Malaria is a debilitating blood-borne disease caused by apicomplexan parasites of the genus Plasmodium. The most virulent of the five species that infect humans is P. falciparum, which causes over 600,000 deaths per year. A critical stage in the infectious life-cycle is the invasion of the human red blood cell by the parasite. Gliding motility, invasion into and egress from the infected host cell, are all powered by the glideosome, a multi-protein assembly anchored in the inner membrane complex. At the heart of the invasion machinery is the class XIV myosin motor MyoA (PfMyoA), which is anchored to the membrane via its light chain subunit called PfMTIP (myosin tail interacting protein), and an adapter protein called PfGAP45. We are the first to succeed in expressing milligram quantities of functional P. falciparum MyoA in a heterologous system. This was accomplished by co-expressing the myosin heavy and light chain(s) with an apicomplexan myosin co-chaperone.
Aim 1 is to reconstitute a minimal Plasmodium falciparum motor protein complex in vitro, consisting of PfMyoA and its bound light chain(s), interacting with P. falciparum actin (PfACT1), which is quite divergent from mammalian actin. We propose a new domain structure for PfMyoA: following the motor domain there is a lever arm that is stabilized by two bound light chains. Binding of a second light chain, in addition to the well-characterized light chain PfMTIP, will allow PfMyoA to move actin faster and produce more force than if it had only one light chain. Functional assays include actin-activated ATPases, ensemble in vitro motility assays, and force-velocity measurements using a laser trap to determine power output. We will express PfACT1 to assess its interaction with PfMyoA. We predict that the motor properties of PfMyoA will be enhanced when interacting with its native actin.
Aim 2 is to understand how glideosome motor activity is regulated. We propose that the motor activity of PfMyoA is regulated by PfMTIP or heavy chain phosphorylation, and potentially by associated proteins. The impact of phosphorylation on PfMyoA motor activity and force production will be determined using actin-activated ATPases, in vitro motility assays, and force-velocity measurements using a laser trap to determine power output. We will test if PfGAP45, the adapter protein that links the motor to the membrane, is a passive player or if it affects PfMyoA activity. Successful completion of this project will increas our understanding of glideosome structure, function, and regulation. Moreover, by being the first to express a key component of the glideosome, namely the motor that powers invasion, we provide a promising next generation druggable target against malaria.

Public Health Relevance

Malaria infection is a major global health challenge afflicting over 500 million people worldwide. Plasmodium falciparum is the most virulent species and causes over 600,000 deaths per year. There is a need to develop next generation druggable targets, because the malaria parasite is becoming resistant to current therapies. Red blood cell invasion by the parasite is driven by a molecular motor called myosin as it engages with a filamentous track called actin. We are the first to express this motor in quantities that allows us to understand how it works at a molecular level, and to use it as a target for drug screening.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI117476-01
Application #
8871052
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Mcgugan, Glen C
Project Start
2015-01-01
Project End
2016-12-31
Budget Start
2015-01-01
Budget End
2015-12-31
Support Year
1
Fiscal Year
2015
Total Cost
$228,750
Indirect Cost
$78,750
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Bookwalter, Carol S; Tay, Chwen L; McCrorie, Rama et al. (2017) Reconstitution of the core of the malaria parasite glideosome with recombinant Plasmodium class XIV myosin A and Plasmodium actin. J Biol Chem 292:19290-19303