Life cycle of Plasmodium
- Exo-erythrocytic stages
- Erythrocytic stages
- Sexual stages
- Sporozoites
Adhesins are located in the apical organelles of the parasite, which bind to receptors on the surface of the erythrocyte [32]. The actin-myosin motor is located in the parasite's inner membrane complex (IMC) and mediates motility [ 38 , 39 ]. The zygote transforms into a motile ookinete within 16-25 hours after gamete fusion in the midgut lumen of the mosquito.
These two domains ensure the interaction of the sporozoites with different cell types, i.e. the salivary gland cells of mosquitoes and hepatocytes of liver in mammals [79].
Control measures of malaria
Prophylaxis and treatment
To treat malaria, antimalarial drugs such as azithromycin and clindamycin are used which inhibit apicoplast formation thus giving rise to a generation of non-infectious merozoites that are blocked midway through intra-erythrocytic development, a phenomenon referred to as delayed phenotype. dead. [97]. Artimisinin is another antimalarial drug used in combination with other drugs to treat malaria [99]. In addition to pre-erythrocytic vaccines and drugs that target intra-erythrocytic parasites, transmission-blocking vaccines are gaining prominence to prevent transmission of malaria to mosquitoes.
Genes that play a key role in gamete, zygote, and ookinete formation have been targeted for the creation of vaccines to block transmission by inhibiting sexual development in the mosquito [100].
Challenges and Current research on malaria
Thus, preerythrocyte vaccines can eliminate parasites before the clinical manifestations of disease associated with blood-stage infections. For example, RTS, S is one of the recombinant vaccines developed from the central tandem repeats of P. Vaccine development against blood stages presents tremendous challenges due to the occurrence of antigenic variation associated with mature asexual blood stages.
By constantly switching the expression of variants, the parasites provide several waves of asexual cycles, each with its own antigenic composition, making it very impossible to develop effective vaccines against these stages.
Plasmodium berghei (P. berghei) – model organism
Introduction
The diversity of the parasite's life cycle is most evident from the point of view of gene regulation, as distinct stages have different regulatory mechanisms to achieve the desired gene expression leading to the manifestation of stage-specific function. Transcriptomic analysis of sequenced GFP expressing infected hepatocytes and genome-wide liver stage gene expression profiling and comparison to other life cycle stages revealed approximately 2000 genes that were active in liver stages. Referred to as sporozoite stages, major transcriptional changes occur in these forms of the parasite while they reside in the mosquito salivary gland—a site that causes them to achieve enhanced infectivity [133].
Unexpectedly, there was an enhanced expression of chorismate synthase, an enzyme of the shikimate pathway, which was earlier reported to be functional in blood stages, making these stages a vulnerable target of the herbicide glyphosate [135].
Materials and Methods
- Retrieval of target genes sequences
- Construction of the transfection /knockout vector
- Sub cloning into targeting vector pBC-GFP-hDHFR
- Preparation of the transfection plasmid by maxi preparation method
- Release of targeting cassette by double restriction digestion
- In vitro culture of schizonts
- Schizont purifications
- Electroporation of schizonts
- Drug selection
- Microscopic enumeration of P. berghei infections by Giemsa staining
- Cryopreservation of P. berghei infected blood
- Observation of GFP parasites under fluorescent microscope
- Genomic DNA isolation from transfected P. berghei population
- Confirmation of targeted gene knockout by site specific integration PCR
- Limiting dilution of transfected/ knockout parasites
- Maintenance of Anopheles stephensi colony
- Transmission of malaria into Anopheles mosquitoes
- Observing the midgut oocyst
- Dissection and purification of salivary gland sporozoites
- HepG2 cell culturing
- Infection of HepG2 cells for obtaining exo erythrocytic forms
- Immunofluorescence assay
- RNA isolation from P. berghei infected HepG2 cultures
- RNA isolation from all P. berghei life cycle stages to study expression of Pb SSPELD
- cDNA synthesis
- Expression analysis of Pb SSPELD by quantitative real time PCR
- Microarray of P. berghei
- RNA Extraction and RNA Quality Control
- Labeling and microarray hybridization
- Hybridization and Scanning
- Feature Extraction: Image Analysis
- Microarray Data Analysis
- Generation of Pb SSPELD mCherry transgenic parasites for studying the localization of SSPLED
- Immuno Fluorescence Assay to confirm the sporozoite membrane localization of Pb SSPELD
Part of the blood was taken for genomic DNA isolation, while the rest was frozen for future use. The anesthetized rabbit was placed on top of the mosquito cages to facilitate blood meal uptake by the female mosquitoes. Fig. 6. Maintenance of half a pen. The maintenance of a second pen includes activities such as preparing breeding cages, where the pups are placed in water bowls that mate and mate within 24-36 hours (A). After the second blood meal, a bowl of water is placed in the breeding cage and the eggs are collected for four consecutive days. amalgamation of adult mosquitoes. Preparation of infection cages and female mosquitoes are separated using a vacuum pump. CandRH80% to facilitate the completion of sexual reproduction of Plasmodiumberghei(E). Mosquitoes were sectioned under dissecting microscope (F) at D14(G) and D18(H) postinfection to observe the oocytes and sporozoites in the salivary glands, respectively, under a fluorescence microscope. Schematic representation of TogiriJ's thesis (2015).
All female mosquitoes (inside the cage) collected in the palm area were collected in a tube attached to a vacuum pump.
Results
- Pb SSPELD is conserved amongst other Plasmodium species
- Gene expression analysis by quantitative real time PCR revealed maximal expression of Pb SSPELD in salivary gland sporozoite stages
- Successful replacement of Pb SSPELD locus by GFP-hDHFR cassette by double homologous recombination method
- Pb SSPELD is not essential for asexual blood stages
- Pb SSPELD is not essential for mosquito stages
- Pb SSPELD sporozoites fail to initiate blood stage infection when malaria is transmitted though natural mosquito bite
- Pb SSPELD exhibit growth arrest at 36 hour time point
- Successful replacement of Pb SSPELD 3’ UTR with mCherry -hDHFR cassette by double homologous recombination method
- Pb SSPELD mCherry transgenics express reporter in oocyst, salivary gland sporozoite and EEF stage
- Immunisation with Pb SSPELD KO generates pre erythrocytic immunity
- Microarray of Pb SSPELD KO reveals dramatic changes in the global gene expression
Discussion
Considering the important role of Pb SSPELD in liver stage development, we generated mCherry transgene of Pb SSPELD and studied its localization in all life cycle stages by visualization of the reporter expression. The sporozoite membrane localization of Pb SSPELD was unexpected, as there were no predicted membrane targeting or attachment motifs in the coding sequence. Interestingly, the orthologue of Pb SSPLD was reported in this study and detected in the proteomics analysis of P.
To unravel the biological process and pathways affected after depletion of Pb SSPELD, we performed microarray analysis of WT and Pb SSPELD mutants at the 36 h time point. Downregulation of genes central to these processes in Pb SSPELD KO may be a consequence of premature interruption of liver stage development. Given the lack of EEF maturation in PbSSPELD mutants together with the likely localization to the PVM membrane, a speculative role of PbSSPELD in membrane biogenesis required for liver-stage merozoite formation [18], nutrient uptake and protein export or - exit cannot be ruled out and deserves further investigation.
Because of the defect of Pb SSPELD mutants in completing liver stage development, we analyzed its potential to generate preerythrocyte immunity. A prime-boost regimen with 2X104 Pb SSPELD mutants in C57BL6 mice induced protective immunity with nearly 50% efficacy. This may explain the observed activation of CD4+ and CD8+ T cells after immunization with Pb SSPELD mutants.
Thus, the role of Pb SSPELD in liver stage development is not surprising given that in SAGE analysis of the sporozoite transcriptome, it clusters with well-characterized transcripts such as UIS-4 [27] and Pbs36p [29] its essential role which in liver stage development has been demonstrated through KO gene experiments. In conclusion, our studies highlight the importance of Pb SSPELD in the termination of liver stage development and these mutants as a complement of preerythrocytic immunogens capable of eliciting protective immunity.
Materials and Methods
- Retrieval of target genes sequences
- Drug selection
- Confirmation of targeted gene knockout by site specific integration PCR
- Maintenance of Anopheles stephensi colony and transmission of Pb SCOT-3 KO to mosquitoes
- Transmission of Pb SCOT3 KO sporozoites to mice through natural mosquito bite
- In vitro development of EEF and IFA to reveal to growth of EEFs
- RNA isolation from all P. berghei life cycle stages to study expression of Pb SCOT3
- cDNA synthesis
- Pb SCOT3 is conserved amongst other Plasmodium species
- Successful replacement of Pb SCOT3 locus by GFP-hDHFR cassette by double homologous recombination method
- Pb SCOT3 is not essential for asexual blood stages
- Pb SCOT3 KO exhibit better growth than WT EEFs at 62h time points
These mosquitoes were allowed to obtain a blood meal from the gametocyte stages of WT or Pb SCOT3 KO mice. Data normalization was performed by obtaining the copy number ratio of Pb SCOT3 and Pb 18S rRNA for each sample. 94 3.3.2 Gene expression analysis by quantitative real-time PCR showed the highest expression of Pb SCOT3 in midgut sporozoite stages and late liver stages.
Normalized data revealed the highest expression of Pb SCOT3 in midgut oocyst stages and late liver stages at 50 h (Fig. 30). Normalized data were expressed as a ratio of absolute copy numbers of Pb SCOT3 versus Pb 18S rRNA (internal control) for each stage of the Plasmodium life cycle. Pb SCOT3 ORF specific primers were used to confirm deletion of the Pb SCOT3 locus, which yielded a PCR product with only WT P .
The sporulation pattern within the oocyst (Figures 33C and D) and the ability of sporozoites to egress to reach the salivary gland (Figures 33E and F) were also comparable to those of WT parasites, suggesting that Pb SCOT3 KO did not show any defect in stages P mosquitoes. Inoculation of Pb SCOT3 KO sporozoites by natural mosquito bite did not initiate blood stage infection in 2 of 3 independent experiments using 3 mice per experiment. After obtaining a clonal line of the Pb SCOT3 mutant, we assessed its ability to transmit malaria to mosquitoes.
To possibly explain the inability of Pb SCOT3 mutants to initiate the blood stage, we studied the development of the EEFs in HepG2 cells at different time points. The inability of Pb SCOT3 mutants to initiate a timely breakthrough of infection, despite the full development of EEFs, likely points to a defect in merozoite exit from the hepatic schizonts.
SUMMARY
Pb SPELD played an important role in the completion of liver stage development, as Pb SPELD mutants were arrested in mid-liver development. The transcriptomic analysis of 36 h liver stage cultures showed downregulation of several late liver stage transcripts. Consistent with the role of PbSSPELD in the completion of EEF development, PbSSPELD mCherry transgenes showed maximal expression from sporulating oocyst stages, in salivary gland sporozoites, and continued to be expressed in the 36-h EEFs.
Pb SSPELD mCherry colocalized with CSP, a major sporozoite surface protein, and also localized to the PVM membrane in 36-h EEFs. Pb SSPELD mutants were unable to initiate blood-stage infection after malaria transmission through a natural mosquito bite. A prime booster regimen with 2X104 Pb SSPELD mutants provided protection that was 50% effective, and protection cognate was mediated by anti-sporozoite antibodies and parasite-specific T cells.
Taken together, we propose the role of PbSSPELD in the completion of liver stage development and these mutants as a complement of pre-erythrocytic antigens capable of eliciting protective immunity. Analysis of the salivary gland sporozoite transcriptome provided information on the upregulation of several genes that have already been extensively studied in human and rodent models. Investigation of the gene function by reverse genetics approach revealed the indispensable role of Pb SCOT3 in asexual blood stage propagation and in the mosquito stages.
However, we identified a role for PbSCOT3 in liver-stage parasite egress, as 62-h EEFs that successfully completed development failed to initiate blood-stage infection in time. Our study demonstrates the feasibility of revealing the function of liver-stage-specific genes of P.
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