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.

Analysis of the health product pipeline for poverty-related and neglected diseases using the Portfolio-to-Impact (P2I) modeling tool.

Background: To estimate how much additional funding is needed for poverty-related and neglected disease (PRND) product development and to target new resources effectively, policymakers need updated information on the development pipeline and estimated costs to fill pipeline gaps. Methods: We previously conducted a pipeline review to identify candidates for 35 neglected diseases as of August 31, 2017 ("2017 pipeline"). We used the Portfolio-to-Impact (P2I) tool to estimate costs to move these candidates through the pipeline, likely launches, and additional costs to develop "missing products." We repeated this analysis, reviewing the pipeline to August 31, 2019 to get a time trend. We made a direct comparison based on the same 35 diseases ("2019 direct comparison pipeline"), then a comparison based on an expanded list of 45 diseases ("2019 complete pipeline"). Results: In the 2017 pipeline, 538 product candidates met inclusion criteria for input into the model; it would cost $16.3 billion (B) to move these through the pipeline, yielding 128 launches. In the 2019 direct comparison pipeline, we identified 690 candidates, an increase of 152 candidates from 2017; the largest increase was for Ebola.  The direct comparison 2019 pipeline yields 196 launches, costing $19.9B. In the 2019 complete pipeline, there were 754 candidates, an increase of 216 candidates from 2017, of which 152 reflected pipeline changes and 64 reflected changes in scope. The complete pipeline 2019 yields 207 launches, costing $21.0B. There would still be 16 "missing products" based on the complete 2019 pipeline; it would cost $5.5B-$14.2B (depending on product complexity) to develop these products. Conclusion: The PRNDs product development pipeline has grown by over a quarter in two years. The number of expected new product launches based on the 2019 pipeline increased by half compared to 2017; the cost of advancing the pipeline increased by a quarter.

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.

HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium.

NMR spectroscopy can detect biomolecules like lipopolysaccharide directly on the surface of the cell, thus avoiding isolation and purification, and providing a more realistic description than the one derived from in vitro studies. Here we present a high-resolution magic-angle spinning NMR study of the O-antigen of Salmonella enterica serovar Typhimurium (S. Typhimurium) performed directly on the cells showing the alteration of its acetylation state over time. The O-antigen region of S. Typhimurium consists of the repeating unit [→2)-α-d-Manp-(1→4)-α-l-Rhap-(1→3)-α-d-Galp-(1→] where Man stands for mannose, Rha for rhamnose, and Gal for galactose. Man is substituted with abequose (Abe) O-acetylated at carbon 2. Our studies revealed that the appearance of de-O-acetylated O-antigen in the stationary growth phase is due to the de-O-acetylation of already synthesized O-acetylated O-antigen and that this reaction is caused by the metabolism-induced basic pH of the growth medium. The labile O-acetylation of the O-antigen we observed in S. Typhimurium generates non-stoichiometric O-acetylation states and therefore changes the nature of an immunogenic epitope.