The Toxoplasma Proteins MIC2 and M2AP Form a Hexameric Complex Necessary for Intracellular Survival

Abstract

Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2⅐M2AP complex participates in host cell attachment and invasion. The short cytoplasmic Cdomain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2⅐M2AP complex of ϳ450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three ␣␤ dimers. Resolution of this complex has implications for how MIC2⅐M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.Apicomplexan parasites actively gain entry into host cells by a unique mechanism that depends on the cohesive interaction of cytoskeletal proteins and surface adhesins (1). Host cell invasion is fundamental to diseases caused by this phylum of pathogenic organisms that includes Plasmodium falciparum, the etiological agent of severe malaria and Toxoplasma gondii, the causative agent of toxoplasmosis. Tachyzoites (Toxoplasma) and sporozoites (Plasmodium) employ similar adhesins for active entry into host cells. These adhesins define a family of thrombospondin-related anonymous proteins (TRAPs) 1 (2, 3). TRAP family members are type 1 transmembrane proteins that are comprised of adhesive N-terminal domains that harbor integrin-like A-domains and at least one thrombospondin like domain and a short C-terminal cytoplasmic domain (C-domain) (2, 3). Toxoplasma gondii contains a single TRAP family member called MIC2, which is expressed by all of the invasive stages of the parasite. MIC2 is stored in the apically located microneme secretory organelles. Following host cell contact, microneme vesicles fuse with the proximal plasma membrane depositing MIC2 on the surface of the parasite (4 -6). Images of T. gondii captured at various stages of invasion indicate that MIC2 is translocated to the posterior end of the parasite during the rapid process of entry (7). Ultimately, MIC2 is removed from the parasite surface by a protease that cleaves the MIC2 protein within the transmembrane domain (8, 9). Amino acids within and just external to the transmembrane domain are implicated in this processing event (9, 10). Recently, a Drosophila rhomboid protease was found to cleave a chimeric substrate containing the M...

A Cleavable Propeptide InfluencesToxoplasmaInfection by Facilitating the Trafficking and Secretion of the TgMIC2–M2AP Invasion Complex

“…In RH and 1C4 parasites, a band of ϳ400 kDa was present, which corresponds to the TgMIC2-M2AP heterohexamer (Figure 8). A band of ϳ230 kDa, corresponding to a trimer of TgMIC2, was the primary species in m2apKO lysates, in agreement with previous results (Jewett and Sibley, 2003b). As mentioned above, despite being abnormally retained in the secretory system, TgMIC2 and TgM2AP⌬pro formed a heterohexamer as evidenced by a band of ϳ400 kDa.…”

“…Extensive studies of the TgMIC2-M2AP complex suggest that TgMIC2 is a central component of the invasion machinery (Carruthers and Sibley, 1997;Brossier et al, 2003;Huynh et al, 2003;Jewett and Sibley, 2003b;Huynh et al, 2004;Huynh et al, 2006). TgMIC2 is a member of the thrombospondin-related anonymous protein (TRAP) family of adhesive proteins, which is conserved across the phylum.…”

“…However, more recent studies have implicated additional factors in trafficking to the micronemes, because genetic disruption of nonanchored microneme proteins results in retention of their TM protein partner in intermediate compartments (Reiss et al, 2001;Meissner et al, 2002a;Huynh et al, 2003). These studies indicate that nonanchored proteins also play a role in trafficking.Extensive studies of the TgMIC2-M2AP complex suggest that TgMIC2 is a central component of the invasion machinery (Carruthers and Sibley, 1997;Brossier et al, 2003;Huynh et al, 2003;Jewett and Sibley, 2003b;Huynh et al, 2004;Huynh et al, 2006). TgMIC2 is a member of the thrombospondin-related anonymous protein (TRAP) family of adhesive proteins, which is conserved across the phylum.…”

Molecular and functional aspects of parasite invasion

“…More recently, PTRAMP has been identified as a conserved Plasmodium thrombospondin-related apical merozoite protein [60]. In T. gondii, the TRAP homolog TgMIC2 forms a macromolecular hexamer complex with MIC2-associated protein (M2AP) [61]. Disruption of the gene encoding TgM2AP provided indirect evidence that TgMIC2 is crucial for host cell invasion [62].…”

The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa

“…Plasmodium studies were the first to demonstrate the crucial role played by the microneme protein TRAP in sporozoite gliding motility in vitro and in infection of the mosquito salivary glands and the rat liver in vivo [15,16]. In T. gondii the homologue of TRAP, TgMIC2, also has an essential role during invasion [17], and is produced as a hexameric complex of TgMIC2 and MIC2-associated protein (MIC2AP) [18] (Figure 2b).…”