Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer.

Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey's proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Integrating artificial intelligence-based epitope prediction in a SARS-CoV-2 antibody discovery pipeline: caution is warranted.

BACKGROUND: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes. METHODS: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs. FINDINGS: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner. INTERPRETATION: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection. FUNDING: Full list of funders is provided at the end of the manuscript.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Structure and function of the EA1 surface layer of Bacillus anthracis.

The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis.

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding.

Various bacteria are suggested to contribute to colorectal cancer (CRC) development, including pks+ E. coli which produce the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells. It remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells and its DNA to cause harm. Using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC, we found that pks+ E. coli drives CRC exacerbation and tissue invasion in a colibactin-dependent manner. Using isogenic mutant strains, we further demonstrate that CRC exacerbation critically depends on expression of the E. coli type-1 pilus adhesin FimH and the F9-pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. Together, we show that the oncogenic potential of pks+ E. coli critically depends on bacterial adhesion to host epithelial cells and is critically mediated by specific bacterial adhesins. Adhesin-mediated epithelial binding subsequently allows production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic avenues for the development of anti-adhesive therapies aiming at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC.

Identification of small molecule antivirals against HTLV-1 by targeting the hDLG1-Tax-1 protein-protein interaction.

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.

Structural analysis and architectural principles of the bacterial amyloid curli.

Two decades have passed since the initial proposition that amyloids are not only (toxic) byproducts of an unintended aggregation cascade, but that they can also be produced by an organism to serve a defined biological function. That revolutionary idea was borne out of the realization that a large fraction of the extracellular matrix that holds Gram-negative cells into a persistent biofilm is composed of protein fibers (curli; tafi) with cross-ß architecture, nucleation-dependent polymerization kinetics and classic amyloid tinctorial properties. The list of proteins shown to form so-called functional amyloid fibers in vivo has greatly expanded over the years, but detailed structural insights have not followed at a similar pace in part due to the associated experimental barriers. Here we combine extensive AlphaFold2 modelling and cryo-electron transmission microscopy to propose an atomic model of curli protofibrils, and their higher modes of organization. We uncover an unexpected structural diversity of curli building blocks and fibril architectures. Our results allow for a rationalization of the extreme physico-chemical robustness of curli, as well as earlier observations of inter-species curli promiscuity, and should facilitate further engineering efforts to expand the repertoire of curli-based functional materials.

A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

The Bacillus anthracis S-layer is an exoskeleton-like structure that imparts mechanical and osmotic stabilization to the cell wall.

Surface layers (S-layers) are 2D paracrystalline protein monolayers covering the cell envelope of many prokaryotes and archaea. Proposed functions include a role in cell support, as scaffolding structure, as molecular sieve, or as virulence factor. Bacillus anthracis holds two S-layers, composed of Sap or EA1, which interchange in early and late exponential growth phase. We previously found that acute disruption of B. anthracis Sap S-layer integrity, by means of nanobodies, results in severe morphological cell surface defects and cell collapse. Remarkably, this loss of function is due to the destruction of the Sap lattice structure rather than detachment of monomers from the cell surface. Here, we combine force nanoscopy and light microscopy observations to probe the contribution of the S-layer to the mechanical, structural, and functional properties of the cell envelope, which have been so far elusive. Our experiments reveal that cells with a compromised S-layer lattice show a decreased compressive stiffness and elastic modulus. Furthermore, we find that S-layer integrity is required to resist cell turgor under hypotonic conditions. These results present compelling experimental evidence indicating that the S-layers can serve as prokaryotic exoskeletons that support the cell wall in conferring rigidity and mechanical stability to bacterial cells.

Efficient long-range conduction in cable bacteria through nickel protein wires.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli.

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by ß-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

ß-Barrels covalently link peptidoglycan and the outer membrane in the α-proteobacterium Brucella abortus.

Gram-negative bacteria are surrounded by a cell envelope that comprises an outer membrane (OM) and an inner membrane that, together, delimit the periplasmic space, which contains the peptidoglycan (PG) sacculus. Covalent anchoring of the OM to the PG is crucial for envelope integrity in Escherichia coli. When the OM is not attached to the PG, the OM forms blebs and detaches from the cell. The Braun lipoprotein Lpp1 covalently attaches OM to the PG but is present in only a small number of γ-proteobacteria; the mechanism of OM-PG attachment in other species is unclear. Here, we report that the OM is attached to PG by covalent cross-links between the N termini of integral OM ß-barrel-shaped proteins (OMPs) and the peptide stems of PG in the α-proteobacteria Brucella abortus and Agrobacterium tumefaciens. Cross-linking is catalysed by L,D-transpeptidases and attached OMPs have a conserved alanyl-aspartyl motif at their N terminus. Mutation of the aspartate in this motif prevents OMP cross-linking and results in OM membrane instability. The alanyl-aspartyl motif is conserved in OMPs from Rhizobiales; it is therefore feasible that OMP-PG cross-links are widespread in α-proteobacteria.

The Bacillus anthracis Cell Envelope: Composition, Physiological Role, and Clinical Relevance.

Anthrax is a highly resilient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. The bacterium presents a complex and dynamic composition of its cell envelope, which changes in response to developmental and environmental conditions and host-dependent signals. Because of their easy to access extracellular locations, B. anthracis cell envelope components represent interesting targets for the identification and development of novel therapeutic and vaccine strategies. This review will focus on the novel insights regarding the composition, physiological role, and clinical relevance of B. anthracis cell envelope components.

Synthesis of Fucose Derivatives with Thiol Motifs towards Suicide Inhibition of Helicobacter pylori.

The syntheses of six thiol-exhibiting monosaccharides towards suicide inhibition of Helicobacter pylori are reported. Blood group Antigen Binding Adhesin (BabA), a bacterial membrane-bound lectin, binds to human ABO and Lewis b blood group structures displayed on the surface of host epithelial cells. Crystal structures of the carbohydrate-recognition domain revealed a conserved disulfide bonded loop that anchors a critical fucose residue in these blood group structures. Disruption of this loop by N-acetylcysteine results in reduced BabA-mediated adherence to human gastric tissue sections and attenuated virulence in Lewis b-expressing transgenic mice. With a view of creating specific inhibitors of the lectin, we designed and successfully synthesised six fucose-derived compounds with thiol motifs to engage in a thiol-disulfide exchange with this disulfide bond of BabA and form a glycan-lectin disulfide linkage. Branching and extending the fucose backbone with 2- and 3-carbon thiol motifs delivered a range of candidates to be tested for biological activity against BabA.

An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity.

Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity.

Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and base-calling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the ß-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation during single-stranded DNA translocation. DNA sequencing using a prototype CsgG-CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70% in homopolymers up to 9 nucleotides long.

Structural insight into the formation of lipoprotein-ß-barrel complexes.

The ß-barrel assembly machinery (BAM) inserts outer membrane ß-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export following conformational cycling of BamA, and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.

Cysteine Residues in Helicobacter pylori Adhesin HopQ are Required for CEACAM-HopQ Interaction and Subsequent CagA Translocation.

Attachment to the host gastric mucosa is a key step in Helicobacter pylori infection. Recently, a novel adhesin, HopQ, was shown to bind distinct host CEACAM proteins-an interaction that was found to be essential for the translocation of CagA, a key virulence factor of H. pylori. The HopQ-CEACAM1 co-crystal structure revealed a binding mode dependent on loops in HopQ that are clasped by disulfide bonds. In this study, we investigated the importance of these cysteine residues for CEACAM1 engagement by H. pylori. We observed a loss of CEACAM1 binding and CagA translocation upon disruption of the disulfide bond in loop CL1 (connecting C103 to C132 in HopQ). Deletion of the Dsb-like oxidoreductase HP0231 did not affect cell surface expression of HopQ or alter the interaction of H. pylori with target cells. Although HP0231 deletion was previously described to impede CagA translocation, our results indicate that this occurs through a HopQ-independent mechanism. Together, our results open up new avenues to therapeutically target the HopQ-CEACAM1 interaction and reduce the burden of pathogenic H. pylori.