An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Comprehensive mapping of immune tolerance yields a regulatory TNF receptor 2 signature in a murine model of successful Fel d 1-specific immunotherapy using high-dose CpG adjuvant.

BACKGROUND: The prevalence of allergy to cat is expanding worldwide. Allergen-specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long-lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety, and adherence to the treatment. Here, we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti-inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT. METHODS: Together with CpG, we used endotoxin-free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry (CyTOF) was applied to analyze AIT-specific immune signatures. RESULTS: We show that AIT with high-dose CpG in combination with endotoxin-free Fel d 1 reverts all major hallmarks of allergy. High-dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC-Treg and B-cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti-inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT. CONCLUSION: Our results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen-specific tolerance in patients with cat allergy or other allergic diseases.

A Toxoplasma gondii patatin-like phospholipase contributes to host cell invasion.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.