Insights into dynamics and gating properties of T2SS secretins.

Secretins are outer membrane (OM) channels found in various bacterial nanomachines that secrete or assemble large extracellular structures. High-resolution 3D structures of type 2 secretion system (T2SS) secretins revealed bimodular channels with a C-module, holding a conserved central gate and an optional top gate, followed by an N-module for which multiple structural organizations have been proposed. Here, we perform a structure-driven in vivo study of the XcpD secretin, which validates one of the organizations of the N-module whose flexibility enables alternative conformations. We also show the existence of the central gate in vivo and its required flexibility, which is key for substrate passage and watertightness control. Last, functional, genomic, and phylogenetic analyses indicate that the optional top gate provides a gain of watertightness. Our data illustrate how the gating properties of T2SS secretins allow these large channels to overcome the duality between the necessity of preserving the OM impermeability while simultaneously promoting the secretion of large, folded effectors.

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw-EPR and Nitroxide-GdIII Distance Measurements.

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes.

Structural lessons on bacterial secretins.

To exchange and communicate with their surroundings, bacteria have evolved multiple active and passive mechanisms for trans-envelope transport. Among the pore-forming complexes found in the outer membrane of Gram-negative bacteria, secretins are distinctive homo-oligomeric channels dedicated to the active translocation of voluminous structures such as folded proteins, assembled fibers, virus particles or DNA. Members of the bacterial secretin family share a common cylinder-shaped structure with a gated pore-forming part inserted in the outer membrane, and a periplasmic channel connected to the inner membrane components of the corresponding nanomachine. In this mini-review, we will present what recently determined 3D structures have told us about the mechanisms of translocation through secretins of large substrates to the bacterial surface or in the extracellular milieu.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases.

Pseudomonas aeruginosa secretes diverse proteins via its type 2 secretion system, including a 39 kDa chitin-binding protein, CbpD. CbpD has recently been shown to be a lytic polysaccharide monooxygenase active on chitin and to contribute substantially to virulence. To date, no structure of this virulence factor has been reported. Its first two domains are homologous to those found in the crystal structure of Vibrio cholerae GbpA, while the third domain is homologous to the NMR structure of the CBM73 domain of Cellvibrio japonicus CjLPMO10A. Here, the 3.0 Šresolution crystal structure of CbpD solved by molecular replacement is reported, which required ab initio models of each CbpD domain generated by the artificial intelligence deep-learning structure-prediction algorithm RoseTTAFold. The structure of CbpD confirms some previously reported substrate-specificity motifs among LPMOAA10s, while challenging the predictive power of others. Additionally, the structure of CbpD shows that post-translational modifications occur on the chitin-binding surface. Moreover, the structure raises interesting possibilities about how type 2 secretion-system substrates may interact with the secretion machinery and demonstrates the utility of new artificial intelligence protein structure-prediction algorithms in making challenging structural targets tractable.

Structural interactions define assembly adapter function of a type II secretion system pseudopilin.

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The type II secretion system (T2SS), a virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in understanding of the T2SS is the molecular role of a small "pseudopilin" protein. Using multiple biophysical techniques, we have deciphered how this missing component of the Xcp T2SS architecture is structurally integrated, and thereby unlocked its function. We demonstrate that low-abundance XcpH is the adapter that bridges a trimeric initiating tip complex, XcpIJK, with a periplasmic filament of XcpG subunits. Each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with dimensions of the periplasm and the outer membrane-spanning secretin through which substrates pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with XcpG subunits. We provide an experimentally validated three-dimensional structural model of a complete type IV filament.

Involvement of the Pseudomonas aeruginosa MexAB-OprM efflux pump in the secretion of the metallophore pseudopaline.

To overcome the metal restriction imposed by the host's nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB-OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Direct interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion.

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpYL and XcpZM, of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa Competition experiments indicated that CbpD binding to XcpYL is XcpZM-dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZM homodimerization and increases the affinity between XcpYL and XcpZM The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.

Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates.

In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we performed a combination of in silico analysis and experimental validation to identify a core set of Tat substrates in the opportunistic pathogen Pseudomonas aeruginosa. In silico analysis predicted 44 putative Tat signal peptides in the P. aeruginosa PA14 proteome. We developed an improved amidase-based Tat reporter assay to show that 33 of these are real Tat signal peptides. In addition, in silico analysis of the full translated genome revealed a Tat candidate with a missassigned start codon. We showed that it is a new periplasmic protein in P. aeruginosa. Altogether we discovered and validated 34 Tat substrates. These show little overlap with Escherichia coli Tat substrates, and functional analysis points to a general role for the P. aeruginosa Tat system in the colonization of environmental niches and pathogenicity.

Pseudomonas aeruginosa LasB Subverts Alveolar Macrophage Activity by Interfering With Bacterial Killing Through Downregulation of Innate Immune Defense, Reactive Oxygen Species Generation, and Complement Activation.

Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, in particular, in hospital patients undergoing ventilation and in patients with cystic fibrosis. Among the virulence factors secreted or injected into host cells, the physiopathological relevance of type II secretions system (T2SS) is less studied. Although there is extensive literature on the destructive role of LasB in vitro on secreted innate immune components and on some stromal cell receptors, studies on its direct action on myeloid cells are scant. Using a variety of methods, including the use of bacterial mutants, gene-targeted mice, and proteomics technology, we show here, using non-opsonic conditions (thus mimicking resting and naïve conditions in the alveolar space), that LasB, an important component of the P.a T2SS is highly virulent in vivo, and can subvert alveolar macrophage (AM) activity and bacterial killing, in vitro and in vivo by downregulating important secreted innate immune molecules (complement factors, cytokines, etc.) and receptors (IFNAR, Csf1r, etc.). In particular, we show that LasB downregulates the production of C3 and factor B complement molecules, as well as the activation of reactive oxygen species production by AM. In addition, we showed that purified LasB impaired significantly the ability of AM to clear an unrelated bacterium, namely Streptococcus pneumoniae. These data provide a new mechanism of action for LasB, potentially partly explaining the early onset of P.a, alone, or with other bacteria, within the alveolar lumen in susceptible individuals, such as ventilated, chronic obstructive pulmonary disease and cystic fibrosis patients.

Multiple Structures Disclose the Secretins' Secrets.

Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homo-oligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.

Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline.

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics.

The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. In Pseudomonas aeruginosa, the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that secretin flexibility at interdimer interfaces is mandatory for its function.IMPORTANCE Bacterial secretins are large homooligomeric proteins constituting the outer membrane pore-forming element of several envelope-embedded nanomachines essential in bacterial survival and pathogenicity. They comprise a well-defined membrane-embedded C-terminal domain and a modular periplasmic N-terminal domain involved in substrate recruitment and connection with inner membrane components. We are studying the XcpQ secretin of the T2SS present in the pathogenic bacterium Pseudomonas aeruginosa Our data highlight the ability of the XcpQ N-terminal domain to spontaneously oligomerize into a hexamer of dimers. Further in vivo experiments revealed that this domain adopts different conformations essential for the T2SS secretion process. These findings provide new insights into the functional understanding of bacterial T2SS secretins.

Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell. However, the full range of proteins that are conveyed by Tat is unknown, despite the importance of Tat for the adaptability and full virulence of P. aeruginosa. In this work, we explored the P. aeruginosa Tat-dependent exoproteome under phosphate starvation by two-dimensional gel analysis. We identified the major secreted proteins and new Tat-dependent exoproteins. These exoproteins were further analyzed by a combination of in silico analysis, regulation studies, and protein localization. Altogether we reveal that the absence of the Tat system significantly affects the composition of the exoproteome by impairing protein export and affecting gene expression. Notably we discovered three new Tat exoproteins and one novel type II secretion substrate. Our data also allowed the identification of two new start codons highlighting the importance of protein annotation for subcellular predictions. The new exoproteins that we identify may play a significant role in P. aeruginosa pathogenesis, host interaction and niche adaptation.

Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus.

Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.

Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

DI-ICR-FT-MS-based high-throughput deep metabotyping: a case study of the Caenorhabditis elegans-Pseudomonas aeruginosa infection model.

In metabolomics there is an ever-growing need for faster and more comprehensive analysis methods to cope with the increasing size of biological studies. Direct-infusion ion-cyclotron-resonance Fourier-transform spectrometry (DI-ICR-FT-MS) is used in non-targeted metabolomics to obtain high-resolution snapshots of the metabolic state of a system. We applied this technology to a Caenorhabditis elegans-Pseudomonas aeruginosa infection model and optimized times needed for cultivation and mass-spectrometric analysis. Our results reveal that DI-ICR-FT-MS is a promising tool for high-throughput in-depth non-targeted metabolomics. We performed whole-worm metabolomics and recovered markers of the induced metabolic changes in C. elegans brought about by interaction with pathogens. In this investigation, we reveal complex metabolic phenotypes enabling clustering based upon challenge. Specifically, we observed a marked decrease in amino-acid metabolism with infection by P. aeruginosa and a marked increase in sugar metabolism with infection by Salmonella enterica. We were also able to discriminate between infection with a virulent wild-type Pseudomonas and with an attenuated mutant, making it possible to use this method in larger genetic screens to identify host and pathogen effectors affecting the metabolic phenotype of infection.

Gene transfer: transformation/electroporation.

Since Pseudomonas aeruginosa is a non-naturally competent bacterium, various methods have been developed to transfer exogenous DNA. Alternatively to transduction and conjugation, electroporation can also be used to transfer exogenous DNA molecules into Pseudomonas. Electroporation uses an electric field which generates pores in bacterial membranes allowing the entry of the exogenous DNA molecule. In contrast to conjugation which is restricted to the transfer of DNA from one bacterial cell to another, electroporation can be used to transfer all types of DNA resuspended in water.