Dissecting the role(s) of Toxoplasma gondii microneme protein protease 1 activity and its substrates

Host-cell invasion by Toxoplasma gondii is associated with the shedding of microneme-derived adhesins (MICs) from the parasite surface. MICs play diverse roles during host cell invasion and MICs shedding modulates these processes. The intramembrane shedding of MICs was described to be mediated by an anonymous proteolytic activity called microneme protein protease 1 (MPP1). In order to unravel the precise function of MPP1 activity and to define the responsible enzyme(s), we generated parasite lacking the candidate enzymes, rhomboid protein proteases 4 and 5 (ROM4 and/or ROM5). We demonstrate that ROM4 mediates the cleavage of MIC2, MIC6 and a proportion of AMA1, while ROM5 cleaves AMA1 family of proteins. ROM4 null parasites possessed enhanced attachment and were impaired in host-cell invasion; however, intracellular growth was not impaired. Moreover, the deletion of ROM5 revealed no major phenotypic consequence during in vitro growth. We also analyzed the essentiality of the AMA1 family of proteins by generating respective parasite mutants, and show that compensatory mechanisms exist in coordinating host-cell invasion process

Analysis of substrate specificity of Trypanosoma brucei oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeast

N-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single-protein OSTs called TbSTT3A, TbSTT3B, and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST, making it an ideal experimental system to study the fundamental properties of OST activity. We characterized the LLO and polypeptide specificity of all three TbOST isoforms and their chimeric forms in the heterologous expression host S. cerevisiae where we were able to apply yeast genetic tools and newly developed glycoproteomics methods. We demonstrated that TbSTT3A accepted LLO substrates ranging from Man5GlcNAc2 to Man7GlcNAc2 In contrast, TbSTT3B required more complex precursors ranging from Man6GlcNAc2 to Glc3Man9GlcNAc2 structures, and TbSTT3C did not display any LLO preference. Sequence differences between the isoforms cluster in three distinct regions. We have swapped the individual regions between different OST proteins and identified region 2 to influence the specificity toward the LLO and region 1 to influence polypeptide substrate specificity. These results provide a basis to further investigate the molecular mechanisms and contribution of single amino acids in OST interaction with its substrates

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

Host 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

Estimating Risk of Introduction of Ebola Virus Disease from the Democratic Republic of Congo to Tanzania: A Qualitative Assessment

Between April 2018 and November 2020, the Democratic Republic of Congo (DRC) experienced its 11th Ebola virus disease (EVD) outbreak. Tanzania’s cross-border interactions with DRC through regular visitors, traders, and refugees are of concern, given the potential for further spread to neighboring countries. This study aimed to estimate the risk of introducing EVD to Tanzania from DRC. National data for flights, boats, and car transport schedules from DRC to Tanzania covering the period of May 2018 to June 2019 were analyzed to describe population movement via land, port, and air travel and coupled with available surveillance data to model the risk of EVD entry. The land border crossing was considered the most frequently used means of travel and the most likely pathway of introducing EVD from DRC to Tanzania. High probabilities of introducing EVD from DRC to Tanzania through the assessed pathways were associated with the viability of the pathogen and low detection capacity at the ports of entry. This study provides important information regarding the elements contributing to the risk associated with the introduction of EBV in Tanzania. It also indicates that infected humans arriving via land are the most likely pathway of EBV entry, and therefore, mitigation strategies including land border surveillance should be strengthened

Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites

Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii, respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ