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Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction.

ABSTRACT: NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

SUBMITTER: Sharaf NG

PROVIDER: S-EPMC8416018 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Escherichia coli metD locus encodes an ABC transporter which includes Abc (MetN), YaeE (MetI), and YaeC (MetQ).

Project description:We report that the genes abc, yaeC, and yaeE comprise metD, an Escherichia coli locus encoding a DL-methionine uptake system. MetD is an ABC transporter with Abc the ATPase, YaeE the permease, and YaeC the likely substrate binding protein. Expression of these genes is regulated by L-methionine and MetJ, a common repressor of the methionine regulon. We propose to rename abc, yaeE, and yaeC as metN, metI, and metQ, respectively.

| S-EPMC135343 | biostudies-literature

Structures of the Neisseria meningitides methionine-binding protein MetQ in substrate-free form and bound to l- and d-methionine isomers.

Project description:The bacterial periplasmic methionine-binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate-free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l- and d-methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate-free form and in complexes with l-methionine and with d-methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate-free (N238A) and substrate-bound N. meningitides MetQ are related by a "Venus-fly trap" hinge-type movement of the two domains accompanying methionine binding and dissociation. l- and d-methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand-free MetQ associates with the ATP-bound form of MetNI ∼40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.

| S-EPMC6739813 | biostudies-literature

Molecular Mechanisms of the Methionine Sulfoxide Reductase System from Neisseria meningitidis.

Project description:Neisseria meningitidis, an obligate pathogenic bacterium in humans, has acquired different defense mechanisms to detect and fight the oxidative stress generated by the host's defense during infection. A notable example of such a mechanism is the PilB reducing system, which repairs oxidatively-damaged methionine residues. This review will focus on the catalytic mechanism of the two methionine sulfoxide reductase (MSR) domains of PilB, which represent model enzymes for catalysis of the reduction of a sulfoxide function by thiols through sulfenic acid chemistry. The mechanism of recycling of these MSR domains by various "Trx-like" disulfide oxidoreductases will also be discussed.

| S-EPMC6210582 | biostudies-literature

Structural and biochemical characterization of free methionine-R-sulfoxide reductase from Neisseria meningitidis.

Project description:A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys(118) is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.

| S-EPMC2915739 | biostudies-literature

Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM L-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells.

Project description:We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular L-glutamate via the GltT-GltM L-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(-S)]. The alleviation disappeared again in AM(-S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the L-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC.

| S-EPMC4534661 | biostudies-literature

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation.

Project description:The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

| S-EPMC2527972 | biostudies-literature

Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells.

Project description:Galectin-3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signalling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin-3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin-3 is expressed during meningococcal disease and colocalizes with bacterial colonies in infected tissues from patients. We show that galectin-3 binds to N.?meningitidis and we demonstrate that this interaction requiresfull-length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin-3 contributes to phagocytosis of N.?meningitidis; instead exogenous galectin-3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin-3 deficient (Gal-3(-/-) ) mice to evaluate the contribution of galectin-3 to meningococcal bacteraemia. We found that Gal-3(-/-) mice had significantly lower levels of bacteraemia compared with wild-type mice after challenge with live bacteria, indicating that galectin-3 confers an advantage to N.?meningitidis during systemic infection.

| S-EPMC3749814 | biostudies-literature

Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

Project description:Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.

| S-EPMC4423775 | biostudies-literature

Zur regulon of Neisseria meningitidis

Project description:The zur regulon in Neisseria meningitidis was elucidated in the strain MC58 using a zur knockout strain and conditions which activate Zur ( zinc supplementation in the medium)

2012-10-05 | GSE38033 | GEO

The number of Neisseria meningitidis type IV pili determines host cell interaction.

Project description:As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers.

| S-EPMC4195760 | biostudies-literature

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