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The Glideosome, a Conserved Machinery for Gliding Motility and Host-Cell Invasion in Apicomplexans
Summary: Dominique Soldati wants to identify and characterize the proteases that enable a family of parasitic protozoa called Apicomplexa to attach themselves to and actively invade host cells. Proteases are exzymes that break down proteins into peptides and amino acids. The parasite Toxoplasma gondii, which causes toxoplasmosis, is being used to do functional, biochemical, and structural analyses of candidate proteases. Validation of the proteases as potential drug targets is being conducted in Plasmodium falciparum, the malaria parasite.
Pathogens have evolved a variety of smart strategies to establish infection in their host while evading the immune system; one strategy is to adopt an intracellular lifestyle. While bacterial pathogens can easily enter nonprofessional phagocytes by induced phagocytosis, such a task is more challenging for the considerably larger protozoan parasites. Apicomplexans are obligate intracellular parasites that have elaborated a unique and active mode of host-cell entry. In the absence of locomotive organelles such as cilia and flagella, the invasive forms of these parasites exhibit a substrate-dependent motility that is essential for invasion and migration across biological barriers.
Genetics studies have established that gliding motility and cell penetration by Toxoplasma gondii need intact parasite actin filaments and are powered by the action of an essential class XIV myosin motor, which propels the parasite into the host's cells. Drugs that interfere with actin polymerization or stabilize actin filaments have profound effects on motility, yet the mechanism by which Apicomplexa control actin polymerization is unknown. Gliding is associated with the discharge of several complexes of adhesive proteins (MICs) that are stored in secretory organelles called micronemes. MIC complexes are released on the parasite's surface upon an increase in the parasite's intracellular calcium level and they bind to host-cell receptors, critically contributing to attachment, motility, and invasion. MICs are thought to mediate high-affinity interactions between the parasite and host surfaces. During invasion, a “tight junction” is formed between parasite and host-plasma membrane, which translocates toward the rear of the parasite via interactions between some of the MIC cytoplasmic tails and a cortical parasite actomyosin system. Remarkably, most MICs are proteolytically cleaved during their biogenesis and/or postexocytosis. The rhomboid protease microneme protein protease 1 (MPP1) is responsible for intramembrane proteolytic cleavage that leads to the release of the MICs from the parasite surface, an activity apparently essential for invasion in T. gondii and in Plasmodium falciparum. Our aim is to dissect the machinery in Apicomplexa that powers gliding motility and host-cell invasion by tackling specific issues: (1) how parasite F-actin dynamics is regulated; (2) the number of myosin motors contributing to motility and conoid protrusion; (3) the molecular basis for host-cell recognition and attachment; and (4) the significance of microneme proteins shedding during invasion.
Invasion factors such as regulators of actin dynamics, myosin motors, microneme proteins, and proteases are conserved and exhibit features that are restricted to the Apicomplexans. Understanding their mode of action at the molecular level not only will shed light on basic cellular processes such as cell motility and cell-cell interactions but also may reveal new targets for therapeutic interventions.
Actin dynamics: F-actin has been difficult to detect in T. gondii and other Apicomplexans, but the susceptibility of the parasite to actin-polymerizing and -depolymerizing drugs as well as molecular-genetic studies confirm that actin nucleation and polymerization are critical for motility. Apicomplexans lack the ARP2/3 complex but likely use a formin/profilin system to orchestrate actin polymerization. We have shown that profilin plays an essential role in gliding motility and invasion and, in collaboration with M-F Carlier and Stephen Matthews, respectively, have characterized biochemically and structurally several apicomplexan profilins. The dual role for profilin in T. gondii motility and host immunomodulation was genetically dissected in collaboration with Alan Sher. T. gondii contains three putative formins that feature a typical FH2 and a weakly conserved FH1 domain. We are investigating the function of these formins in T. gondii and in the rodent malaria parasite Plasmodium berghei.
Repertoire of myosin motors: Toxoplasma exhibits three distinct forms of motility that depend on TgMyoA. We have assembled the repertoire of apicomplexan myosin motors and subjected them to a comprehensive phylogenetic analysis. Two novel myosin motors caught our attention: TgMyoG, which contains a microtubule-binding domain in its tail region (TH4) and potentially connects both types of cytoskeleton; and TgMyoF, which contains WD40 domains and, like TgMyoA, is restricted to and conserved in all Apicomplexa.
Myosin light chains are associated with myosin heavy chains and play a role in regulating motor function and/or in stabilizing the neck domain to produce steps of a definite size. TgMLC1 binds to TgMyoA and brings this motor to its site of action. We have assembled the four additional genes coding for putative myosin light chains in T. gondii and their subcellular localizations suggest that additional myosin motors are likely to participate in gliding and invasion. Ultimately, we want to assign a myosin light chain and a function to MyoF and MyoG.
Microneme proteins: Extracellular parasites release the MICs, which are critical for host-cell attachment and invasion. To accommodate the broad host-range specificity of T. gondii, adhesion may involve the recognition of ubiquitous surface-exposed host molecules and/or the presence of various parasite attachment molecules able to recognize different host-cell receptors. The four major MIC complexes characterized so far indicate that these complexes are fulfilling important and non-overlapping functions.
MIC4-1-6 was the first complex composed of an escorter and adhesins to be characterized; we have undertaken a detailed structure/function analysis in collaboration with Stephen Matthews. MIC1 is a remarkable, multifunctional protein, which is essential for proper folding and transport of the whole complex through the secretory pathways. The NMR structure revealed a novel galectin domain within the C-terminus of TgMIC1 that recruits correctly folded TgMIC6 to the complex; the structure of the N-terminal domain of MIC1 revealed a novel fold (MAR). The screening of carbohydrates chips with MAR identified sialic acid as a binding entity. MAR is one of few characterized adhesive motif in T. gondii; such knowledge will help shed light on the nature of the of host receptor(s) involved in this interaction.
Rhomboid-like proteases: Most MICs are cleaved during transport to the micronemes. The significance of such processing, which occurs along the secretory pathway, is linked to the specificity of complex formation, biogenesis, targeting to the organelle, and masking of enzymatically active sites. After discharge by the organelle, further cleavages cause the dissolution of MIC complexes and their release from the parasite surface by intramembrane cleavage. The protease responsible for this cleavage is likely to be a member of the rhomboid proteases, a family of polytopic serine proteases that are well conserved in Apicomplexa. MPP1 is constitutively active at the parasite surface where two of the six T. gondii rhomboid-like proteases have been localized (TgROM4 and TgROM5). The exact contribution of intramembrane cleavage to the invasion process would be best elucidated in T. gondii, in which the phenotypic consequences of a conditional knockout can be selectively inspected for gliding, moving junction formation, penetration, sealing of the parasitophorous vacuole, and egress.
Importance of ROM4 in the three invasive stages of Plasmodium: The life cycle of Plasmodium includes three invasive stages. The motility and invasiveness of the ookinetes and sporozoites can be best studied in the rodent malarial parasite P. berghei. Antibodies raised against the conserved C-terminal extracellular domain of PfROM4 indicate that ROM4 is constitutively expressed on the surface of the three invasion forms; these antibodies block motility and invasion. Given that PfROM4 is presumed to cleave all members of the EBA family responsible for the alternative invasion pathways of the red blood cell, this unique gene may represent the Achilles heel of malaria parasites. Our aim is to validate ROM4 as a potential target for drug or vaccine development by generating a conditional knockout in P. falciparum using the inducible system recently established. In parallel, we are exploiting a cell-based cleavage assay to examine substrate specificities of the protease.
Last updated September 2008
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