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Endosomal sorting complex required transport (ESCRT) III proteins are essential for membrane remodeling and repair across all domains of life. Eukaryotic ESCRT-III and the cyanobacterial homologs PspA and Vipp1/Imm30 remodel membrane into vesicles, rings, filaments and tubular rods structures. Here our microscopy analysis showed that multicellular bacteria, referred to as magnetoglobules, possess multiple compartments including magnetosome organelles, polyphosphate granules, vesicles, rings, tubular rods, filaments and MVB-like structures. Therefore, membrane remodeling protein PspA might be required for the formation of these compartments, and contribute to the morphogenesis and evolution of multicellularity. To assess these hypotheses, we sequenced nine genomes of magnetoglobules and found a significant genome expansion compared to unicellular magnetotactic bacteria. Moreover, PspA was ubiquitous in magnetoglobules and formed a distinct clade on the tree of eubacterial and archaeal ESCRT-III. The phylogenetic feature suggested the evolution of magnetoglobules from a unicellular ancestor of deltaproteobacterium. Hetero-expression of ellipsoidal magnetoglobulepspA2gene alone inEscherichia coliresulted in intracellular membrane aggregation. GFP fusion labeling revealed polar location of PspA2 in rod-shaped unicells and regular interval location in filamentous cells. Cryo-electron tomography analysis showed filament bundle, membrane sacculus, vesicles and MVB-like structure in the cells expressing PspA2. Moreover, electron-dense area with a similar distribution as GFP-PspA2 foci in filamentous cells changed the inward orientation of the septum, which might interfere with the cell division. Collectively, these results show the membrane remodeling function of magnetoglobule PspA proteins, which may contribute to morphogenesis and the evolution of multicellularity of magnetotactic bacteria.

International audience; Rapid, yet sensitive and accurate testing concepts are critical in the control of spreading diseases. With the COVID-19 pandemic still ongoing, the need for efficient, fast and accurate testing of the infection state of children and the elderly traveling as well as people with symptoms has not declined. Most current methods, which are highly sensitive, are rather slow and cannot be applied at the point of care. While rapid antigenic tests ascertain only high viral burden, here, we demonstrate an alternative, rapid point-of-care diagnostics with the ability to sense low viral loads. The goal of a portable and fast quantitative diagnostic device has been achieved via the use of VHH-72-Fc, a nanobody featuring high binding strength to the spike 1 glycoprotein of the SARS-CoV-2 viral envelope, a surface plasmon resonance sensing approach, and machine learning for predicting the cut-off value between positive and negative nasopharyngeal swab samples. The concept was validated on 119 nasopharyngeal samples, 50 positive and 69 negative, as identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, showing a 88% positive percentage agreement and a 92% negative percentage agreement, as compared to RT-qPCR. Simple artificial neural network data processing revealed the influence in the sampling time to achieve unique performance in terms of speed, specificity and sensitivity. These sensing features combined with no sample preparation and portability of the diagnostic device suggest that this approach is well-adapted to be operated in hospital or laboratory located diagnostic centres.

Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector's CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector's CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.

Background An ongoing need during the COVID-19 pandemic has been the requirement for accurate and efficient point-of-care testing platforms to distinguish infected from non-infected people, and to differentiate SARS-CoV-2 infections from other viruses. Electrochemical platforms can detect the virus via its envelope spike protein by recording changes in voltammetric signals between samples. However, this remains challenging due to the limited sensitivity of these sensing platforms. Methods Here, we report on a nanobody-functionalized electrochemical platform for the rapid detection of whole SARS-CoV-2 viral particles in complex media such as saliva and nasopharyngeal swab samples. The sensor relies on the functionalization of gold electrode surface with highly-oriented Llama nanobodies specific to the spike protein receptor binding domain (RBD). The device provides results in 10 min of exposure to 200 µL of unprocessed samples with high specificity to SARS-CoV-2 viral particles in human saliva and nasopharyngeal swab samples. Results The developed sensor could discriminate between different human coronavirus strains and other respiratory viruses, with 90% positive and 90% negative percentage agreement on 80 clinical samples, as compared to RT-qPCR. Conclusions We believe this diagnostic concept, also validated for RBD mutants and successfully tested on Delta variant samples, to be a powerful tool to detect patients’ infection status, easily extendable to other viruses and capable of overcoming sensing-related mutation effects.

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.

18 pags., 4 figs. -- This article has been updated, a correction to this article was published on 27 April 2021, Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week’s training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein–protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction., JBC is supported by ARC program (SLS220120605310), French Infrastructure for Integrated Structural Biology (FRISBI) ANR10-INSB-05, ANR-18-CE44-0008, ANR-20-CE11-0026, INCA 2016- 1-PL BIO-11 and INCA SLX4 INCA 2016-159.

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

International audience; Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.

International audience; Point-of-care (POC) technologies and testing programs hold great potential to significantly improve diagnosis and disease surveillance. POC tests have the intrinsic advantage of being able to be performed near the patient or treatment facility, owing to their portable character. With rapid results often in minutes, these diagnostic platforms have a high positive impact on disease management. POC tests are, in addition, advantageous in situations of a shortage of skilled personnel and restricted availability of laboratory-based analytics. While POC testing programs are widely considered in addressing health care challenges in low-income health systems, the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could largely benefit from fast, efficient, accurate, and cost-effective point-of-care testing (POCT) devices for limiting COVID-19 spreading. The unrestrained availability of SARS-CoV-2 POC tests is indeed one of the adequate means of better managing the COVID-19 outbreak. A large number of novel and innovative solutions to address this medical need have emerged over the last months. Here, we critically elaborate the role of the surface ligands in the design of biosensors to cope with the current viral outbreak situation. Their notable effect on electrical and electrochemical sensors' design will be discussed in some given examples.

Correction to: European Biophysics Journal https://doi.org/10.1007/s00249-021-01517-5

International audience; Keywords Extracellular vesicle production Membrane protein expression Membrane protein-ligand interaction Cell-surface receptor Electron microscopy Immunization Highlights Membrane protein interaction studies using extracellular vesicles Extracellular vesicles as a tool for structure determination of membrane proteins Extracellular vesicles use for immunization Abstract Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most procedures developed involve isolating the protein by detergent solubilization and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.

International audience; Molecular CellArticleCas9 Allosteric Inhibitionby the Anti-CRISPR Protein AcrIIA6Olivier Fuchsbauer,1,2,9Paolo Swuec,3,4,9Claire Zimberger,1,2Be ́atrice Amigues,1,2Se ́bastien Levesque,5Daniel Agudelo,5Alexis Duringer,5Antonio Chaves-Sanjuan,4Silvia Spinelli,1,2Genevie`ve M. Rousseau,6,7Minja Velimirovic,5Martino Bolognesi,3,4Alain Roussel,1,2Christian Cambillau,1,2Sylvain Moineau,6,7,8Yannick Doyon,5and Adeline Goulet1,2,10,*1Architecture et Fonction des Macromole ́cules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case932, 13288 Marseille Cedex 09, France2Architecture et Fonction des Macromole ́cules Biologiques, Aix-Marseille Universite ́, Campus de Luminy, Case 932, 13288 Marseille Cedex09, France3Dipartimento di Bioscienze, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy4Centro di Ricerca Pediatrica Romeo ed Enrica Invernizzi, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy5Centre Hospitalier Universitaire de Que ́bec–Universite ́Laval Research Center, Que ́bec City, QC G1V 4G2, Canada6De ́partement de biochimie, de microbiologie, et de bio-informatique, Faculte ́des sciences et de ge ́nie, Universite ́Laval, Que ́bec City, QC,G1V 0A6, Canada7Groupe de recherche en e ́cologie buccale, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada8Fe ́lix d’He ́relle Reference Center for Bacterial Viruses, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada9These authors contributed equally10Lead Contact*Correspondence:adeline.goulet@afmb.univ-mrs.frhttps://doi.org/10.1016/j.molcel.2019.09.012SUMMARYIn the arms race against bacteria, bacteriophageshave evolved diverse anti-CRISPR proteins (Acrs)that block CRISPR-Cas immunity. Acrs play key rolesin the molecular coevolution of bacteria with theirpredators, use a variety of mechanisms of action,and provide tools to regulate Cas-based genomemanipulation. Here, we present structural and func-tional analyses of AcrIIA6, an Acr from virulentphages, exploring its unique anti-CRISPR action.Our cryo-EM structures and functional data ofAcrIIA6 binding toStreptococcus thermophilusCas9 (St1Cas9) show that AcrIIA6 acts as an allo-steric inhibitor and induces St1Cas9 dimerization.AcrIIA6 reduces St1Cas9 binding affinity for DNAand prevents DNA binding within cells. The PAMand AcrIIA6 recognition sites are structurally closeand allosterically linked. Mechanistically, AcrIIA6 af-fects the St1Cas9 conformational dynamics associ-ated with PAM binding. Finally, we identify a naturalSt1Cas9 variant resistant to AcrIIA6 illustratingAcr-driven mutational escape and molecular diversi-fication of Cas9 proteins

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents productive DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Summary: Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer. : The production of adenosine via CD39 and CD73 ectoenzymes participates in an immunosuppressive tumor microenvironment. Perrot et al. generated two antibodies, IPH5201 and IPH5301, targeting human CD39 and CD73, respectively. In vitro and in vivo data support the use of anti-CD39 and anti-CD73 mAbs in combination cancer therapies. Keywords: CD39, CD73, cancer immunotherapies, therapeutic antibodies, adenosine pathway, tumor micro-environment, immunosuppression

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.

International audience; The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM. However, the structural characterisation of the baseplate lags behind and should be the target of future studies. Finally, cryo-tomography should provide low/medium resolution maps allowing to assemble the different parts ultimately leading to a complete structural description of T6SS.

Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior., No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the periplasmic domains from two major T9SS components PorM and GldM, which span most of the periplasmic space, and propose a putative model of the T9SS core membrane complex.

In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. The precise role of PGRP-SC is less well defined because the PGRP-SC genes (PGRP-SC1a, PGRP-SC1b and PGRP-SC2) lie very close on the chromosome and have been studied using a deletion encompassing the three genes. By generating PGRP-SC-specific mutants, we reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2, respectively, during immune responses. We showed that these genes are expressed in different gut domains and that they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. We also demonstrated that PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, we showed that mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, we demonstrated that PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria.

The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.

PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

International audience; The transport of proteins at the cell surface of Bacteriodetes depends on a secretory apparatus known as Type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins such as adhesins required for gliding motility in Flavobacteria, S-layer components in Tannerella forsythia and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. While a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed, and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.

Summary Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer. Video Abstract

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.

The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae . In Salmonella enterica serovar Typhimurium ( S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P ramA ). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P ramA , including the −10 conserved region, the transcriptional start site of ramA , and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P ramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity ( K D [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P ramA of an MDR S. Typhimurium clinical isolate than for the wild-type P ramA ( K D = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.

International audience; Previous studies have shown that the trypsin inhibitors LMPI-1, LMPI-3 and SGTI from locusts display an unusual species selectivity. They inhibit locust, crayfish and fungal trypsins several orders of magnitude more efficiently than bovine trypsin. In contrast, the chymotrypsin inhibitors from the same family, LMPI-2 and SGCI, are active towards mammalian enzymes. The crystal structures of a variant of LMPI-1 and of LMPI-2 in complex with bovine chymotrypsin have revealed subtle structural differences between the trypsin and chymotrypsin inhibitors. In a previous report, it was proposed that Pro173 of bovine trypsin is responsible for the weak inhibitory activity of LMPI-1 and LMPI-3. A fungal trypsin from Fusarium oxysporum contains Gly173 instead of Pro173 and has been shown to be strongly inhibited by LMPI-1 and LMPI-3. To explore the structural features that are responsible for this property, the crystal structure of the complex between LMPI-3 and F. oxysporum trypsin was determined at 1.8 angstrom resolution. This study indicates that this small inhibitor interacts with the protease through the reactive site P3-P4' and the P10-P6 residues. Comparison of this complex with the SGTI-crayfish trypsin and BPTI-bovine trypsin complexes reinforces this hypothesis on the role of residue 173 of trypsin in species selectivity.

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.