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

Abstract

Micronemal proteins of the thrombospondin-related anonymous Background: protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against , the causative agent Plasmodium falciparum of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in . We performed a Toxoplasma gondii Methods: careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and virulence. We in vivoResults: verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of causes a mic2 slightly delayed infection leading only to mild attenuation of virulence; in vivo, like with wildtype parasites, inoculation with even low numbers of KO mic2 parasites causes lethal disease in mice. However, deletion of causes mic2 delayed host cell egress , possibly via disrupted signal transduction in vitro pathways.We confirm a critical role of MIC2 in parasite Conclusions: attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite. Amendments from Version 1In response to the reviewers comments, we modified figure 5 and 6 (error bars, size bars have been added). We also provide more information regarding the in vivo experiments and tuned down the interpretation regarding virulence of mic2KO parasites. Furthermore, supplementary information for the RNAseq analysis has been added. New data added: RH FPKM, MIC2 KO FPKM and AMA1 FPKM, contain the FPKM of the three triplicates for all Toxoplasma genes for RH, mic2 KO and ama1 KO strains. MIC2KO-RH and AMA1KO-RH total results obtained after Cutdiff analysis between RH and mutants. The addition of these data will allow to access the whole RNA sequencing results and perform independent analysis.

O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

“…However, the egress defect in ⌬mic2 was found to be significantly more severe than in either ⌬pofut2 or ⌬nst2 ( Table 2). The increased egress defect in the ⌬mic2 strain may explain the markedly reduced infectivity in culture of the ⌬mic2 strain, reported by Gras et al (13), versus the more modest reduction in the ⌬pofut2 and ⌬nst2 strains shown here.…”

“…Comparison between the two O-fucosylation-deficient strains generated in this study and the previously described ⌬mic2 (13) indicates that glycosylation of TSRs has the strongest effects on attachment and invasion (Fig. 10).…”

“…Our results also suggest that the M2AP/MIC2 complex is not affected in either ⌬pofut2 or ⌬nst2, as we observed mislocalization of MIC2 and M2AP to the early/mid-secretory pathway in a statistically comparable number of vacuoles. This is unlike the ⌬mic2, where M2AP is redirected to the constitutive secretion pathway (13). Previous work showed that M2AP binds MIC2 TSR6 through its galectin-like domain, and structural studies indicated that the recognition is due to protein-protein, not carbohydrate-protein, interactions (16).…”

“…Cell culture of T. gondii tachyzoites was performed as described previously (39,61). The ⌬mic2 cell line was a kind gift of Dr. Markus Meissner (Ludwig-Maximilians-University, Munich, Germany) (13). RH ⌬ku80 was used as parental strain for all T. gondii transgenic cell lines generated in this study (39).…”

“…Although knockout of the mic2 gene strongly affects motility and invasion, MIC2 is not essential for parasite viability. Tachyzoites lacking this protein still glide, attach to, and invade fibroblasts, albeit inefficiently (13). Numerous studies have shown that correct trafficking of the M2AP/ MIC2 complex to the micronemes is important for its function.…”