Identification and Characterization of an Escorter for Two Secretory Adhesins in Toxoplasma gondii

Abstract

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion

“…2(A) and results not shown), suggesting a microneme localisation based on the predominant colocalisation with the microneme marker TgMIC4. Additional labelling in the late secretory pathway is possibly an effect of protein overexpression as a similar distribution with a significant accumulation in the secretory pathway was previously observed with some microneme proteins (Reiss et al, 2001). Since N-and C-terminal tagging of TgROM1 resulted in the same localisation, the other ROMs were tagged only with an N-terminal myc-epitope.…”

“…3,4-Dichloroisocoumarin may act directly or indirectly on MPP1. The C-terminal MPP1 cleavage also occurs on several other transmembrane proteins such as TgMIC6, TgMIC12, TgMIC8 (Reiss et al, 2001) and TgAMA-1 (Donahue et al, 2000) as well as members of the TRAP family in Plasmodium and other apicomplexan species. The cleavage site of TgMIC6 by MPP1 was recently mapped by mass spectrometry, and shown to be within the transmembrane (TM) domain at the site IAYGG, a sequence conserved in several apicomplexan transmembrane microneme proteins (Opitz et al, 2002).…”

Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding

“…The stage of the parasite responsible for dissemination of acute infection, termed the tachyzoite, is promiscuous in vitro, invading every mammalian cell line. While evidence that molecules secreted from the microneme organelles direct the process of host cell attachment is accumulating (Garcia-Réguet et al 2000 ;Reiss et al 2001 ;Brecht et al 2001 ;Lourenco et al 2001), the role of parasite surface molecules in this process remains equivocal.…”

Molecular dissection of host cell invasion by the Apicomplexans: the glideosome

“…Three complexes have been described to date in T. gondii. TgMIC1/MIC4/MIC6 (Reiss et al, 2001) TgMIC3/MIC8 (Meissner et al, 2002a) and TgMIC2/M2AP (Rabenau et al, 2001) (Fig. 2A).…”