Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion

Abstract

SummaryHost cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre-recombinase and CRISPR-Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4-ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii

“…Following secretion from the micronemes to the parasite plasma membrane, AMA1 is cleaved within its TM domain by the rhomboid protease ROM4 (Rugarabamu et al, 2015; Shen et al, 2014) and its ectodomain released from the parasite. As Cys504 lies within the AMA1 TM domain, we hypothesized that palmitoylation on this site might regulate AMA1 intramembrane cleavage and shedding.…”

Gliding motility powers invasion and egress in Apicomplexa

“…Mutations in the tail domain of MIC2 and TRAP that are facing the glideosome complex impair the motility of sporozoites and tachyzoites, respectively 85 . However, the deletion of TgMIC2 does not completely abolish motility 23,86 , which suggests that other microneme proteins may participate in gliding.…”

“…Interestingly, the deletion of TgROM4 has no effect on parasite survival in vitro and is not compensated for by other rhomboid proteases 86,91,92 . However, the non-cleaved ligand-receptor complexes are translocated rearwards and accumulate at the posterior pole of the parasite, leading to tachyzoites exhibiting stationary twirling motility (visualized as upright parasites twirling on their posterior pole; Supplementary information S4 (movie)) instead of productive helical motility (Supplementary information S2 (movie)) 86,92 . The non-cleaved, secreted microneme proteins accumulate on the surface of the parasites, and this results in increased attachment and a defect in apical reorientation.…”

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

“…Indeed, microneme secretion has been demonstrated in several studies to be linked to efficient parasite invasion and gliding motility, and it has been suggested that micronemal proteins act as force transmitters for the acto-myosin system, similar to the role of integrins in amoeboid cells ( Bargieri et al , 2014; Tardieux & Baum, 2016). These proteins are then cleaved by rhomboid proteases (ROMs) to release the force ( Rugarabamu et al , 2015; Shen et al , 2014a). …”