RESUMO
The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.
Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Externa Bacteriana/fisiologia , Proteínas de Escherichia coli/metabolismo , Lipídeos/química , Transporte Biológico/fisiologia , Escherichia coli , Proteínas de Escherichia coli/genética , ImidazóisRESUMO
The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Assuntos
Parede Celular/genética , Proteínas de Escherichia coli/genética , Lipopolissacarídeos/genética , Oxirredutases/genética , Peptidoglicano/genética , Divisão Celular/genética , Membrana Celular/genética , Membrana Celular/microbiologia , Parede Celular/microbiologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Lipopolissacarídeos/biossíntese , Mutagênese , Fosfolipídeos/biossíntese , Fosfolipídeos/genéticaRESUMO
The conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It is proposed to mediate OM constriction during cell division via cell wall tethering. Yet, recent studies suggest the complex has additional roles in OM lipid homeostasis and septal wall separation. How Tol-Pal facilitates all these processes is unclear. To gain insights into its function(s), we applied transposon-insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and > 20 negative strong interactions based on fitness. Disrupting osmoregulated-periplasmic glucan biosynthesis restores fitness and OM barrier function, but not proper division, in tol-pal mutants. In contrast, deleting genes involved in OM homeostasis and cell wall remodeling causes synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or division phenotypes. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even when single mutants divided normally; this highlights the possibility for OM phenotypes to indirectly impact cell division. Overall, our work underscores the intricate nature of Tol-Pal function, and reinforces its key roles in cell wall-OM tethering, cell wall remodeling, and in particular, OM homeostasis.
Assuntos
Proteínas de Escherichia coli , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismoRESUMO
Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Auranofina/farmacologia , Auranofina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fatores de Virulência/metabolismo , Fatores de Virulência/farmacologia , Fatores de Virulência/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Biofilmes , Percepção de Quorum , Proteínas de Bactérias/farmacologiaRESUMO
The outer membrane (OM) is a formidable permeability barrier that protects Gram-negative bacteria from detergents and antibiotics. It possesses exquisite lipid asymmetry, requiring the placement and retention of lipopolysaccharides (LPS) in the outer leaflet, and phospholipids (PLs) in the inner leaflet. To establish OM lipid asymmetry, LPS are transported from the inner membrane (IM) directly to the outer leaflet of the OM. In contrast, mechanisms for PL trafficking across the cell envelope are much less understood. In this review, we summarize and discuss recent advances in our understanding of PL transport, making parallel comparisons to well-established pathways for OM lipoprotein (Lol) and LPS (Lpt). Insights into putative PL transport systems highlight possible connections back to the 'Bayer bridges', adhesion zones between the IM and the OM that had been observed more than 50 years ago, and proposed as passages for export of OM components, including LPS and PLs.
Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Externa Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Homeostase , Lipopolissacarídeos/metabolismo , Chaperonas Moleculares/metabolismo , Fosfolipídeos/metabolismoRESUMO
The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Membrana Externa Bacteriana/fisiologia , Escherichia coli/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular , Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Homeostase , Mutação , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismoRESUMO
The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metaloproteases/química , Metaloproteases/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Cristalografia por Raios X , Proteínas de Escherichia coli/isolamento & purificação , Metaloproteases/isolamento & purificação , Permeabilidade , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry, with lipopolysaccharides (LPS) residing in the outer leaflet and phospholipids (PLs) in the inner leaflet. This asymmetric bilayer protects the bacterium against intrusion of many toxic substances, including antibiotics and detergents, yet allows acquisition of nutrients necessary for growth. To build the OM and ensure its proper function, the cell produces OM constituents in the cytoplasm or inner membrane and transports these components across the aqueous periplasmic space separating the two membranes. Of note, the processes by which the most basic membrane building blocks, i.e. PLs, are shuttled across the cell envelope remain elusive. This review highlights our current understanding (or lack thereof) of bacterial PL trafficking, with a focus on recent developments in the field. We adopt a mechanistic approach and draw parallels and comparisons with well-characterized systems, particularly OM lipoprotein and LPS transport, to illustrate key challenges in intermembrane lipid trafficking. Pathways that transport PLs across the bacterial cell envelope are fundamental to OM biogenesis and homeostasis and are potential molecular targets that could be exploited for antibiotic development.
Assuntos
Membrana Externa Bacteriana/metabolismo , Bactérias Gram-Negativas/metabolismo , Fosfolipídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Homeostase , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Piperidinas/farmacologia , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Fatores Corda/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Piperidinas/química , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.
Assuntos
Proteínas de Bactérias/metabolismo , Fatores Corda/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium smegmatis/enzimologia , Ácidos Micólicos/metabolismo , Metabolismo dos Lipídeos , Piperazinas , Pirróis , EsferoplastosRESUMO
The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photo-cross-linking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial intermembrane lipid transport important for the maintenance of OM lipid asymmetry.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfolipídeos/metabolismo , Transporte Biológico , Cromatografia em Camada Fina , Reagentes de Ligações Cruzadas , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Porinas/metabolismo , Mapas de Interação de Proteínas , Espectrometria de Massas em TandemRESUMO
A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Porinas/metabolismo , Transporte Biológico , Escherichia coli/química , Escherichia coli/citologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/análise , Humanos , Bicamadas Lipídicas/análise , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/análise , Fosfolipídeos/análise , Porinas/análise , Mapas de Interação de Proteínas , Multimerização ProteicaRESUMO
Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.
Assuntos
Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Divisão Celular , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Homeostase , Lipopolissacarídeos/metabolismo , Transporte Proteico/fisiologiaRESUMO
Gram-negative bacteria can survive in harsh environments in part because the asymmetric outer membrane (OM) hinders the entry of toxic compounds. Lipid asymmetry is established by having phospholipids (PLs) confined to the inner leaflet of the membrane and lipopolysaccharides (LPS) to the outer leaflet. Perturbation of OM lipid asymmetry, characterized by PL accumulation in the outer leaflet, disrupts proper LPS packing and increases membrane permeability. The multi-component Mla system prevents PL accumulation in the outer leaflet of the OM via an unknown mechanism. Here, we demonstrate that in Escherichia coli, the Mla system maintains OM lipid asymmetry with the help of osmoporin OmpC. We show that the OM lipoprotein MlaA interacts specifically with OmpC and OmpF. This interaction is sufficient to localize MlaA lacking its lipid anchor to the OM. Removing OmpC, but not OmpF, causes accumulation of PLs in the outer leaflet of the OM in stationary phase, as was previously observed for MlaA. We establish that OmpC is an additional component of the Mla system; the OmpC-MlaA complex may function to remove PLs directly from the outer leaflet to maintain OM lipid asymmetry. Our work reveals a novel function for the general diffusion channel OmpC in lipid transport.
Assuntos
Escherichia coli/metabolismo , Lipídeos de Membrana/metabolismo , Porinas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Bicamadas Lipídicas , Fosfolipídeos/metabolismoRESUMO
The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane ß-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD ß-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the ß-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Periplasma/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Membrana Celular/química , Membrana Celular/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Lipopolissacarídeos/química , Lipopolissacarídeos/genética , Viabilidade Microbiana/genética , Modelos Moleculares , Periplasma/química , Periplasma/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two-amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the ß-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the ß-barrel assembly machine.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/patologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Complexos Multiproteicos/metabolismo , Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Transporte Biológico/fisiologia , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Lipopolissacarídeos/genética , Complexos Multiproteicos/genética , Deleção de SequênciaRESUMO
Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.
Assuntos
Dissulfetos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Isomerases de Dissulfetos de Proteínas/deficiência , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína/química , Cisteína/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de ProteínaRESUMO
The Gram-negative bacterial envelope is bounded by two membranes. Disulfide bond formation and isomerization in this oxidizing environment are catalyzed by DsbA and DsbC, respectively. It remains unknown when and how the Dsb proteins participate in the biogenesis of outer membrane proteins, which are transported across the cell envelope after their synthesis. The Escherichia coli protein LptD is an integral outer membrane protein that forms an essential complex with the lipoprotein LptE. We show that oxidation of LptD is not required for the formation of the LptD/E complex but it is essential for function. Remarkably, none of the cysteines in LptD are essential because either of two nonconsecutive disulfide bonds suffices for function. Oxidation of LptD, which is efficiently catalyzed by DsbA, does not involve the isomerase DsbC, but it requires LptE. Thus, oxidation is completed only after LptD interacts with LptE, an interaction that occurs at the outer membrane and seems necessary for LptD folding.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Dissulfetos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Immunoblotting , Proteínas de Membrana/genética , Mutação , Oxirredução , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismoRESUMO
Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1:1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Membrana Celular/metabolismo , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , TripsinaRESUMO
Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter associated with distinct heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers a molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies.