Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites

Abstract

SummaryHost cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with fulllength TgSUB1 restores processing, complementation of Dsub1 parasites with TgSUB1 lacking the GPI anchor (Dsub1::DGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Dsub1 and Dsub1::DGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

“…1, A-C). The heteronuclear 15 N NOE showed that NOE values for the C-terminal six residues are significantly lower than for the main globular domain (Fig. 1D).…”

“…The protein fractions were pooled together, concentrated to ϳ500 M, and exchanged into an NMR buffer (25 mM KH 2 PO 4 , pH 7.2, 100 mM NaCl). 15 N, 13 C-labeled samples of WT MIC5 were produced in minimal medium containing 0.07% 15 NH 4 Cl and 0.2% 13 C 6 -glucose. Solution Structure Determination of TgMIC5-Backbone and side chain assignments were completed using our in-house, semiautomated assignment algorithms and standard triple-resonance assignment methodology (19).…”

“…Based on these events we assessed the abundance of TgMIC4 50 in the ESA as a measure of TgSUB1 activity and inhibition thereof. Recombinant TgMIC5 was added to the parasites prior to secretion, and its inhibitory activity was compared with that of ALLN, a previously described inhibitor of TgSUB1 (15). Lysozyme, a globular protein of a size similar to TgMIC5, was included as a negative control.…”

“…TgSUB1 is a glycosylphosphatidylinositol (GPI)-anchored microneme protein that shows subtilisin-like serine protease activity. Lagal et al (15) recently showed that TgSUB1 mediates the necessary surface processing of certain MICs for efficient host recognition and invasion, such as TgMIC2-M2AP and MIC4.…”

Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives

“…Chrompodellids also share an expansion in subtilisin proteases (SI Appendix, Fig. S7), which are secreted from micronemes and important for adhesion to host cell during infection (40). Lastly, two groups of proteins are uniquely shared between apicomplexans and their closest relatives and potentially linked to shared morphologies (SI Appendix, Table S6).…”