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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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joseph Verdi reports financial support was provided by National Institute on Drug Abuse. Nina Papavasiliou reports financial support was provided by Helmholtz Foundation. Erec Stebbins reports financial support was provided by Helmholtz Foundation. Joseph Verdi reports a relationship with Hepione Therapeutics Inc. that includes: employment and equity or stocks. Nina Papavasiliou reports a relationship with Hepione Therapeutics Inc. that includes: equity or stocks. Erec Stebbins reports a relationship with Hepione Therapeutics Inc. that includes: board membership and equity or stocks. Yosip Kelemen reports a relationship with Hepione Therapeutics Inc. that includes: employment. Joseph Verdi reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Nina Papavasiliou reports a relationship with Panosome GmbH that includes: board membership and equity or stocks. Erec Stebbins reports a relationship with Panosome GmbH that includes: board membership and equity or stocks. Anastasia Gkeka reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Katharina Urban reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Monica Chandra reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Dennis Greiner reports a relationship with Panosome GmbH that includes: employment. Sandra Ruf reports a relationship with Panosome GmbH that includes: equity or stocks. Selina Pollich reports a relationship with Panosome GmbH that includes: employment. Joseph Verdi has patent pending to Hepione Therapeutics Inc. Nina Papavasiliou has patent pending to Hepione Therapeutics Inc. Erec Stebbins has patent pending to Hepione Therapeutics Inc. Yosip Kelemen has patent pending to Hepione Therapeutics Inc. Marco Pravetoni has patent pending to University of Minnesota. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2023 Đaković et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of interests F.N.P., C.E.S., and J.P.V. report being shareholders of Panosome GmbH. F.N.P. and C.E.S. report being the managing directors of Panosome GmbH., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests G.T., C.E.S., F.N.P., and J.P.V. report being shareholders of Panosome GmbH and Hepione Therapeutics and having patents planned, pending, or issued broadly relevant to the work. C.E.S. and F.N.P. report being the managing directors of Panosome GmbH. S.K. reports being a shareholder of Panosome GmbH and Hepione Therapeutics. S.R. and K.U. report being employees of Panosome GmbH. P.V., K.U., M.v.S., J.P.Z., Y.K., A.S., A.K.M, A.B., and H.H. report having patents planned, pending, or issued broadly relevant to the work. M.P. and C.B. have patents pending related to fentanyl haptens, conjugates, and mAbs against fentanyl-like compounds., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chandra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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 Å resolution 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.

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., Competing Interests: The authors declare no conflict of interest.

We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 μM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria.

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.

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 β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pseudomonas syringae injects numerous bacterial proteins into host plant cells through a type 3 secretion system (T3SS). One of the first such bacterial effectors discovered, HopA1, is a protein that has unknown functions in the host cell but possesses close homologs that trigger the plant hypersensitive response in resistant strains. Like the virulence factors in many bacterial pathogens of animals, HopA1 depends upon a cognate chaperone in order to be effectively translocated by the P. syringae T3SS. Herein, we report the crystal structure of a complex of HopA1(21-102) with its chaperone, ShcA, determined to 1.56-Å resolution. The structure reveals that three key features of the chaperone-effector interactions found in animal pathogens are preserved in the Gram-negative pathogens of plants, namely, (i) the interaction of the chaperone with a nonglobular polypeptide of the effector, (ii) an interaction centered on the so-called β-motif, and (iii) the presence of a conserved hydrophobic patch in the chaperone that recognizes the β-motif. Structure-based mutagenesis and biochemical studies have established that the β-motif is critical for the stability of this complex. Overall, these results show that the β-motif interactions are broadly conserved in bacterial pathogens utilizing T3SSs, spanning an interkingdom host range.

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.

SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G₁/S and G₂/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.

Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events.

Virulence associated protein type III secretion systems (T3SSs) are intricately structured organic nanosyringes that achieve the translocation of bacterial proteins from the prokaryotic cytoplasm across three membranes into the host cytosol. The substrates for these systems number in the hundreds, with remarkably diverse biological activities, modulating host cell biology for the benefit of the pathogen. Although there has been tremendous progress on the structure and function of the T3SS substrates, there has been comparatively little progress on the much more highly conserved secretion apparatus itself. This review summarizes recent advances in the field of structural microbiology that have begun to address this shortcoming, finally bringing to bear the power of structural biology to this central virulence system of Gram-negative bacterial pathogens., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.

Yersinia spp. is currently an antibiotic resistance concern and a re-emerging disease. The essential virulence factor Yersinia protein kinase A (YpkA) contains a Ser/Thr kinase domain whose activity modulates pathogenicity. Here, we present an approach integrating a machine learning method, homology modeling, and multiple conformational high-throughput docking for the discovery of YpkA inhibitors. These first reported inhibitors of YpkA may facilitate studies of the pathogenic mechanism of YpkA and serve as a starting point for development of anti-plague drugs.

Many animal models of bacterial diseases are hampered by differences in tissue tropism and the course of pathogenesis. In a recent issue of Cell, by rationally mutating a surface invasion protein (InlA) to have higher binding affinity for its cognate host receptor (E-cadherin), Wollert et al. were able to "murinize"Listeria monocytogenes, creating a strain capable of invading intestinal epithelial cells in mice, mimicking the route of infection in humans.

The Rho family of small GTPases represent well characterized signaling molecules that regulate many cellular functions such as actin cytoskeletal arrangement and the cell cycle by acting as molecular switches. A Rac1-GDP-Zn complex has been crystallized in space group P3(2)21 and its crystal structure has been solved at 1.9 A resolution. These trigonal crystals reveal the unexpected ability of Rac1 to coordinate Zn atoms in a tetrahedral fashion by use of its biologically relevant switch I and switch II regions. Upon coordination of zinc, the switch I region is stabilized in the GDP-bound conformation and contributes to a Rac1 trimer in the asymmetric unit. Zinc coordination causes switch II to adopt a novel conformation with a symmetry-related molecule. Additionally, zinc was found to displace magnesium from its octahedral coordination at switch I, although GDP binding remained stable. This structure represents the first reported Rac1-GDP-Zn complex, which further underscores the conformational flexibility and versatility of the small GTPase switch regions.

The cytolethal distending toxin (CDT) is a widespread bacterial toxin that consists of an active subunit CdtB with nuclease activity and two ricin-like lectin domains, CdtA and CdtC, that are involved in the delivery of CdtB into the host cell. The three subunits form a tripartite complex that is required to achieve the fully active holotoxin. In the present study we investigate the assembly and dynamic properties of the CDT holotoxin using molecular dynamics simulations and binding free energy calculations. The results have revealed that CdtB likely adopts a different conformation in the unbound state with a closed DNA binding site. The two characterized structural elements of the aromatic patch and groove on the CdtA and CdtC protein surfaces exhibit high mobility, and free energy calculations show that the heterodimeric complex CdtA-CdtC, as well as the CdtA-CdtB and CdtB-CdtC sub-complexes are less energetically stable as compared to the binding in the tripartite complex. Analysis of the dynamical cross-correlation map reveals information on the correlated motions and long-range interplay among the CDT subunits associated with complex formation. Finally, the estimated binding free energies of subunit interactions are presented, together with the free energy decomposition to determine the contributions of residues for both binding partners, providing insight into the protein-protein interactions in the CDT holotoxin., ((c) 2006 Wiley-Liss, Inc.)

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia spp., causing gastrointestinal diseases and the plague. Like eukaryotic PTPases, YopH catalyzes the hydrolysis of the phosphate moiety of phosphotyrosine within a highly conserved binding pocket, which is also characterized by the closure of the so-called "WPD loop" upon ligand binding. In this study, we investigate the conformational changes and dynamics of the WPD loop by molecular dynamics simulations. Consistent with experimental observations, our simulations show that the WPD loop of YopH is intrinsically flexible and fluctuates between the open and closed conformation with a frequency of approximately 4 ns for the apo, native protein. The region of helix alpha4 spanning loop 384-392, which has been revealed experimentally as a second substrate-binding site in YopH, is found to be highly associated with the WPD loop, stabilizing it in the closed, active conformation, and providing a structural basis for the cooperation of the second-substrate binding site in substrate recognition. Loop L4 (residues 323-327) is shown to be involved in a parallel, correlated motion mode with the WPD loop that contributes the stabilization of a more extended open conformation. In addition, we have simulated the loop reopening in the ligand-bound protein complex by applying the locally enhanced sampling method. Finally, the dynamic behavior of the WPD loop for the C403S mutant differs from the wild-type YopH remarkably. These results shed light on the role of the WPD loop in PTPase-mediated catalysis, and are useful in structure-based design for novel, selective YopH inhibitors as antibacterial drugs.

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

Microbial pathogens of both plants and animals employ virulence factors that suppress the host immune response. The tomato pathogen Pseudomonas syringae injects the AvrPtoB type III effector protein into the plant cell to suppress programmed cell death (PCD) associated with plant immunity. AvrPtoB also inhibits PCD in yeast, indicating that AvrPtoB manipulates a conserved component of eukaryotic PCD. To identify host targets of AvrPtoB, we performed a yeast two-hybrid screen and identified tomato ubiquitin (Ub) as a strong AvrPtoB interactor. AvrPtoB is ubiquitinated in vitro and exhibits E3 Ub ligase activity in the presence of recombinant E1 activating enzyme and specific E2 Ub-conjugating enzymes. The C terminus of AvrPtoB is sufficient for both anti-PCD and E3 Ub ligase activities, suggesting the two functions are associated. Indeed, mutation of AvrPtoB lysine residues in the C terminus, between K512 and K529, disrupts AvrPtoB-Ub interactions, decreases AvrPtoB-mediated anti-PCD activity, and abrogates P. syringae pathogenesis of susceptible tomato plants. Remarkably, quantitative decreases in AvrPtoB anti-PCD activity are correlated with decreases in AvrPtoB ubiquitination and E3 Ub ligase activity. Overall, these data reveal a unique bacterial pathogenesis strategy, where AvrPtoB manipulates the host Ub system and requires intrinsic E3 Ub ligase activity to suppress plant immunity.

Cytolethal distending toxins (CDTs) constitute a family of bacterial proteins that enter eukaryotic cells with genotoxic activity leading to cell cycle arrest and apoptosis. CDTs are widespread, having been found in a variety of Gram-negative pathogens with a broad tissue tropism. The recently determined crystal structure of the Haemophilus ducreyi CDT provides a powerful starting point for analysis of the structure and function in this toxin family. In this study, we apply comparative modeling and structural analysis to extend the experimental structural information to multiple CDT toxins from a diverse species. Analysis of structurally and functionally important residues in the active subunit, CdtB, and putative cell delivery elements, CdtA and CdtC, begins to establish the fundamental, mechanistic elements of this unique holotoxin. The results reveal that key structural features with important functional consequences are highly conserved across different CDTs, providing a blueprint for directed examination of functional hypotheses in a variety of pathogenic contexts., (2005 Wiley-Liss, Inc.)

The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

Many bacterial pathogens that cause different illnesses employ the cytolethal distending toxin (CDT) to induce host cell DNA damage, leading to cell cycle arrest or apoptosis. CDT is a tripartite holotoxin that consists of a DNase I family nuclease (CdtB) bound to two ricin-like lectin domains (CdtA and CdtC). Through the use of structure-based mutagenesis, biochemical and cellular toxicity assays, we have examined several key structural elements of the CdtA and CdtC subunits for their importance to toxin assembly, cell surface binding, and activity. CdtA and CdtC possess N- and C-terminal nonglobular polypeptides that extensively interact with each other and CdtB, and we have determined the contribution of each to toxin stability and activity. We have also functionally characterized two key binding elements of the holotoxin revealed from its crystal structure. One is an aromatic cluster in CdtA, and the other is a long and deep groove that is formed at the interface of CdtA and CdtC. We demonstrate that mutations of the aromatic patch or groove residues impair toxin binding to HeLa cells and that cell surface binding is tightly correlated with intoxication of cultured cells. These results establish several structure-based hypotheses for the assembly and function of this toxin family.

Bacterial pathogens achieve the internalization of a multitude of virulence factors into eukaryotic cells. Some secrete extracellular toxins which bring about their own entry, usually by hijacking cell surface receptors and endocytic pathways. Others possess specialized secretion and translocation systems to directly inject bacterial proteins into the host cytosol. Recent advances in the structural biology of these virulence factors has begun to reveal at the molecular level how these bacterial proteins are delivered and modulate host activities ranging from cytoskeletal structure to cell cycle progression.

Three-dimensional quantitative structure-activity relationship (QSAR) studies were conducted on two classes of recently explored compounds with known YopH inhibitory activities. Docking studies were employed to position the inhibitors into the YopH active site to determine the probable binding conformation. Good correlations between the predicated binding free energies and the inhibitory activities were found for two subsets of phosphate mimetics: alpha-ketocarboxylic acid and squaric acid (R2=0.70 and 0.68, respectively). The docking results also provided a reliable conformational alignment scheme for 3D-QSAR modeling. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed based on the docking conformations, giving q2 of 0.734 and 0.754 for CoMFA and CoMSIA models, respectively. The 3D-QSAR models were significantly improved after removal of an outlier (q2=0.829 for CoMFA and q2=0.837 for CoMSIA). The predictive ability of the models was validated using a set of compounds that were not included in the training set. Mapping the 3D-QSAR models to the active site of YopH provides new insight into the protein-inhibitor interactions for this enzyme. These results should be applicable to the prediction of the activities of new YopH inhibitors, as well as providing structural implications for designing potent and selective YopH inhibitors as antiplague agents.

Many bacterial pathogens manipulate the host cell cytoskeleton during infection. Such cytoskeletal modulation can occur at several points of contact between the pathogen and the host, and involves extracellular receptors, intracellular signal transduction and cytoskeletal proteins themselves. The field of bacterial pathogenesis has progressed dramatically over the past decade, such that structural knowledge is both timely and essential for a full appreciation of the biology at the pathogen-host interface. Several recent examples involving bacterial proteins that target actin, Rho family GTPases and extracellular receptors have contributed to a structural understanding of eukaryotic cytoskeletal modulation by pathogens.

A common theme in bacterial disease is the manipulation of the eukaryotic cytoskeleton by pathogenic factors. Understanding how virulence factors operate to achieve these alterations is a growing and important research effort. This review focuses on several virulence factors from the pathogen Salmonella, and considers the contributions of structural biology to our appreciation of bacterial modulation of cytoskeletal dynamics.