Structural similarities between the metacyclic and bloodstream form variant surface glycoproteins of the African trypanosome.

During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of several thousand genes and pseudogenes, and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts by the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We present here three novel metacyclic form VSG N-terminal domain crystal structures (mVSG397, mVSG531, and mVSG1954) and show that they mirror closely in architecture, oligomerization, and surface diversity the known classes of bloodstream form VSGs. These data suggest that the mVSGs are unlikely to be a specialized subclass of VSG proteins, and thus could be poor candidates as the major components of prophylactic vaccines against trypanosomiasis.

A structural classification of the variant surface glycoproteins of the African trypanosome.

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the adaptive immune system of the host. The diversity space of the "VSGnome" at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography (presenting structures of N-terminal domains of coat proteins VSG11, VSG21, VSG545, VSG558, and VSG615) with deep-learning prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection.

A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl.

Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens.

Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome.

The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.