Molecular Crowding Alters the Interactions of Polymyxin Lipopeptides within the Periplasm of E. coli: Insights from Molecular Dynamics.

The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the E. coli periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.

Novel Synthesis of Phytosterol Ferulate Using Acidic Ionic Liquids as a Catalyst and Its Hypolipidemic Activity.

Phytosterol ferulate (PF) is quantitively low in rice, corn, wheat, oats, barley, and millet, but it is potentially effective in reducing plasma lipids. In this study, PF was synthesized for the first time using acidic ionic liquids as a catalyst. The product was purified, characterized using Fourier transform infrared, mass spectroscopy, and nuclear magnetic resonance, and ultimately confirmed as the desired PF compound. The conversion of phytosterol surpassed an impressive 99% within just 2 h, with a selectivity for PF exceeding 83%. Plasma lipid-lowering activity of PF was further investigated by using C57BL/6J mice fed a high-fat diet as a model. Supplementation of 0.5% PF into diet resulted in significant reductions in plasma total cholesterol, triacylglycerols, and nonhigh-density lipoprotein cholesterol by 13.7, 16.9, and 46.3%, respectively. This was accompanied by 55.8 and 36.3% reductions in hepatic cholesterol and total lipids, respectively, and a 22.9% increase in fecal cholesterol excretion. Interestingly, PF demonstrated a higher lipid-lowering activity than that of its substrates, a physical mixture of phytosterols and ferulic acid. In conclusion, an efficient synthesis of PF was achieved for the first time, and PF had the great potential to be developed as a lipid-lowering dietary supplement.

An ode on the odyssey of lipids.

The poem Ode on the Odyssey of lipoproteins describes the structure, functions and metabolism of lipoproteins namely Chylomicrons, LDL, VLDL and HDL. This poem is a triolet with eight lines in each stanza. Odyssey is the travel experience of an adventurous journey when someone travels far and wide. This poem describes the transport adventures of Lipids when they travel in the form of lipoproteins. The poetic form of describing the metabolism of lipoproteins was intended to kindle the interest of the learners and to gain an imaginary experience in the metabolism of lipoproteins.

Nuclear Magnetic Resonance-Based Metabolomics and Risk of CKD.

RATIONALE & OBJECTIVE: Chronic kidney disease (CKD) leads to lipid and metabolic abnormalities, but a comprehensive investigation of lipids, lipoprotein particles, and circulating metabolites associated with the risk of CKD has been lacking. We examined the associations of nuclear magnetic resonance (NMR)-based metabolomics data with CKD risk in the UK Biobank study. STUDY DESIGN: Observational cohort study. SETTING & PARTICIPANTS: A total of 91,532 participants in the UK Biobank Study without CKD and not receiving lipid-lowering therapy. EXPOSURE: Levels of metabolites including lipid concentration and composition within 14 lipoprotein subclasses, as well as other metabolic biomarkers were quantified via NMR spectroscopy. OUTCOME: Incident CKD identified using ICD codes in any primary care data, hospital admission records, or death register records. ANALYTICAL APPROACH: Cox proportional hazards regression models were used to estimate hazard ratios and 95% confidence intervals. RESULTS: We identified 2,269 CKD cases over a median follow-up period of 13.1 years via linkage with the electronic health records. After adjusting for covariates and correcting for multiple testing, 90 of 142 biomarkers were significantly associated with incident CKD. In general, higher concentrations of very-low-density lipoprotein (VLDL) particles were associated with a higher risk of CKD whereas higher concentrations of high-density lipoprotein (HDL) particles were associated with a lower risk of CKD. Higher concentrations of cholesterol, phospholipids, and total lipids within VLDL were associated with a higher risk of CKD, whereas within HDL they were associated with a lower risk of CKD. Further, higher triglyceride levels within all lipoprotein subclasses, including all HDL particles, were associated with greater risk of CKD. We also identified that several amino acids, fatty acids, and inflammatory biomarkers were associated with risk of CKD. LIMITATIONS: Potential underreporting of CKD cases because of case identification via electronic health records. CONCLUSIONS: Our findings highlight multiple known and novel pathways linking circulating metabolites to the risk of CKD. PLAIN-LANGUAGE SUMMARY: The relationship between individual lipoprotein particle subclasses and lipid-related traits and risk of chronic kidney disease (CKD) in general population is unclear. Using data from 91,532 participants in the UK Biobank, we evaluated the associations of metabolites measured using nuclear magnetic resonance testing with the risk of CKD. We identified that 90 out of 142 lipid biomarkers were significantly associated with incident CKD. We found that very-low-density lipoproteins, high-density lipoproteins, the lipid concentration and composition within these lipoproteins, triglycerides within all the lipoprotein subclasses, fatty acids, amino acids, and inflammation biomarkers were associated with CKD risk. These findings advance our knowledge about mechanistic pathways that may contribute to the development of CKD.

Pal power: Demonstration of the functional association of the Helicobacter pylori flagellar motor with peptidoglycan-associated lipoprotein (Pal) and its preliminary crystallographic analysis.

The bacterial flagellar motor is a molecular nanomachine, the assembly and regulation of which requires many accessory proteins. Their identity, structure and function are often discovered through characterisation of mutants with impaired motility. Here, we demonstrate the functional association of the Helicobacter pylori peptidoglycan-associated lipoprotein (HpPal) with the flagellar motor by analysing the motility phenotype of the ∆pal mutant, and present the results of the preliminary X-ray crystallographic analysis of its globular C-terminal domain HpPal-C. Purified HpPal-C behaved as a dimer in solution. Crystals of HpPal-C were grown by the hanging drop vapour diffusion method using medium molecular weight polyethylene glycol (PEG) Smear as the precipitating agent. The crystals belong to the primitive orthorhombic space group P1 with unit cell parameters a = 50.7, b = 63.0, c = 75.1 Å. X-ray diffraction data were collected to 1.8 Å resolution on the Australian Synchrotron beamline MX2. Calculation of the Matthews coefficient (VM=2.24 Å3/Da) and molecular replacement showed that the asymmetric unit contains two protein subunits. This study is an important step towards elucidation of the non-canonical role of H. pylori Pal in the regulation, or function of, the flagellar motor.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Serum lipoprotein subfractions are associated with the periodontal status: Results from the population-based cohort SHIP-TREND.

AIM: To investigate the medium-term associations of serum protein subfractions derived from proton nuclear magnetic resonance (1 H-NMR) spectroscopy with periodontitis and tooth loss. MATERIALS AND METHODS: A total of 3031 participants of the cohort Study of Health in Pomerania (SHIP-TREND) were included. In addition to conventional serum testing, serum lipoprotein contents and subfractions were analysed by 1 H-NMR spectroscopy. Confounder-adjusted associations of lipoprotein variables with periodontitis and the number of missing teeth variables were analysed using mixed-effects models with random intercepts for time across individuals, accounting for multiple testing. RESULTS: While only spurious associations between lipoprotein levels from conventional blood tests were found-that is, triglycerides were associated with mean clinical attachment level (CAL) and low-density lipoprotein cholesterol/high-density lipoprotein cholesterol (LDL-C/HDL-C) ratio with the number of missing teeth - several associations emerged from serum lipoprotein subfractions derived from 1 H-NMR analysis. Specifically, elevated LDL triglycerides were associated with higher levels of mean probing depth (PD), mean CALs, and increased odds of having <20 teeth. HDL-4 cholesterol levels were inversely associated with mean PD. Systemic inflammation (C-reactive protein) might mediate the effects of LDL and HDL triglyceride contents on periodontitis severity. CONCLUSIONS: Several associations between serum lipoprotein subfractions and periodontitis were observed. As the underlying biochemical mechanisms remain unclear, further research is needed.

Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.

Identification of a novel transport system in Borrelia burgdorferi that links the inner and outer membranes.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a diderm organism that is similar to Gram-negative organisms in that it contains both an inner and outer membrane. Unlike typical Gram-negative organisms, however, B. burgdorferi lacks lipopolysaccharide (LPS). Using computational genome analyses and structural modeling, we identified a transport system containing six proteins in B. burgdorferi that are all orthologs to proteins found in the lipopolysaccharide transport (LPT) system that links the inner and outer membranes of Gram-negative organisms and is responsible for placing LPS on the surface of these organisms. While B. burgdorferi does not contain LPS, it does encode over 100 different surface-exposed lipoproteins and several major glycolipids, which like LPS are also highly amphiphilic molecules, though no system to transport these molecules to the borrelial surface is known. Accordingly, experiments supplemented by molecular modeling were undertaken to determine whether the orthologous LPT system identified in B. burgdorferi could transport lipoproteins and/or glycolipids to the borrelial outer membrane. Our combined observations strongly suggest that the LPT transport system does not transport lipoproteins to the surface. Molecular dynamic modeling, however, suggests that the borrelial LPT system could transport borrelial glycolipids to the outer membrane.

In situ visualization of Braun's lipoprotein on E. coli sacculi.

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli, as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.

Transcription factor DegU-mediated multi-pathway regulation on lichenysin biosynthesis in Bacillus licheniformis.

Lichenysin, producted by Bacillus licheniformis, is an important cyclic lipopeptide biosurfactant, which has potential applications in oil exploitation, drug development, biological control of agriculture and bioremediation. While studies are lacking on metabolism regulation of lichenysin biosynthesis, which limits metabolic engineering and large-scale production of lichenysin. In this study, the yield of lichenysin was improved obviously by 13.6 folds to 2.18 ± 0.03 g/L in degU deletion strain (WX02△degU) compared with the wild-type strain (WX02) and completely inhibited in degU overexpressed strain (WX02/pHY-degU). We further proved that DegU, a transcription factor plays a significant role in multicellular behavior, is a key negative regulator of lichenysin synthesis lchA operon. But interestingly, lichenysin yield was still inhibited by overexpressing DegU in the promoter-substituted strain (WX02-PP43lch), in which promoter of lchA operon cannot be controlled by DegU. Thus, through 13C-metabolic flux analysis, we found that deletion of degU also enhanced glucose uptake, branched chain amino acid synthesis, and fatty acid synthesis, while decrease acetoin synthesis, which is beneficial for the supply of lichenysin precursors. Further experiments demonstrate that DegU regulates these pathways by binding to the promoter regions of related genes. Overall, we systematically investigated the multi-pathway regulation network mediated by DegU on lichenysin biosynthesis, which not only contributes to the further metabolic engineering for lichenysin high-production, but sheds light on studies of transcription factor regulation.

Identification and transcriptional profile of Culex quinquefasciatus Say (Diptera: Culicidae) lipophorin in different stages.

Lipophorin is a major hemolymph lipoprotein found in insects with a molecular native mass of 700 kDa. In mosquitoes, two different types of apolipoproteins are characterized, apolipophorin-I (ApoLp-I, ~250 kDa) and apolipophorin-II (ApoLp-II, ~80 kDa). This concentration depends on the stage of development and the age of the insects. Lipophorins are best studied in mosquitoes of the genus Aedes and Anopheles. In this study, we analyze the lipophorin sequence and show the lipophorin purification of the Culex quinquefasciatus and the transcriptional profile of the lipophorin gene in different life cycle stages. Similar amino acid composition and molecular weights are founded in three mosquitoes species lipophorins amino acid sequence. The two subunits of purified lipophorin (Apo I and Apo II) showed molecular masses of approximately 248 and 93 kDa, like that found in other mosquitoes. A gradual increase in the lipophorin expression gene was obtained during the previtellogenic period and after feeding we obtained peak expression at 24 h after feeding. With our results, we conclude that C. quinquefasciatus protein sequence has the same characteristics as those observed in other mosquitoes and that the expression of its apolipophorins is induced by blood feeding.

The Association of Lipids and Lipoproteins with Hip Fracture Risk: The Cardiovascular Health Study.

BACKGROUND: It is uncertain if lipids or lipoproteins are associated with osteoporotic fractures. In this study, incident hip fracture risk according to conventional lipid levels and lipoprotein levels and sizes was examined. METHODS: We followed 5832 participants aged ≥65 years from the Cardiovascular Health Study for hip fracture for a mean of 13.5 (SD 5.7) years. Standard enzymatic methods were used to determine lipid levels (ie, high-density lipoprotein-cholesterol [HDL-c], low-density lipoprotein-cholesterol [LDL-c], and triglycerides). Nuclear magnetic resonance spectroscopy was used to measure lipoprotein fractions (ie, very-low-density lipoprotein-particle [VLDL-P], low-density lipoprotein-particle [LDL-P], high-density lipoprotein-particle [HDL-P]) in a subset of 1849 participants. RESULTS: We documented 755 incident hip fractures among women (1.19 fractures per 100 participant years [95% confidence interval, 1.04, 1.35]) and 197 among men (0.67 fractures per 100 participant years [95% CI, 0.41, 1.10]) over an average follow-up. HDL-c and LDL-c levels had statistically significant nonlinear U-shaped relationships with hip fracture risk (HDL-c, P = .009; LDL-c, P = .02). Triglyceride levels were not significantly associated with hip fracture risk. In fully adjusted conjoint models, higher VLDL-P concentration (hazard ratio [HR] per 1 standard deviation [SD] increment 1.47 [1.13, 1.91] and size [HR per 1 SD increment 1.24 [1.05, 1.46]) and higher high-density lipoprotein particle size (HR per 1 SD increment 1.81 [1.25, 2.62]) were all associated with higher hip fracture risk. CONCLUSIONS: Lipids and lipoproteins are associated with hip fracture risk in older adults. The associations are complex. Mechanistic studies are needed to understand these findings.

[Lipoprotein apheresis in patients with familial hypercholesterolemia: a single center research].

Objective: We evaluated the safety and efficacy of lipoprotein apheresis (LA) in patients with familial hypercholesterolemia (FH) who can't reach low-density lipoprotein cholesterol(LDL-C) target goals with the maximal tolerated dose of lipid-lowering agents. Methods: This was a retrospective cross-sectional study. Between February 2015 and November 2019, patients with FH who were admitted in Fuwai hospital and treated with LA were consecutively enrolled. Based on intensive lipid-lowering agents, these patients received LA by double filtration plasma pheresis (DFPP) method. The changes of lipid levels such as LDL-C and lipoprotein(a)[Lp(a)] were compared before and after LA treatment, and the changes of immunoglobulin (Ig) concentration and LA-related adverse effects were also discussed. Results: A total of 115 patients with FH were enrolled in this study, of which 8 cases were homozygous FH and 107 cases were heterozygous FH. The age was (43.9±12.2) years and there were 75 (65.2%) males, and 108 (93.8%) with coronary artery disease. For pre-and immediately after LA treatment, the LDL-C was (5.20±2.94) mmol/L vs. (1.83±1.08) mmol/L, Lp(a) concentration was 428.70(177.00, 829.50)mg/L vs. 148.90(75.90, 317.00) mg/L (P<0.001), with a decrease of 64.2% and 59.8% respectively. The levels of IgG and IgA measured 1 day after LA treatment were both in the normal range and IgM concentration was below the reference value, the reductions of which were 15.1%, 25.0% and 58.7% respectively (P<0.001). Six patients had mild symptoms of nausea, hypotension dyspnea and palpitation, the symptoms were relieved by symptomatic treatment. Conclusion: For patients with FH who do not achieve LDL-C target goal with the maximal tolerated lipid-lowering agents, especially those with elevated Lp(a) levels, LA, which can significantly further reduce LDL-C and Lp(a) levels, is an effective and safe option.

Evolutionary origin and sequence signatures of the heterodimeric ABCG5/ABCG8 transporter.

ATP-binding cassette (ABC) systems, characterized by ABC-type nucleotide-binding domains (NBDs), play crucial roles in various aspects of human physiology. Human ABCG5 and ABCG8 form a heterodimeric transporter that functions in the efflux of sterols. We used sequence similarity search, multiple sequence alignment, phylogenetic analysis, and structure comparison to study the evolutionary origin and sequence signatures of ABCG5 and ABCG8. Orthologs of ABCG5 and ABCG8, supported by phylogenetic analysis and signature residues, were identified in bilaterian animals, Filasterea, Fungi, and Amoebozoa. Such a phylogenetic distribution suggests that ABCG5 and ABCG8 could have originated in the last common ancestor of Amorphea (the unikonts), the eukaryotic group including Amoebozoa and Opisthokonta. ABCG5 and ABCG8 were missing in genomes of various lineages such as snakes, jawless vertebrates, non-vertebrate chordates, echinoderms, and basal metazoan groups. Amino-acid changes in key positions in ABCG8 Walker A motif and/or ABCG5 C-loop were observed in most tetrapod organisms, likely resulted in the loss of ATPase activity at one nucleotide-binding site. ABCG5 and ABCG8 in Ecdysozoa (such as insects) exhibit elevated evolutionary rates and accumulate various changes in their NBD functional motifs. Alignment inspection revealed several residue positions that show different amino-acid usages in ABCG5/ABCG8 compared to other ABCG subfamily proteins. These residues were mapped to the structural cores of transmembrane domains (TMDs), the NBD-TMD interface, and the interface between TMDs. They serve as sequence signatures to differentiate ABCG5/ABCG8 from other ABCG subfamily proteins, and some of them may contribute to substrate specificity of the ABCG5/ABCG8 transporter.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

The viability of Leptospira is related to physicochemical properties of the surface water surrounding an agricultural area and HemO and LipL32 gene expression in response to iron in water.

BACKGROUND: The pathogenic Leptospira can survive and contaminate surface water based on physicochemical factors. This study aimed to determine how the physicochemical properties of water sources influence the growth and effect of iron on the gene expression of Leptospira spp. P47. METHODS: Surface water samples (n=55) were collected and used for Leptospira spp. P47 cultivation. Physicochemical factors, including iron, calcium, magnesium and pH, were analyzed. The association between Leptospira spp. P47 viability at days 5, 10 and 15 with the physicochemical factors were analyzed. In addition, this bacterium was cultured in six selected water samples. The effect of iron in water on HemO and LipL32 gene expression was determined by relative quantification real-time PCR. RESULTS: Leptospira viability at day 5 was not significantly correlated with physicochemical factors, while Leptospira viability at day 10 was associated with both pH and iron. The Leptospira viability rate at day 15 had a significantly positive association with pH and iron and a negative association with calcium. HemO expression was significantly increased, mostly in selected water samples and under iron-depleted conditions. Conversely, LipL32 expression was significantly decreased in all water samples. CONCLUSIONS: Physicochemical factors in natural surface waters are key factors for bacterial survival in the environment, which may increase the chance of Leptospira infection in humans.

Transport Across Two Membrane Bilayers in E. coli by Efflux Pumps of Different Dimensions.

AcrAB-TolC and CusBAC are two of the most well-studied Resistance-Nodulation-Division (RND) family tripartite efflux pumps in E. coli. AcrAB-TolC is a multidrug efflux system, while CusBAC transports Cu(I), Cu(II) and Ag(I). The RND pump complexes span both the inner membrane (IM) and the outer membrane (OM). The long axis dimension of the fully assembled AcrAB-TolC is ∼3 nm longer than that of CusBAC. To probe if these two efflux systems with different dimensions affect each other when they need to work simultaneously in the same cell, two real-time assays were used to monitor the efflux activities of these two pumps and their impact on each other. The results showed that the presence of AcrAB-TolC substrates accelerated the accumulation of Cu(I) in BW25113 but not in BW25113ΔcusBA or BW25113ΔtolC strains. Similarly, the presence of Ag(I) slowed down the Nile red efflux in the parent strain more significantly than in the CusBA deficient mutant. To further investigate the impact of the OM/IM distance on the function of these tripartite complexes, we experimented with strains lacking the lipoprotein Lpp or containing Lpp mutant of different lengths. Data from efflux/accumulation assays and susceptibility tests revealed that mutation of Lpp resulted in functional deficiency of both AcrAB-TolC and CusBAC. In conclusion, this study demonstrated that when AcrAB-TolC and CusBAC functioned simultaneously, it took the cell a few minutes to adjust. Furthermore, the presence of Lpp of proper length is important to support full efflux activity of transporters spanning both membrane layers in E. coli.

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution.

The tripartite AcrAB-TolC assembly, which spans both the inner and outer membranes in Gram-negative bacteria, is an efflux pump that contributes to multidrug resistance. Here, we present the in situ structure of full-length Escherichia coli AcrAB-TolC determined at 7 Å resolution by electron cryo-tomography. The TolC channel penetrates the outer membrane bilayer through to the outer leaflet and exhibits two different configurations that differ by a 60° rotation relative to the AcrB position in the pump assembly. AcrA protomers interact directly with the inner membrane and with AcrB via an interface located in proximity to the AcrB ligand-binding pocket. Our structural analysis suggests that these AcrA-bridged interactions underlie an allosteric mechanism for transmitting drug-evoked signals from AcrB to the TolC channel within the pump. Our study demonstrates the power of in situ electron cryo-tomography, which permits critical insights into the function of bacterial efflux pumps.