Cyclic nucleotide signalling in malaria parasite differentiation
Lead Research Organisation:
London School of Hygiene & Tropical Medicine
Department Name: Department of Pathogen Molecular Biology
Abstract
Strategic Research Priority: World Class Biosciences
Abstract
Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) are important signalling molecules which regulate critical events across all stages of the malaria parasite lifecycle. To further our understanding of how this signalling pathway governs important events involved in egress and invasion, this study investigates two different members of this signalling cascade in Plasmodium falciparum parasites.
Project
Guanylyl Cyclase a (GCa) is a large predicted bifunctional enzyme which is a key component of the signalling pathway. It consists of an ATPase domain, which is thought to flip phospholipids; and a GC domain, which is responsible for generating cGMP. Using the dimerisable Cre recombinase system (DiCre), an inducible GCa knockout line was generated, confirming that GCa is essential and required for egress. GCa-deficient parasites can be rescued by chemically complementing with a cGMP analogue. Although the exact role of the ATPase remains unknown, mutagenesis of this domain indicate that it performs an essential function.
Activation of this signalling cascade culminates in the phosphorylation of several effector proteins, many of which form part of the glideosome. This includes the actomyosin motor protein, Myosin A (MyoA), which is phosphorylated at a single site, Ser19. Since the glideosome generates the force required for merozoites to invade host cells, these cGMP-dependent phosphorylation events may be involved in modulating motor activity. A DiCre MyoA knockout line was generated, revealing that MyoA is essential for red blood cell invasion. This conditional KO line was then used to investigate whether phosphorylation of Ser19 is important for motor function by complementing with wild type or mutant versions of the protein that either mimic or ablate phosphorylation of Ser19. In vitro motility assays using material derived from parasites expressing either wildtype or mutant Myosin A were also performed in order to understand the effects of this phosphorylation event on motor activity.
Abstract
Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) are important signalling molecules which regulate critical events across all stages of the malaria parasite lifecycle. To further our understanding of how this signalling pathway governs important events involved in egress and invasion, this study investigates two different members of this signalling cascade in Plasmodium falciparum parasites.
Project
Guanylyl Cyclase a (GCa) is a large predicted bifunctional enzyme which is a key component of the signalling pathway. It consists of an ATPase domain, which is thought to flip phospholipids; and a GC domain, which is responsible for generating cGMP. Using the dimerisable Cre recombinase system (DiCre), an inducible GCa knockout line was generated, confirming that GCa is essential and required for egress. GCa-deficient parasites can be rescued by chemically complementing with a cGMP analogue. Although the exact role of the ATPase remains unknown, mutagenesis of this domain indicate that it performs an essential function.
Activation of this signalling cascade culminates in the phosphorylation of several effector proteins, many of which form part of the glideosome. This includes the actomyosin motor protein, Myosin A (MyoA), which is phosphorylated at a single site, Ser19. Since the glideosome generates the force required for merozoites to invade host cells, these cGMP-dependent phosphorylation events may be involved in modulating motor activity. A DiCre MyoA knockout line was generated, revealing that MyoA is essential for red blood cell invasion. This conditional KO line was then used to investigate whether phosphorylation of Ser19 is important for motor function by complementing with wild type or mutant versions of the protein that either mimic or ablate phosphorylation of Ser19. In vitro motility assays using material derived from parasites expressing either wildtype or mutant Myosin A were also performed in order to understand the effects of this phosphorylation event on motor activity.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1618511 | Studentship | BB/M009513/1 | 01/10/2015 | 30/09/2019 | Stephanie Nofal |
| Description | I have generated a transgenic Plasmodium falciparum line where an essential protein, Myosin A, can be conditionally knocked out. We have shown that complementation of the conditional knockout line with mutant versions of Myosin A which either mimic or block phosphorylation of Ser19 leads to a growth defect, indicating that this cGMP-dependendent phosphorite is important for parasite growth. Using an in vitro motility assay we have also shown that the S19A mutation leads to reduced myosin motor motility. I have also generated a transgenic Plasmodium falciparum line where a key player in cyclic nucleotide signalling, Guanylyl Cyclase alpha, can be conditionally knocked out. This has revealed that GCalpha is the sole quanylyl cyclase responsible for generating cGMP during the blood stages of the life cycle and that it is required for egress and calcium mobilisation. Furthermore we have been able to chemically complement GCalpha knockout parasites with PET-cGMP, a cGMP analogue. We have also shown that the ATPase domain is essential for parasite survival and that loss of this domain can also be compensated for by addition of PET-cGMP. |
| Exploitation Route | The findings once published will shed a further light on the importance of cyclic nucleotide signalling and will inform on potential novel drug targets in the parasite. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Francis Crick Institute in vitro motility |
| Organisation | Francis Crick Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Stephanie Nofal has generated transgenic parasite lines designed to investigate the role of phosphorylation of P. falciparum Myosin A in blood stage development. |
| Collaborator Contribution | Justin Molloy at the Francis Crick has collaborated with us on investigation of the role of phosphorylation of P. falciparum Myosin A in blood stage development providing facilities, expertise and lab visits for Stephanie Nofal. |
| Impact | PhD thesis by Stephanie Nofal (2019) |
| Start Year | 2018 |
| Description | LSHTM Mosquito Day |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Hosted the Malaria Centre Malaria Day where we discussed our work with the general public and came up with a time line of important events that occurred during the history of the disease. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Malaria Centre PhD Student Showcase |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Chaired and organised the Student Showcase for LSHTM students working on malaria. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Malaria Journal Club |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | A bi-monthly journal club meeting with around 20 participants from UCL, LSHTM, Birkbeck and the Francis Crick institute. One person would present a malaria paper and we would discuss the paper . |
| Year(s) Of Engagement Activity | 2016,2017,2018 |