Cyclic nucleotide-mediated ion homeostasis in the malaria parasite

NISHITH GUPTA (HUB) in partnership with Kiaran Kirk (ANU) and Alexander Maier (ANU)

 

Cyclic nucleotide-gated (CNG) channel-mediated ion homeostasis is involved in a myriad of processes across the tree of life. CNG channels are non-selective channels, the function of which is regulated by binding of cGMP or cAMP and/or polarization events. Even though cyclic nucleotides (cNMP) signalling as well as ion homeostasis are known to regulate several aspects of Plasmodium biology [1, 2], their co-regulation has not been studied yet.  This is mainly due to lack of specific methods to perturb and monitor cyclic nucleotides and major ions (Ca2+, H+, Na+) within intracellular parasites without influencing host cells. Recent work by Nishith Gupta’s group has pioneered the application of optogenetics in infection research using the related model parasite, Toxoplasma gondii [3, 4]. The group has been able to achieve specific, efficient, spatiotemporal and reversible modulation as well as monitoring of parasite/host pathways independently of one other. This development overcomes a major roadblock, which remained insurmountable when using chemical agonists and antagonists in conjunction with fluorescent sensors. For instance, it has now become feasible to photo-activate cAMP or cGMP signalling and quantify cytosolic Ca2+ oscillations simultaneously by co-expressing a light-regulated cyclase and a gene-encoded Ca2+indicator (GECI) in the parasite cytoplasm. The work constitutes a sound basis for the integrated utility of optogenetic and parasitology methods to investigate cNMP-mediated ion homeostasis in Plasmodium.

The proposed project employs Plasmodium falciparum to photo-activate cAMP or cGMP cascade, and determine real-time fluctuation of major ions (Ca2+, H+, Na+) in the parasite cytosol during its asexual reproduction in erythrocytes. Together with the group of Alexander Maier, two transgenic parasite lines expressing GECI along with a photo-activated guanylate or adenylate cyclase will be engineered. The resultant optogenetically-modified parasite strains will permit concurrent induction of signalling and dynamic monitoring of cytosolic Ca2+ in response to light exposure. Cytosolic Na+ and pH will be monitored using ion-sensitive fluorophores. The phenotypic consequences of light-induced ion flux on invasion, replication and egress of the parasite will be examined in cooperation with Kiaran Kirk. Compounds known to inhibit the ion channels and transporters will also be utilized to establish the co-regulation of cNMP signalling and ion transport. In parallel, phosphoproteomics and transcriptomics of light-exposed and dark-adapted cultures will be performed to identify the core mediators (e.g. phosphorylated proteins and modulated genes). These datasets will be used for hierarchical clustering to construct an integrated model of cNMP signalling and ion homeostasis in P. falciparum. We will then validate the initial model by generating and phenotyping the selected parasite mutants, each lacking a component positioned along the signalling cascade (i.e. sensors, actuators, effectors). Upon successful completion, this project will reveal the mechanistic regulation of signalling and ion transport, which can eventually be exploited to develop antimalarials.

 

Interlinkages: Giel van Dooren (ANU), Edda Klipp (HUB), Adele Lehane (ANU)

 

References:

(1) Baker, D.A. (2011) Cell. Microbiol. 13: 331-339

(2) Kirk, K. (2015). Ann. Rev. Microbiol. 69: 341–359

(3) Hartmann, A. et al. (2013) J. Biol. Chem. 288: 13705-13717

(4) Kuchipudi, A. et al. (2016) Microbial Cell 3:215-223

(5) Lehane, A.M. et al. (2014) Mol. Microbiol. 94: 327-339