The fentanyl-specific antibody FenAb024 can shield against carfentanil effects.

The surge in opioid-related deaths, driven predominantly by fentanyl and its synthetic derivatives, has become a critical public health concern, which is particularly evident in the United States. While the situation is less severe in Europe, the European Monitoring Centre for Drugs and Drug Addiction reports a rise in drug overdose deaths, with emerging concerns about the impact of fentanyl-related molecules. Synthetic opioids, initially designed for medical use, have infiltrated illicit markets due to their low production costs and high potency, with carfentanil posing additional threats, including potential chemical weaponization. Existing overdose mitigation heavily relies on naloxone, requiring timely intervention and caregiver presence, while therapeutic prevention strategies face many access challenges. To provide an additional treatment option, we propose the use of a fentanyl-specific monoclonal antibody (mAb), as a non-opioid method of prophylaxis against fentanyl and carfentanil. This mAb shows protection from opioid effects in a pre-clinical murine model. mAbs could emerge as a versatile countermeasure in civilian and biodefense settings, offering a novel approach to combat opioid-associated mortality.

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.

Immunodominant surface epitopes power immune evasion in the African trypanosome.

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.

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 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.

Dynamic, variable oligomerization and the trafficking of variant surface glycoproteins of Trypanosoma brucei.

African trypanosomes cause disease in humans and livestock, avoiding host immunity by changing the expression of variant surface glycoproteins (VSGs); the major glycosylphosphatidylinositol (GPI) anchored antigens coating the surface of the bloodstream stage. Proper trafficking of VSGs is therefore critical to pathogen survival. The valence model argues that GPI anchors regulate progression and fate in the secretory pathway and that, specifically, a valence of two (VSGs are dimers) is critical for stable cell surface association. However, recent reports that the MITat1.3 (M1.3) VSG N-terminal domain (NTD) behaves as a monomer in solution and in a crystal structure challenge this model. We now show that the behavior of intact M1.3 VSG in standard in vivo trafficking assays is consistent with an oligomer. Nevertheless, Blue Native Gel electrophoresis and size exclusion chromatography-multiangle light scattering chromatography of purified full length M1.3 VSG indicates a monomer in vitro. However, studies with additional VSGs show that multiple oligomeric states are possible, and that for some VSGs oligomerization is concentration dependent. These data argue that individual VSG monomers possess different propensities to self-oligomerize, but that when constrained at high density to the cell surface, oligomeric species predominate. These results resolve the apparent conflict between the valence hypothesis and the M1.3 NTD VSG crystal structure.

Structure of trypanosome coat protein VSGsur and function in suramin resistance.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

Mechanistic Similarities between Antigenic Variation and Antibody Diversification during Trypanosoma brucei Infection.

Trypanosoma brucei, which causes African trypanosomiasis, avoids immunity by periodically switching its surface composition. The parasite is coated by 10 million identical, monoallelically expressed variant surface glycoprotein (VSG) molecules. Multiple distinct parasites (with respect to their VSG coat) coexist simultaneously during each wave of parasitemia. This substantial antigenic load is countered by B cells whose antigen receptors (antibodies or immunoglobulins) are also monoallelically expressed, and that diversify dynamically to counter each variant antigen. Here we examine parallels between the processes that generate VSGs and antibodies. We also discuss current insights into VSG mRNA regulation that may inform the emerging field of Ig mRNA biology. We conclude by extending the parallels between VSG and Ig to the protein level.

African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat.

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.

The Structure and Function of Type III Secretion Systems.

Type III secretion systems (T3SSs) afford Gram-negative bacteria an intimate means of altering the biology of their eukaryotic hosts--the direct delivery of effector proteins from the bacterial cytoplasm to that of the eukaryote. This incredible biophysical feat is accomplished by nanosyringe "injectisomes," which form a conduit across the three plasma membranes, peptidoglycan layer, and extracellular space that form a barrier to the direct delivery of proteins from bacterium to host. The focus of this chapter is T3SS function at the structural level; we will summarize the core findings that have shaped our understanding of the structure and function of these systems and highlight recent developments in the field. In turn, we describe the T3SS secretory apparatus, consider its engagement with secretion substrates, and discuss the posttranslational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS.

A common assembly module in injectisome and flagellar type III secretion sorting platforms.

Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

Structural and enzymatic characterization of a host-specificity determinant from Salmonella.

GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65 Šresolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine protease inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.

Structure of the Helicobacter pylori CagA oncoprotein bound to the human tumor suppressor ASPP2.

The Cytotoxin associated gene A (CagA) protein of Helicobacter pylori is associated with increased virulence and risk of cancer. Recent proteomic studies have demonstrated an association of CagA with the human tumor suppressor Apoptosis-stimulating Protein of p53-2 (ASPP2). We present here a genetic, biochemical, and structural analysis of CagA with ASPP2. Domain delineation of the 120-kDa CagA protein revealed a stable N-terminal subdomain that was used in a yeast two-hybrid screen that identified the proline-rich domain of ASPP2 as a host cellular target. Biochemical experiments confirm this interaction. The cocrystal structure to 2.0-Å resolution of this N-terminal subdomain of CagA with a 7-kDa proline-rich sequence of ASPP2 reveals that this domain of CagA forms a highly specialized three-helix bundle, with large insertions in the loops connecting the helices. These insertions come together to form a deep binding cleft for a highly conserved 20-aa peptide of ASPP2. ASPP2 forms an extended helix in this groove of CagA, burying more than 1,000 Å(2) of surface area. This interaction is disrupted in vitro and in vivo by structure-based, loss-of-contact point mutations of key residues in either CagA or ASPP2. Disruption of CagA and ASPP2 binding alters the function of ASPP2 and leads to the decreased survival of H. pylori-infected cells.

Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.

Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called ß-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the ß-motif. Biochemical studies establish that the ß-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.

Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches.

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase.

Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.