Microneme protein 8--a new essential invasion factor in Toxoplasma gondii.

Apicomplexan parasites rely on sequential secretion of specialised secretory organelles for the invasion of the host cell. First, micronemes release their content upon contact with the host cell. Second, rhoptries are discharged, leading to the formation of a tight interaction (moving junction) with the host cell, through which the parasite invades. The functional characterisation of several micronemal proteins in Toxoplasma gondii suggests the occurrence of a stepwise process. Here, we show that the micronemal protein MIC8 of T. gondii is essential for the parasite to invade the host cell. When MIC8 is not present, a block in invasion is caused by the incapability of the parasite to form a moving junction with the host cell. We furthermore demonstrate that the cytosolic domain is crucial for the function of MIC8 and can not be functionally complemented by any other micronemal protein characterised so far, suggesting that MIC8 represents a novel, functionally distinct invasion factor in this apicomplexan parasite.

Rapid control of protein level in the apicomplexan Toxoplasma gondii.

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

Functional and biophysical analyses of the class XIV Toxoplasma gondii myosin D.

The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.