Structure and Dynamics of Type 4a Pili and Type 2 Secretion System Endopili.

Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail.

Structure and function of minor pilins of type IV pili.

Type IV pili are versatile and highly flexible fibers formed on the surface of many Gram-negative and Gram-positive bacteria. Virulence and infection rate of several pathogenic bacteria, such as Neisseria meningitidis and Pseudomonas aeruginosa, are strongly dependent on the presence of pili as they facilitate the adhesion of the bacteria to the host cell. Disruption of the interactions between the pili and the host cells by targeting proteins involved in this interaction could, therefore, be a treatment strategy. A type IV pilus is primarily composed of multiple copies of protein subunits called major pilins. Additional proteins, called minor pilins, are present in lower abundance, but are essential for the assembly of the pilus or for its specific functions. One class of minor pilins is required to initiate the formation of pili, and may form a complex similar to that identified in the related type II secretion system. Other, species-specific minor pilins in the type IV pilus system have been shown to promote additional functions such as DNA binding, aggregation and adherence. Here, we will review the structure and the function of the minor pilins from type IV pili.

Target Engagement and Binding Mode of an Antituberculosis Drug to Its Bacterial Target Deciphered in Whole Living Cells by NMR.

Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc1:aa3, the major respiratory terminal oxidase in mycobacteria. The most advanced IPA called Q203 is currently in clinical trial. Using its derivative IPA317, we identified the atoms of the drug interacting with the cytochrome b in whole cells. NMR data and the self-organizing map algorithm were used to cluster a large set of drug-target complex models. The selected ensemble revealed IPA317 in a transient cavity of the cytochrome b, interacting directly with the residue T313, which is the site of spontaneous mutation conferring resistance to the IPA series. Our approach constitutes a pipeline to obtain atomic information on hit-target interactions in the cellular context.

Dynamics of a type 2 secretion system pseudopilus unraveled by complementary approaches.

Secretion pili, bacterial fibers responsible for transporting proteins to the extracellular milieu in some secretion systems, are very strong structures but at the same time highly flexible. Their flexibility and helical symmetry make structure determination at atomic resolution a challenging task. We have previously used an integrative structural biology approach including liquid-state NMR, cryo-electron microscopy (cryo-EM), and modeling to determine the pseudo-atomic resolution structure of the type 2 secretion system pseudopilus in a mutant form, where we employed NMR to determine the high resolution structure of the pilin (the monomer building block of the pilus). In this work, we determine the pseudo-atomic structure of the wild type pilus, and compare the dynamics of wild type and mutant pili by normal mode analysis. We present a detailed NMR analysis of the dynamics of the pilin in isolation, and compare dynamics and solvent accessibility of isolated and assembled pilins by Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS). These complementary approaches provide a comprehensive view of internal and overall dynamics of pili, crucial for their function.

Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach.

Bacteria use diverse signalling pathways to adapt gene expression to external stimuli. In Gram-negative bacteria, the binding of scarce nutrients to membrane transporters triggers a signalling process that up-regulates the expression of genes of various functions, from uptake of nutrient to production of virulence factors. Although proteins involved in this process have been identified, signal transduction through this family of transporters is not well understood. In the present study, using an integrative approach (EM, SAXS, X-ray crystallography and NMR), we have studied the structure of the haem transporter HasR captured in two stages of the signalling process, i.e. before and after the arrival of signalling activators (haem and its carrier protein). We show for the first time that the HasR domain responsible for signal transfer: (i) is highly flexible in two stages of signalling; (ii) extends into the periplasm at approximately 70-90 Å (1 Å=0.1 nm) from the HasR ß-barrel; and (iii) exhibits local conformational changes in response to the arrival of signalling activators. These features would favour the signal transfer from HasR to its cytoplasmic membrane partners.

Ton motor conformational switch and peptidoglycan role in bacterial nutrient uptake.

Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane, but the mechanism of energy transfer remains enigmatic. Here, we study the periplasmic domain of ExbD, a crucial component of the proton channel of the Ton system. We show that this domain is a dynamic dimer switching between two conformations representing the proton channel's open and closed states. By in vivo phenotypic assays we demonstrate that this conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. We also reveal the anchoring role of the peptidoglycan layer in this mechanism. Herein, we propose a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity. Our study fills important gaps in understanding bacterial motor mechanism and proposes novel antibacterial strategies.

A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

Mechanism of action of phthalazinone derivatives against rabies virus.

Rabies, a viral zoonosis, is responsible for almost 59,000 deaths each year, despite the existence of an effective post-exposure prophylaxis. Indeed, rabies causes acute encephalomyelitis, with a case-fatality rate of 100 % after the onset of neurological clinical signs. Therefore, the development of therapies to inhibit the rabies virus (RABV) is crucial. Here, we identified, from a 30,000 compound library screening, phthalazinone derivative compounds as potent inhibitors of RABV infection and more broadly of Lyssavirus and even Mononegavirales infections. Combining in vitro experiments, structural modelling, in silico docking and in vivo assays, we demonstrated that phthalazinone derivatives display a strong inhibition of lyssaviruses infection by acting directly on the replication complex of the virus, and with noticeable effects in delaying the onset of the clinical signs in our mouse model.

Role of the iron axial ligands of heme carrier HasA in heme uptake and release.

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Ton Motor Conformational Switch and Peptidoglycan Role in Bacterial Nutrient Uptake.

Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane, but the mechanism of energy transfer remains enigmatic. Here, we study the periplasmic domain of ExbD, a crucial component of the proton channel of the Ton system. We show that this domain is a dynamic dimer switching between two conformations representing the proton channel's open and closed states. By in vivo phenotypic assays we demonstrate that this conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. We also reveal the anchoring role of the peptidoglycan layer in this mechanism. Herein, we propose a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity. Our study fills important gaps in understanding bacterial motor mechanism and proposes novel antibacterial strategies.

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system.

Type II secretion systems (T2SSs) allow diderm bacteria to secrete hydrolytic enzymes, adhesins, or toxins important for growth and virulence. To promote secretion of folded proteins, T2SSs assemble periplasmic filaments called pseudopili or endopili at an inner membrane subcomplex, the assembly platform (AP). Here, we combined biophysical approaches, nuclear magnetic resonance (NMR) and X-ray crystallography, to study the Klebsiella AP components PulL and PulM. We determined the structure and associations of their periplasmic domains and describe the structure of the heterodimer formed by their ferredoxin-like domains. We show how structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and crosslinking data provided additional constraints to build a structural model of the PulL-PulM assembly in the cellular context. Our structural and functional insights, together with the relative cellular abundance of its components, support the role of AP as a dynamic hub that orchestrates pilus polymerization.

The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope bridge for phospholipid trafficking.

E. coli and most other diderm bacteria (those with two membranes) have an inner membrane enriched in glycerophospholipids (GPLs) and an asymmetric outer membrane (OM) containing GPLs in its inner leaflet and primarily lipopolysaccharides in its outer leaflet. In E. coli, this lipid asymmetry is maintained by the Mla system which consists of six proteins: the OM lipoprotein MlaA extracts GPLs from the outer leaflet, and the periplasmic chaperone MlaC transfers them across the periplasm to the inner membrane complex MlaBDEF. However, GPL trafficking still remains poorly understood, and has only been studied in a handful of model species. Here, we investigate GPL trafficking in Veillonella parvula, a diderm Firmicute with an Mla system that lacks MlaA and MlaC, but contains an elongated MlaD. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry which can be suppressed by mutations in tamB, supporting that these two systems have opposite GPL trafficking functions across diverse bacterial lineages. Structural modelling and subcellular localisation assays suggest that V. parvula MlaD forms a transenvelope bridge, comprising a typical inner membrane-localised MCE domain and, in addition, an outer membrane ß-barrel. Phylogenomic analyses indicate that this elongated MlaD type is widely distributed across diderm bacteria and likely forms part of the ancestral functional core of the Mla system, which would be composed of MlaEFD only.

Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex.

Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 A from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.

Secondary structure and 1H, 15 N & 13C resonance assignments of the periplasmic domain of OutG, major pseudopilin from Dickeya dadantii type II secretion system.

The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by Gram-negative bacteria is to secrete exoprotein substrates via the type II secretion system (T2SS). The T2SS is a proteinaceous complex spanning the bacterial envelope that translocates folded proteins such as toxins and enzymes from the periplasm to the extracellular milieu. In the T2SS, a cytoplasmic ATPase elongates in the periplasm the pseudopilus, a non-covalent polymer composed of protein subunits named pseudopilins, and anchored in the inner membrane by a transmembrane helix. The pseudopilus polymerisation is coupled to the secretion of substrates. The T2SS of Dickeya dadantii secretes more than 15 substrates, essentially plant cell wall degrading enzymes. In D. dadantii, the major pseudopilin or the major subunit of the pseudopilus is called OutG. To better understand the mechanism of secretion of these numerous substrates via the pseudopilus, we have been studying the structure of OutG by NMR. Here, as the first part of this study, we report the 1H, 15N and 13C backbone and sidechain chemical shift assignment of the periplasmic domain of OutG and its NMR derived secondary structure.

Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog.

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.

1H, 15 N and 13C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system.

Type II secretion systems (T2SS) allow Gram-negative bacteria to transport toxins and enzymes from the periplasm to the external milieu, and are thus important for the pathogenicity of bacteria. To drive secretion, T2SS assemble filaments called pseudopili closely related to bacterial type IV pili. These filaments are non-covalent polymers of proteins that are assembled by an inner membrane complex called the assembly platform connected to a cytoplasmic ATPase motor. In the Klebsiella oxytoca T2SS, the PulL protein from the assembly platform is essential for pseudopilus assembly and protein secretion. However, its role in these processes is not well understood. To decipher the molecular basis of PulL function, we used solution NMR to study its structure and interactions with other components of the machinery. Here as a first step, we report the 1H, 15 N and 13C backbone and side-chain chemical shift assignments of the C-terminal periplasmic domain of PulL and its secondary structure based on NMR data.

Computational and biochemical analysis of type IV pilus dynamics and stability.

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend and retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating nuclear magnetic resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability, and function, we performed a total of 108 ms all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH, and ionic strength is complemented with targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments reveal a conserved set of residues defining a calcium-binding site at the interface between three pilin subunits. Calcium binding enhances T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Mapping the interaction between the hemophore HasA and its outer membrane receptor HasR using CRINEPT-TROSY NMR spectroscopy.

The first step of heme acquisition by Gram-negative pathogenic bacteria through the so-called heme acquisition system, Has, requires delivery of the heme from the extracellular hemophore protein HasA to a specific outer membrane receptor, HasR. CRINEPT-TROSY NMR experiments in DPC micelles were here used to obtain information on the intermediate HasA-HasR complex in solution. A stable protein-protein adduct is detected both in the presence and in the absence of heme. Structural information on the complexed form of HasA is obtained from chemical shift mapping and statistical analysis of the spectral fingerprint of the protein NMR spectra obtained under different conditions. This approach shows the following: (i) only three different conformations are possible for HasA in solution: one for the isolated apoprotein, one for the isolated holoprotein, and one for the complexed protein, that is independent of the presence of the heme; (ii) the structure of the hemophore in the complex resembles the open conformation of the apoprotein; (iii) the surface contact area between HasA and HasR is independent of the presence of the heme, involving loop L1, loop L2, and the beta2-beta6 strands; (iv) upon complex formation the heme group is transferred from holoHasA to HasR.

Structure and Assembly of the Enterohemorrhagic Escherichia coli Type 4 Pilus.

Bacterial type 4a pili are dynamic surface filaments that promote bacterial adherence, motility, and macromolecular transport. Their genes are highly conserved among enterobacteria and their expression in enterohemorrhagic Escherichia coli (EHEC) promotes adhesion to intestinal epithelia and pro-inflammatory signaling. To define the molecular basis of EHEC pilus assembly, we determined the structure of the periplasmic domain of its major subunit PpdD (PpdDp), a prototype of an enterobacterial pilin subfamily containing two disulfide bonds. The structure of PpdDp, determined by NMR, was then docked into the density envelope of purified EHEC pili obtained by cryoelectron microscopy (cryo-EM). Cryo-EM reconstruction of EHEC pili at ∼8 Å resolution revealed extremely high pilus flexibility correlating with a large extended region of the pilin stem. Systematic mutagenesis combined with functional and interaction analyses identified charged residues essential for pilus assembly. Structural information on exposed regions and interfaces between EHEC pilins is relevant for vaccine and drug discovery.