Lpt Proteins Research Articles

YdbH and YnbE form an intermembrane bridge to maintain lipid homeostasis in the outer membrane of Escherichia coli

The outer membrane (OM) of didermic gram-negative bacteria is essential for growth, maintenance of cellular integrity, and innate resistance to many antimicrobials. Its asymmetric lipid distribution, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet, is required for these functions. Lpt proteins form a transenvelope bridge that transports newly synthesized LPS from the inner membrane (IM) to OM, but how the bulk of phospholipids are transported between these membranes is poorly understood. Recently, three members of the AsmA-like protein family, TamB, YhdP, and YdbH, were shown to be functionally redundant and were proposed to transport phospholipids between IM and OM in Escherichia coli. These proteins belong to the repeating β-groove superfamily, which includes eukaryotic lipid-transfer proteins that mediate phospholipid transport between organelles at contact sites. Here, we show that the IM-anchored YdbH protein interacts with the OM lipoprotein YnbE to form a functional protein bridge between the IM and OM in E. coli. Based on AlphaFold-Multimer predictions, genetic data, and in vivo site-directed cross-linking, we propose that YnbE interacts with YdbH through β-strand augmentation to extend the continuous hydrophobic β-groove of YdbH that is thought to shield acyl chains of phospholipids as they travel through the aqueous intermembrane periplasmic compartment. Our data also suggest that the periplasmic protein YdbL prevents extensive amyloid-like multimerization of YnbE in cells. We, therefore, propose that YdbL has a chaperone-like function that prevents uncontrolled runaway multimerization of YnbE to ensure the proper formation of the YdbH-YnbE intermembrane bridge.

Structural Insights into the Lipopolysaccharide Transport (Lpt) System as a Novel Antibiotic Target.

Lipopolysaccharide (LPS) is a critical component of the extracellular leaflet within the bacterial outer membrane, forming an effective physical barrier against environmental threats in Gram-negative bacteria. After LPS is synthesized and matured in the bacterial cytoplasm and the inner membrane (IM), LPS is inserted into the outer membrane (OM) through the ATP-driven LPS transport (Lpt) pathway, which is an energy-intensive process. A trans-envelope complex that contains seven Lpt proteins (LptA-LptG) is crucial for extracting LPS from the IM and transporting it across the periplasm to the OM. The last step in LPS transport involves the mediation of the LptDE complex, facilitating the insertion of LPS into the outer leaflet of the OM. As the Lpt system plays an essential role in maintaining the impermeability of the OM via LPS decoration, the interactions between these interconnected subunits, which are meticulously regulated, may be potential targets for the development of new antibiotics to combat multidrug-resistant Gram-negative bacteria. In this review, we aimed to provide an overview of current research concerning the structural interactions within the Lpt system and their implications to clarify the function and regulation of LPS transport in the overall process of OM biogenesis. Additionally, we explored studies on the development of therapeutic inhibitors of LPS transport, the factors that limit success, and future prospects.

Conformational investigation of the asymmetric periplasmic domains of E. coli LptB2FGC using SDSL CW EPR spectroscopy.

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptB2FG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptB2FGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within β-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

Discovery, characterization, and redesign of potent antimicrobial thanatin orthologs from Chinavia ubica and Murgantia histrionica targeting E. coli LptA

Podisus maculiventris thanatin has been reported as a potent antimicrobial peptide with antibacterial and antifungal activity. Its antibiotic activity has been most thoroughly characterized against E. coli and shown to interfere with multiple pathways, such as the lipopolysaccharide transport (LPT) pathway comprised of seven different Lpt proteins. Thanatin binds to E. coli LptA and LptD, thus disrupting the LPT complex formation and inhibiting cell wall synthesis and microbial growth. Here, we performed a genomic database search to uncover novel thanatin orthologs, characterized their binding to E. coli LptA using bio-layer interferometry, and assessed their antimicrobial activity against E. coli. We found that thanatins from Chinavia ubica and Murgantia histrionica bound tighter (by 3.6- and 2.2-fold respectively) to LptA and exhibited more potent antibiotic activity (by 2.1- and 2.8-fold respectively) than the canonical thanatin from P. maculiventris. We crystallized and determined the LptA-bound complex structures of thanatins from C. ubica (1.90 Å resolution), M. histrionica (1.80 Å resolution), and P. maculiventris (2.43 Å resolution) to better understand their mechanism of action. Our structural analysis revealed that residues A10 and I21 in C. ubica and M. histrionica thanatin are important for improving the binding interface with LptA, thus overall improving the potency of thanatin against E. coli. We also designed a stapled variant of thanatin that removes the need for a disulfide bond but retains the ability to bind LptA and antibiotic activity. Our discovery presents a library of novel thanatin sequences to serve as starting scaffolds for designing more potent antimicrobial therapeutics.

Bacterial AsmA-Like Proteins: Bridging the Gap in Intermembrane Phospholipid Transport.

In eukaryotic cells, nonvesicular lipid transport between organelles is mediated by lipid-transfer proteins. Recently, a new class of these lipid transporters has been described to facilitate the bulk of inter-organelle lipid transport at contact sites by forming bridge-like structures with a hydrophobic groove through which lipids travel. Because their predicted structure is composed of repeating β-groove (RBG) domains, they have been named the RBG protein superfamily. Early studies on RBG proteins VPS13 and ATG2 recognized the resemblance of their predicted structures to that of the bacterial Lpt system, which transports newly synthesized lipopolysaccharides (LPS) between the inner and the outer membranes (IMs and OMs) of Gram-negative bacteria. In these didermic bacteria, the IMs and OMs are separated by an aqueous periplasmic compartment that is traversed by a bridge-like structure built with β-jelly roll domains from several Lpt proteins that provides a hydrophobic groove for LPS molecules to travel across the periplasm. Despite structural and functional similarities between RBG proteins and the Lpt system, the bacterial AsmA-like protein family has recently emerged as the likely ancestor of RBG proteins and long sought-after transporters that facilitate the transfer of phospholipids from the IM to the OM. Here, we review our current understanding of the structure and function of bacterial AsmA-like proteins, mainly focusing on recent studies that have led to the proposal that AsmA-like proteins mediate the bulk of phospholipid transfer between the IMs and OMs.

Identification of a Small Molecule That Inhibits the Interaction of LPS Transporters LptA and LptC.

The need for novel antibiotics has become imperative with the increasing prevalence of antibiotic resistance in Gram-negative bacteria in clinics. Acting as a permeability barrier, lipopolysaccharide (LPS) protects Gram-negative bacteria against drugs. LPS is synthesized in cells and transported to the outer membrane (OM) via seven lipopolysaccharide transport (Lpt) proteins (LptA–LptG). Of these seven Lpt proteins, LptC interacts with LptA to transfer LPS from the inner membrane (IM) to the OM, and assembly is aided by LptD/LptE. This interaction among the Lpt proteins is important for the biosynthesis of LPS; therefore, the Lpt proteins, which are significant in the assembly process of LPS, can be a potential target for new antibiotics. In this study, a yeast two-hybrid (Y2H) system was used to screen compounds that could block LPS transport by inhibiting LptA/LptC interaction, which finally disrupts the biosynthesis of the OM. We selected the compound IMB-0042 for this study. Our results suggest that IMB-0042 disrupts LptA/LptC interaction by binding to both LptA and LptC. Escherichia coli cells, when treated with IMB-0042, showed filament morphology, impaired OM integrity, and an accumulation of LPS in the periplasm. IMB-0042 inhibited the growth of Gram-negative bacteria and showed synergistic sensitization to other antibiotics, with low cytotoxicity. Thus, we successfully identified a potential antibacterial agent by using a Y2H system, which blocks the transport of LPS by targeting LptA/LptC interaction in Escherichia coli.

Development of a BLI assay-based method for detecting LptA/LptC interaction

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.

Novel Small Molecule Growth Inhibitor Affecting Bacterial Outer Membrane Reduces Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection in Avian Model.

ABSTRACTAvian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly implicated in urinary tract infections and meningitis in humans. A major limitation for the current ExPEC antibiotic therapy is the development of resistance, and antibacterial drugs that can circumvent this problem are critically needed. Here, we evaluated eight novel membrane-affecting anti-APEC small molecule growth inhibitors (GIs), identified in our previous study, against APEC infection in chickens. Among the GIs tested, GI-7 (the most effective), when administered orally (1 mg/kg of body weight), reduced the mortality (41.7%), severity of lesions (62.9%), and APEC load (2.6 log) in chickens. Furthermore, GI-7 administration at an optimized dose (60 mg/liter) in drinking water also reduced the mortality (14.7%), severity of lesions (29.5%), and APEC load (2.2 log) in chickens. The abundances of Lactobacillus and oleate were increased in the cecum and serum, respectively, of GI-7-treated chickens. Pharmacokinetic analysis revealed that GI-7 was readily absorbed with minimal accumulation in the tissues. Earlier, we showed that GI-7 induced membrane blebbing and increased membrane permeability in APEC, suggesting an effect on the APEC membrane. Consistent with this finding, the expression of genes essential for maintaining outer membrane (OM) integrity was downregulated in GI-7-treated APEC. Furthermore, decreased levels of lipopolysaccharide (LPS) transport (Lpt) proteins and LPS were observed in GI-7-treated APEC. However, the mechanism of action of GI-7 currently remains unknown and needs further investigation. Our studies suggest that GI-7 represents a promising novel lead compound that can be developed to treat APEC infection in chickens and related human ExPEC infections.IMPORTANCE APEC is a subgroup of ExPEC, and genetic similarities of APEC with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), have been reported. Our study identified a novel small molecule growth inhibitor, GI-7, effective in reducing APEC infection in chickens with an efficacy similar to that of the currently used antibiotic sulfadimethoxine, notably with an 8-times-lower dose. GI-7 affects the OM integrity and decreases the Lpt protein and LPS levels in APEC, an antibacterial mechanism that can overcome the antibiotic resistance problem. Overall, GI-7 represents a promising lead molecule/scaffold for the development of novel antibacterial therapies that could have profound implications for treating APEC infections in chickens, as well as human infections caused by ExPECs and other related Gram-negative bacteria. Further elucidation of the mechanism of action of GI-7 and identification of its target(s) in APEC will benefit future novel antibacterial development efforts.

Lipopolysaccharide transport involves long-range coupling between cytoplasmic and periplasmic domains of the LptB2FGC extractor.

The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB2FGCADE forms a proteinaceous trans-envelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB2FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB2 is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB2FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB2 can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB2FGC. Together, our genetic data reveal a path for the bi-directional coupling between LptB2 and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter.IMPORTANCEGram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a PEZ candy dispenser. This trans-envelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bi-directional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.

Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC-LptA Interaction and Decreasing LptA Stability.

The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC–LptA and LptA–LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC–LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Structural basis of lipopolysaccharide extraction by the LptB2FGC complex.

Lipopolysaccharide (LPS) in Gram-negative bacteria is essential for outer membrane formation and antibiotic resistance. The LPS transport proteins A-G (LptA-G) move LPS from the inner to outer membrane. The ATP-binding cassette (ABC) transporter LptB2FG, tightly associated with LptC, extracts LPS out of the inner membrane. The mechanism of the entire LptB2FGC complex and the role of LptC are poorly understood. Here, we used single-particle cryo-EM to characterize the structures of LptB2FG and LptB2FGC in the nucleotide-free and vanadate-trapped states. These cryo-EM structures resolve the bound LPS, reveal the transporter-LPS interactions with side-chain details, and uncover the basis of coupling LPS capture and extrusion to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, representing an unprecedented regulatory mechanism for ABC transporters. Our results suggest a role of LptC in coordinating LptB2FG action and the periplasmic Lpt protein interactions to achieve efficient LPS transport.

THE IMPACT OF TREATED LEMON PULP WITH YEAST ON GROWTH PERFORMANCE, NUTRITIVE VALUE, TOTAL ANTI-OXIDANT ENZYME AND IMMUNE RESPONSE OF GROWING RABBITS

The main target of current study was evaluated effect of incorporation of treated lemon pulp by Saccharomyces cerevisiae yeast (LPT) in growing rabbit diets on productive performance, anti-oxidant enzyme and immune response. Forty eight cross bred (New Zealand White (NZW) X California), 6 weeks age with live body weight ranging from 730.00 to 733.30g were divided into four experimental diet groups (twelve rabbits in each). Each treatment group was replicate six times and each replicates (2 rabbits in each replicate).Thesewere divided into basal diet without LPT as a control group, while 3%, 6% and 9%LPT (replacement basal diet protein by three different levels of LPT protein) as the second, third, and fourth group, respectively. The results showed no significant differences in final body weight (FBW, g), daily body weight gain [BWG (g/rabbit/day)], daily feed intake [FI (g/rabbit/day)] and feed conversion ratio [FCR, g feed/g gain] were observed among rabbit groups and control group. Moreover, no significant differences were observed in DM, OM, CP, CF, EE, NFE% digestibility and nutritive value among experimental groups. The total lipid (TL) value increased (P≤0.05) with rabbits fed 3and 6%LPT diets groups when compared to control and 9%LPT diets. While increasing LPT up to 9% decreased (P≤0.05) the concentrate of cholesterol and triglycerides in experimental groups as compared to control group. The lowest (P≤0.05) value of aspartate aminotransferase (AST) was observed with 6%LPT group when compared to control and 3% treated groups. The alanine aminotransferase (ALT) decreased (P≤0.05) with 6% LPT group when compared to 3 and 9% LPT dietary groups, while, no significant differences between treated groups and control group. The rabbit fed 6%LPT diet increased (P≤0.05) in blood urea value when compared to rabbit fed control diet. No significant difference was detected in creatinine between LPT dietary groups and control group. The total anti-oxidant enzyme values were significantly (P ≤ 0.05) increased with rabbits fed on all LPT levels when compared to control group. Immunoglobulin titer IgG significantly (P ≤ 0.05) increased with rabbit fed 9%LPT as compared to control and other experimental groups. Immunoglobulin titer IgM was insignificantly difference between LPT dietary groups and control group. Conclusively, suggesting that can be using LPT in growing rabbit diets up to 9% possible without any adverse effects on performance, digestibility and can be improvement of total anti-oxidant enzyme and immune response.

Cor a 14 is the superior serological marker for hazelnut allergy in children, independent of concomitant peanut allergy.

Hazelnut is the most frequent cause of tree nut allergy, but up to half of all children with hazelnut allergy additionally suffer from peanut allergy. Our aim was to identify diagnostic values of the most promising serological markers (Cor a 9 and Cor a 14) and to address the influence of concomitant peanut allergy and PR10 sensitization. We included 155 children suspected of hazelnut allergy and challenged according to the guidelines. Concomitant allergy to peanuts was verified or ruled out by challenge. Skin prick test, s-IgE and CRD to hazelnut, peanut, PR10 and LPT protein families were measured using ImmunoCAP. Sixty-five children had a positive hazelnut challenge, and 60% of these also had a concomitant peanut allergy. Children allergic to hazelnut were sensitized to Cor a 9 and Cor a 14; peanut-allergic children were sensitized to Ara h 2. Sensitization to PR10 protein components was seen in 45% of all included children, irrelevant to allergy to peanut or hazelnut. A cut-off >0.72 kU/L of IgE towards Cor a 14 diagnosed 87% correctly, making Cor a 14 the superior serology marker. However, nine hazelnut-allergic children were primarily sensitized to Cor a 9. Concomitant peanut allergy is common in hazelnut-allergic children, but decision points as well as diagnostic values for Cor a 14 are not affected. We found three independent and well-characterized serotypes; hazelnut-allergic children were sensitized to Cor a 14, peanut-allergic children were sensitized to Ara h 2, and independently of this were children sensitized to birch pollen (Bet v 1).

Functional properties of LptA and LptD in Anabaena sp. PCC 7120.

Lipopolysaccharides (LPS) are central components of the outer membrane and consist of Lipid A, the core polysaccharide, and the O-antigen. The synthesis of LPS is initiated at the cytosolic face of the cytoplasmic membrane. The subsequent transport to and across the outer membrane involves multiple lipopolysaccharide transport (Lpt) proteins. Among those proteins, the periplasmic-localized LptA and the outer membrane-embedded LptD participate in the last steps of transfer and insertion of LPS into the outer membrane. While the process is described for proteobacterial model systems, not much is known about the machinery in cyanobacteria. We demonstrate that anaLptD (alr1278) of Anabaena sp. PCC 7120 is important for cell wall function and its pore domain shows a Lipid A sensitive cation-selective gating behavior. The N-terminal domain of anaLptD recognizes anaLptA (alr4067), but not ecLptA. Furthermore, anaLptA specifically interacts with the Lipid A from Anabaena sp. PCC 7120 only, while anaLptD binds to Lipid A isolated from Escherichia coli as well. Based on the comparative analysis of proteins from E. coli and Anabaena sp. we discuss the properties of the cyanobacterial Lpt system.

An induced folding process characterizes the partial-loss of function mutant LptAI36D in its interactions with ligands

Lipopolysaccharide (LPS) is an essential glycolipid of the outer membrane (OM) of Gram-negative bacteria with a tripartite structure: lipid A, oligosaccharide core and O antigen. Seven essential LPS-transport proteins (LptABCDEFG) move LPS to the cell surface. Lpt proteins are linked by structural homology, featuring a β-jellyroll domain that mediates protein–protein interactions and LPS binding. Analysis of LptA–LPS interaction by fluorescence spectroscopy is used here to evaluate the contribution of each LPS moiety in protein−ligand interactions, comparing the wild-type (wt) protein to the I36D mutant. In addition to a crucial role of lipid A, an unexpected contribution emerges for the core region in recognition and binding of Lpt proteins.

Structural basis for lipopolysaccharide insertion in the bacterial outer membrane.

One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS). The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface. Of the seven Lpt proteins, the LptD-LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD-LptE from Shigella flexneri at 2.4 Å resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded β-barrel formed by LptD. Importantly, the secondary structures of the first two β-strands are distorted by two proline residues, weakening their interactions with neighbouring β-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD-LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality.

Functional Characterization of E. coli LptC: Interaction with LPS and a Synthetic Ligand

Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt–LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS–LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 μm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 μM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.

Characterization of lipopolysaccharide transport protein complex

Abstract Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.