Identification validation and therapeutic potential of cis-trans interactions that direct coordinated gene expression in Plasmodium falciparum
Lead Research Organisation:
Keele University
Department Name: Inst for Science and Tech in Medicine
Abstract
This research proposal asks a simple question that has wide ranging implications to our understanding of malaria pathology - how does the malarial parasite Plasmodium falciparum control the molecular process that turn its genes on and off as it progresses through its complex life cycle. Malaria kills over two million people each year, yet this devastating statistic only represents the tip of the 'malaria iceberg' - with over 500 million cases each year resulting in significant health and financial burdens to malaria endemic regions. Processes that are essential to the parasite such as infection and development in humans, drug resistance and immune evasion all rely on the concerted control of the 5300 genes present in the parasite's genetic make-up. We already know that the molecular control of malarial gene expression is coordinated and intimately linked to different stages of parasite development; yet the molecular mechanisms (or more likely their combination) that drive this coordinated process still elude us. What we do know is that they are quite distinct to those present in humans. It is the nature of these differences, and how they control parasite-specific adaptations that interest me as they offer an attractive opportunity - differences can be exploited! My hypothesis is that genes that encode proteins that are turned on together and work together are also controlled together. I want to establish how this happens. The research will start with a computer-based investigation that exploits the product of the malaria genome project - we know all the DNA sequences that could possibly contain gene regulatory elements, we just don't know where they are. Modelling experiments in my laboratory have identified areas of the genome most likely to contain regulatory motifs involved in the coordinated control of gene expression (the process of turning a gene 'on'). This enriched pool of DNA sequences will be searched to find common sequence motifs that we predict will direct coordinated expression. At this point, the project moves into the laboratory. Here we will test these computer-based predictions in genetically-modified parasites where we will replace, delete or mutate these proposed regulatory sequences to see what effect they have on gene expression - specifically, what is their impact in altering the level and/or timing of gene expression. We will also characterise the nuclear proteins that bind to these proposed regulatory sequences, thus attempting to complete our understanding of the relative contributions that different molecular mechanisms make in controlling the flow of genetic information in this organism. This study is important. I want to not only to better understand how the molecular mechanisms that control gene expression come together to drive parasite development in humans and mosquitoes, but also help establish the contribution that this programme of gene expression make to the pathogenicity of the host-parasite interaction. But how can this information be translated into a new drug or therapy? The process of rational drug design is fundamentally underpinned by this type of descriptive research, as is perhaps best exemplified by the adage 'know thine enemy'. Towards this end, this proposed investigation aims not only identify the component parts of the gene regulatory process, but also to dissect at the molecular level how they work together. In this way, I want to better understand the dynamic interplay in gene regulation as the parasite infects, colonizes and often ultimately causes pathogenesis of its host, identifying how these processes may be subverted for much needed alternative routes to treat this devastating human disease.
Technical Summary
This proposal integrates bioinformatic, molecular and cell biology approaches to systematically map informative cis-acting regulatory sequences (CRS) and identify their cognate nuclear trans-acting factors (NTF) in order to dissect their role in directing coordinated gene expression in the human malarial parasite Plasmodium falciparum. We will initially utilise an evidenced-based global bioinformatic search for putative CRS using the FIRE (finding informative regulatory elements) algorithm. Using intergenic sequences that our models indicate most likely contain CRS, informative motifs correlating to microarray expression data during intraerythrocytic development will be identified. Functional validation of these predicted CRS will utilise in vivo and ex vivo approaches. These include; (i) genetically modified parasites with integrated luciferase reporter constructs bearing mutated, deleted or replacement CRS to quantitatively measure changes to absolute and temporal activity of modified promoters, (ii) gel shift assays to determine specific and temporal patterns of NTF interaction with CRS and (iii) temporal binding of NTF to CRS will be correlated with histone modifications over promoters using chromatin immunoprecipitation assays. The identity of NTF will be established using either (i) affinity selection of nuclear extract to CRS or (ii) in situ hybridization TRAP (tagging and recovery of associated proteins) followed by tandem mass spectrometry. These latter protocols reflect the adoption of a more direct strategy to identify novel NTF, particularly potentially exciting RNA-binding proteins, in an organism that has evolved an apicomplexan-distinct complement of NTF for which traditional approaches in NTF identification have proven problematic.
People |
ORCID iD |
Paul Horrocks (Principal Investigator) |
Publications
Hasenkamp S
(2012)
Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum.
in Malaria journal
Hasenkamp S
(2013)
A quantitative analysis of Plasmodium falciparum transfection using DNA-loaded erythrocytes.
in Molecular and biochemical parasitology
Hasenkamp S
(2013)
Functional analysis of the 5' untranslated region of the phosphoglutamase 2 transcript in Plasmodium falciparum.
in Acta tropica
Hasenkamp S
(2013)
Evaluation of bioluminescence-based assays of anti-malarial drug activity.
in Malaria journal
Hasenkamp S
(2012)
An improved single-step lysis protocol to measure luciferase bioluminescence in Plasmodium falciparum.
in Malaria journal
Russell K
(2014)
Homopolymer tract organization in the human malarial parasite Plasmodium falciparum and related Apicomplexan parasites.
in BMC genomics
Wong EH
(2011)
Analysis of the molecular mechanisms governing the stage-specific expression of a prototypical housekeeping gene during intraerythrocytic development of P. falciparum.
in Journal of molecular biology
Description | One key outcome from this research was the development of a genetically modified parasite line that now forms the basis of a novel assay being utilized in antimalarial drug development. The assay allows us to ask how quickly is a parasite being killed by a chemical compound. This is important, as any future antimalarial drug needs to be able to quickly kill the parasite - alleviating the parasite burden as quickly as possible to reduce death and morbidity. We have used this assay to rank the rate of kill for an important antimalarial discovery compound library. We are now in the process of extending this work in collaboration with GlaxoSmithKline who are providing some 15000 compounds of particular interest as potential antimalarials. |
Exploitation Route | The assay format offer opportunity to be redefined to explore other stages of parasite development. Moreover, the principle that bioluminescence rapidly reports parasite death has been taken forward in leishmania parasites |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Development of a rapid, simple and sensitive luciferase-based growth assay for the high throughput screening of antimalarial drugs |
Amount | £14,870 (GBP) |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2011 |
End | 05/2012 |
Title | Genetically modified P. falciparum - expresses luciferase |
Description | The human malaria parasite has been modified to express luciferase. The temporal expression of luciferase is a key component to their subsequent use in development of a bioluminescent assay for antimalarial drug activity |
Type Of Material | Cell line |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | These parasites now form the basis of a novel bioluminescence assay that monitors the initial cidal activity of antimalarial compounds, This assay has been used to triage an open access library of discovery antimalarials, identifying potential devbelopment targets. See A validated bioluminescence-based assay for the rapid determination of the initial rate of kill for discovery antimalarials Imran Ullah Raman Sharma Giancarlo A. Biagini Paul Horrocks J Antimicrob Chemother (2017) 72 (3): 717-726. |
Title | Quantitative analysis of transfection efficiency for Plasmodium falciparum |
Description | Utilising reagents generated on this grant, we used bioluminescence as a quantitative tool to explore critical parameters for transfection to provide an improved technique |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | This work validated the efficiency of a methodological improvement: the research has been cited 18 times since publication |
Description | Collaboration with Kenneth Marx, Umass |
Organisation | University of Massachusetts |
Country | United States |
Sector | Academic/University |
PI Contribution | Ongoing collaboration regarding the study of homopolymer tract frequency and organization in P. falciparum - visit to UMass at Lowell in September 2010 and subsequent teleconferences/email links |
Start Year | 2010 |
Title | Quantitative analysis of transfection efficiency for Plasmodium falciparum |
Description | Utilising reagents generated on this grant, we used bioluminescence as a quantitative tool to explore critical parameters for transfection to provide an improved technique |
Type Of Technology | New/Improved Technique/Technology |
Impact | No actual Impacts realised to date |
Description | Invited Seminar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research or patient groups |
Results and Impact | Pasteur Institute, Paris no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |
Description | Presentation to Jeremy Lefroy, MP |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Jeremy Lefroy, MP is a local MP and Chair of the All Party Group on Malaria and Neglected Tropical Diseases Online press information by Keele University and on Jeremy Lefroy's website no actual impacts realised to date |
Year(s) Of Engagement Activity | 2012 |
URL | http://www.independent.co.uk/news/science/birds-hold-key-to-secret-of-human-speech-1932745.html |