Toxoplasma gondii is a protozoan parasite of the Phylum Apicomplexa. This phylum contains over 5,000 species, many of which are of significant medical or veterinary importance, including Plasmodium spp., the causative agents of malaria, Cryptosporidium spp., the causative agents of the diarrheal disease cryptosporidiosis, and Eimeria spp., which cause losses in the US poultry industry exceeding $600 million annually.
Like most apicomplexans, T. gondii
is an obligate intracellular parasite. Host cell invasion is a parasite-driven, multistep process that is necessary for parasite survival (reviewed in 
). Prior to invasion, parasites glide along the surface of the host cell to be invaded, extending and retracting a tubulin-based cytoskeletal structure, the conoid, at their extreme apical tip 
. Invasion is initiated by proteins released onto the parasite surface from apical secretory organelles known as the micronemes; these proteins mediate intimate and irreversible attachment to the host cell 
. At least one microneme protein also interacts with proteins secreted by a second set of apical organelles, the rhoptries, to form a ring-shaped zone of tight contact between the host cell plasma membrane (PM) and the PM of the internalizing parasite 
. As the parasite penetrates through this junction and into the host cell, it becomes enveloped by a parasitophorous vacuole membrane (PVM) that is derived primarily from the host cell PM 
. In the final step of invasion, the PVM pinches off from the host cell PM to surround the fully internalized parasite.
Both gliding motility and host cell penetration are driven by the same unconventional Class XIV myosin motor protein, TgMyoA 
. TgMyoA is a 93kDa protein consisting of a head domain, which contains only 23–34% identity to other myosin heavy chains, and a short neck/tail domain 
. Although TgMyoA lacks a number of generally well conserved sequence features, such as a pair of cysteine residues in the converter domain and a glycine residue that acts as the “pivot-point” for the lever arm in most other myosin heavy chains 
, it has a step size of 5.3nm and moves towards the plus-end of actin filaments at approximately 5 µm/s, a velocity comparable to skeletal muscle myosin 
The short neck/tail domain of TgMyoA binds a single, calmodulin-like myosin light chain, TgMLC1 
. These two proteins associate with two additional proteins, TgGAP45 and TgGAP50, to form the myosin motor complex (
; see ). TgGAP45 contains predicted palmitoylation and myristoylation sites and functions in motor complex assembly 
, while TgGAP50 is a transmembrane protein that is thought to anchor the motor complex into the inner membrane complex (IMC) (; 
). The motor complex is firmly immobilized in the IMC within cholesterol-enriched microdomains 
. Short actin filaments located between the parasite PM and the IMC are connected to ligands on the host cell surface through a number of bridging proteins, including TgMIC2 and aldolase (
; ); these proteins, together with the myosin motor complex, are collectively referred to as the glideosome 
. During invasion, when TgMyoA anchored into the IMC undergoes its power stroke, the parasite is driven through the ring-shaped junction and into the host cell.
Treatment of parasites with tachypleginA induces a shift in the electrophoretic mobility of TgMLC1.
The components of the myosin motor complex are highly conserved across apicomplexan parasites 
, and myosin-based motility is essential not only for invasion but also for penetrating biological barriers and disseminating through tissues during infection 
. While the components of the motor complex have been well characterized, nothing is currently known about how the activity of the complex is regulated to generate the different speeds and types of motility that the parasite is capable of 
. Myosin regulation in other systems can occur through heavy chain phosphorylation, which can alter the actin-activated ATPase activity of the myosin, its localization in the cell or its assembly with other myosin subunits (reviewed in 
). Myosin light chains also play a major role in regulating the ATPase activity and stability of myosin motor proteins 
. The effect of a particular light chain on myosin function is regulated by calcium binding to the light chain and/or phosphorylation of the light chain by myosin light chain kinases, whose activities are themselves regulated by intracellular calcium levels and a variety of other signaling pathways 
. The myosin light chain of P. falciparum
(named MTIP, for m
rotein) was recently shown to be phosphorylated 
, but whether or how the myosin light chains of apicomplexan parasites contribute to the regulation of Class XIV myosin motor function is unknown.
In a recent high-throughput screen, we identified 24 novel small-molecule inhibitors and six enhancers of T. gondii
. Surprisingly, 21 out of the 24 invasion inhibitors inhibited parasite motility and all six enhancers of invasion enhanced parasite motility. This led us to hypothesize that some of these small molecules exert their effects by altering the composition or function of the T. gondii
myosin motor complex. We show here that treatment of parasites with one of the motility inhibitors results in a posttranslational modification to TgMLC1. Furthermore, we show that motor complexes containing the altered form of TgMLC1 have reduced mechanical activity in an in vitro
motility assay. This change in TgMyoA motor activity likely accounts for the motility defects seen in the parasite after compound treatment and provides the first evidence, in any apicomplexan parasite, for the modulation of Class XIV myosin activity by myosin light chain(s).