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The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the variant surface glycoprotein or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory; minor alterations within these exposed epitopes confer antigenically distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross-reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

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.

One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4Cdt2, during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophilacells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophilalines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitinpromoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis, and neoplastic growth.

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 bruceipropulsion 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.

The Z-discs of insect muscle contain kettin, a modular protein of 500–700 kDa. The Drosophilaprotein is made up of a chain of immunoglobulin (Ig) domains separated by linker sequences. Kettin differs from other modular muscle proteins of the Ig superfamily in binding to thin filaments rather than thick filaments. Kettin isolated from Lethocerus(waterbug) muscle is an elongated molecule 180 nm long, which binds to F-actin with high affinity (Kd=1.2 nM) and a stoichiometry of one Ig domain per actin protomer. Competition between kettin and tropomyosin for binding to actin excludes tropomyosin from the Z-disc. In contrast, kettin and α-actinin bind simultaneously to actin, which would reinforce the Z-disc lattice. In vitro, kettin promotes the antiparallel association of actin filaments, and a similar process may occur in the developing sarcomere: actin filaments interdigitate in an antiparallel fashion in the Z-disc with the N terminus of kettin within the Z-disc, and the C terminus some way outside. We propose a model for the association of kettin with actin in which the molecule follows the genetic helix of actin and Ig domains, separated by linker sequences, bind to each actin protomer.

Recent protein engineering studies have confirmed the multidomain nature of protein disulfide isomerase previously suggested on the basis of analysis of its amino acid sequence. The boundaries of three domains, denoted a, a′ and b, have been determined, and each domain has been expressed as an individual soluble folded protein. In this report, the boundaries of the final structural domain, b′, are defined by a combination of restricted proteolysis and protein engineering approaches to complete our understanding of the domain organization of PDI. Using these data an optimized polypeptide construct has been prepared and characterized with a view to further structural and functional studies.

Genetic studies have recently identified DsbG, a new member of the dsbgroup of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli. We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond. There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC. Our observations further elaborate the pathway of disulfide bond formation in E. coli.