Outer Membrane Protein TolC Research Articles

Discovery and Characterization of Two Folded Intermediates for Outer Membrane Protein TolC Biogenesis

TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.

Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.

Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system.

The ABC transporter hemolysin B (HlyB) is the key protein of the HlyA secretion system, a paradigm of type 1 secretion systems (T1SS). T1SS catalyze the one-step substrate transport across both membranes of Gram-negative bacteria. The HlyA T1SS is composed of the ABC transporter (HlyB), the membrane fusion protein (HlyD), and the outer membrane protein TolC. HlyA is a member of the RTX (repeats in toxins) family harboring GG repeats that bind Ca2+ in the C-terminus upstream of the secretion signal. Beside the GG repeats, the presence of an amphipathic helix (AH) in the C-terminus of HlyA is essential for secretion. Here, we propose that a consensus length between the GG repeats and the AH affects the secretion efficiency of the heterologous RTX secreted by the HlyA T1SS. Our in silico studies along with mutagenesis and biochemical analysis demonstrate that there are two binding pockets in the nucleotide binding domain of HlyB for HlyA. The distances between the domains of HlyB implied to interact with HlyA indicated that simultaneous binding of the substrate to both cytosolic domains of HlyB, the NBD and CLD, is possible and required for efficient substrate secretion.

Identity Determinants of the Translocation Signal for a Type 1 Secretion System.

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process.

Colonization of gut microbiota by plasmid-carrying bacteria is facilitated by evolutionary adaptation to antibiotic treatment

Multidrug-resistant plasmid-carrying bacteria are of particular clinical concern as they could transfer antibiotic resistance genes to other bacterial species. However, little is known whether evolutionary adaptation of plasmid-carrying bacteria after long-term antibiotic exposure could affect their subsequent colonization of the human gut. Herein, we combined a long-term evolutionary model based on Escherichia coli K-12 MG1655 and the multidrug-resistant plasmid RP4 with in vivo colonization experiments in mice. We found that the evolutionary adaptation of plasmid-carrying bacteria to antibiotic exposure facilitated colonization of the murine gut and subsequent plasmid transfer to gut bacteria. The evolved plasmid-carrying bacteria exhibited phenotypic alterations, including multidrug resistance, enhanced bacterial growth and biofilm formation capability and decreased plasmid fitness cost, which might be jointly caused by chromosomal mutations (SNPs in rpoC, proQ, and hcaT) and transcriptional modifications. The upregulated transcriptional genes, e.g., type 1 fimbrial-protein pilus (fimA and fimH) and the surface adhesin gene (flu) were likely responsible for the enhanced biofilm-forming capacity. The gene tnaA that encodes a tryptophanase-catalyzing indole formation was transcriptionally upregulated, and increased indole products participated in facilitating the maximum population density of the evolved strains. Furthermore, several chromosomal genes encoding efflux pumps (acriflavine resistance proteins A and B (acrA, acrB), outer-membrane protein (tolC), multidrug-resistance protein (mdtM), and macrolide export proteins A and B (macA, macB)) were transcriptionally upregulated, while most plasmid-harboring genes (conjugal transfer protein (traF) and (trbB), replication protein gene (trfA), beta-lactamase TEM precursor (blaTEM), aminoglycoside 3'-phosphotransferase (aphA) and tetracycline resistance protein A (tetA)) were downregulated. Collectively, these findings demonstrated that evolutionary adaptation of plasmid-carrying bacteria in an antibiotic-influenced environment facilitated colonization of the murine gut by the bacteria and plasmids.

Structural characterization of the EmrAB-TolC efflux complex from E. coli

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB–TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.

Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058

Extraintestinal pathogenic Escherichia coli (ExPEC) shows an enhanced ability to cause infection outside the intestinal tract. Avian pathogenic E. coli (APEC), one type of ExPEC, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide that is characterized by systemic infection. Some ExPEC strains as well as other pathogenic enterobacteria produce enterobactin, a catecholate siderophore used to sequester iron during infection. Here, we showed that disruption of enterobactin efflux via outer membrane protein TolC significantly decreased the pathogenicity of APEC strain E058. Furthermore, colonization and persistence assays performed using a chicken infection model showed that the ΔtolC mutant was obviously attenuated (p˂0.001). In contrast, disruption of enterobactin synthesis gene entE and/or the inner membrane transporter gene entS had little effect on pathogenicity. Analysis of growth kinetics revealed a significant reduction in the growth of triple mutant strain E058ΔentEΔentSΔtolC in iron-deficient medium compared with the wild-type strain (p˂0.001), while no growth impairment was noted for the E058ΔtolC mutant in either Luria-Bertani broth or iron-deficient medium. The E058ΔentEΔentSΔtolC mutant also showed significantly decreased virulence compared with single mutant strain E058ΔtolC. Low-copy complementation of strains E058ΔtolC and E058ΔentEΔentSΔtolC with plasmid-borne tolC restored virulence to wild-type levels in the chicken infection model. Macrophage infection assays showed that ingestion of E058ΔtolC by macrophage cell line HD11 cells was reduced compared with ingestion of the E058ΔentEΔentSΔtolC mutant. However, no significant differences were observed between the mutants and the wild-type in a chicken serum resistance assay. Together, these results suggest that EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058 in an iron-deficient environment.

Contributions of TolC Orthologs to Francisella tularensis Schu S4 Multidrug Resistance, Modulation of Host Cell Responses, and Virulence.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia. TolC is an integral part of efflux pumps that export small molecules and type I secretion systems that export a range of bacterial virulence factors. In this study, we analyzed TolC and its two orthologs, FtlC and SilC, present in the fully virulent F. tularensis Schu S4 strain for their contributions to multidrug efflux, suppression of innate immune responses, and virulence. We found that each TolC ortholog participated in multidrug efflux, with overlapping substrate specificities for TolC and FtlC and a distinct substrate profile for SilC. In contrast to their shared roles in drug efflux, only TolC functioned in the modulation of macrophage apoptotic and proinflammatory responses to Schu S4 infection, consistent with a role in virulence factor delivery to host cells. In agreement with previous results with the LVS, the Schu S4 ΔtolC mutant was highly attenuated for virulence in mice by both the intranasal and intradermal routes of infection. Unexpectedly, FtlC was also critical for Schu S4 virulence, but only by the intradermal route. Our data demonstrate a conserved and critical role for TolC in modulation of host immune responses and Francisella virulence and also highlight strain- and route-dependent differences in the pathogenesis of tularemia.

Identification of outer membrane protein TolC as the major adhesin and potential vaccine candidate for Vibrio harveyi in hybrid grouper, Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂)

Vibrio harveyi is a serious pathogen of scale drop and muscle necrosis disease in marine commercial fishes. Adhesion to and colonization of the host cells surfaces is the first and crucial step for pathogenic bacterial infection, which is usually mediated by outer membrane proteins (Omps). The objectives of this study were to identify the major adhesin in Omps that plays the essential role in adhesion of V. harveyi to the host cells, and to assess the potential of this adhesin as a vaccine candidate for V. harveyi infection. We observed that pathogenic V. harveyi adhered to the surface of grouper embryonic cells (GEM cells) and induced apoptosis of them. Native Omps were extracted from nine different V. harveyi strains, and five common Omp bands were isolated by SDS-PAGE analysis. Western blot analysis and an anti-native Omp antibodies blocking assay indicated that one strong and several weak immunoreactivity Omps bands presence. Next, a total of five Omps, including TolC, Agg (Agglutination protein), Omp47, Fla (Flagellin), and OmpW, were identified and their encoding genes were cloned, characterized, and expressed in E. coli. The purified recombinant TolC could competitively inhibit the invasion of V. harveyi to GEM cells in vitro, and anti-TolC antibody also could significantly block the adhesion of V. harveyi to GEM cells. When used to immunize hybrid groupers, the recombinant TolC could confer significant protection to fish against experimental V. harveyi challenge. These data suggested that outer membrane protein TolC functions as a major adhesin in V. harveyi and could be a potential vaccine candidate for V. harveyi infection.

The ABC Transporter Components HgdB and HgdC are Important for Glycolipid Layer Composition and Function of Heterocysts in Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a filamentous cyanobacterium able to fix atmospheric nitrogen in semi-regularly spaced heterocysts. For correct heterocyst function, a special cell envelope consisting of a glycolipid layer and a polysaccharide layer is essential. We investigated the role of the genes hgdB and hgdC, encoding domains of a putative ABC transporter, in heterocyst maturation. We investigated the subcellular localization of the fusion protein HgdC-GFP and followed the differential expression of the hgdB and hgdC genes during heterocyst maturation. Using a single recombination approach, we created a mutant in hgdB gene and studied its phenotype by microscopy and analytical chromatography. Although heterocysts are formed in the mutant, the structure of the glycolipid layer is aberrant and also contains an atypical ratio of the two major glycolipids. As shown by a pull-down assay, HgdB interacts with the outer membrane protein TolC, which indicates a function as a type 1 secretion system. We show that the hgdB-hgdC genes are essential for the creation of micro-oxic conditions by influencing the correct composition of the glycolipid layer for heterocyst function. Our observations confirm the significance of the hgdB-hgdC gene cluster and shed light on a novel mode of regulation of heterocyst envelope formation.

The outer-membrane protein TolC of Vibrio cholerae serves as a second cell-surface receptor for the VP3 phage

Receptor recognition is a key step in the initiation of phage infection. Previously, we found that VP3, the T7 family phage of the Vibrio cholerae serogroup O1 biotype El Tor, can adsorb the core oligosaccharide (OS) of lipopolysaccharides of V. cholerae However, some wildtype strains of V. cholerae possessing the intact OS gene cluster still have VP3 binding but are resistant to VP3 infection. Moreover, an OS gene-deletion mutant still exhibits weak VP3 binding, suggesting multiple factors are possibly involved in VP3 binding to V. cholerae Here, we report that the outer-membrane protein TolC of V. cholerae is involved in the host adsorption of VP3. We observed that TolC directly interacts with the VP3 tail fiber protein gp44 and its C-terminal domains, and we also found that three amino acid residues in the outside loops of TolC, at positions 78, 290, and 291, are critical for binding to gp44. Among the VP3-resistant wildtype V. cholerae strains, frequent amino acid residue mutations were observed in the loops around the sites 78, 290, and 291, which were predicted to be exposed to the cell surface. These findings reveal a co-receptor-binding mechanism for VP3 infection of V. cholerae and that both outer-membrane TolC and OS are necessary for successful VP3 infection of V. cholerae We conclude that mutations on the outside loops of the receptor may confer V. cholerae strains with VP3 phage resistance, enabling these strains to survive in environments containing VP3 or related phages.

Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt

BackgroundThe tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. MethodsWe utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. ResultsTolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. ConclusionsTolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. General significanceTolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.

TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis.

Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility in Klebsiella pneumoniae.

In Klebsiella pneumoniae, overproduction of RamA and RarA leads to increased MICs of various antibiotics; MarA and SoxS are predicted to perform a similar function. We have compared the relative effects of overproducing these four AraC-type regulators on envelope permeability (a combination of outer membrane permeability and efflux), efflux pump and porin production, and antibiotic susceptibility in K. pneumoniae. Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR. Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific. RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates.

Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc.

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.

Virulence genes in Moraxella spp. isolates from infectious bovine keratoconjunctivitis cases.

Infectious bovine keratoconjunctivitis (IBK) is an important ocular disease which affects cattle worldwide. To advance towards IBK effective prevention and treatment strategies, it is important to define the distribution and genetic diversity of potential virulence factors present in M. bovis and M. bovoculi. The objective of this work was to identify and to analyze Moraxella spp. potential virulence factor genes in a collection of clinical isolates. The presence and diversity of virulence factors in a collection of Moraxella spp. strains isolated since 1983 to 2009 in Uruguay was analyzed by PCR using primers for partial amplification of tolC, omp79, plb, fur and mbxA. The selection criterion of these genes was based on the fact that they encode virulence factors which could be present and conserved within strains, an important issue for the development of vaccines. Differences in PCR amplification were observed within tolC (84%), omp79 (80%), plb (76%) and fur (44%) in M. bovis strains, whereas mbxA was amplified in all M. bovis and M. bovoculi strains. Regarding genetic diversity, the tolC nucleotide sequences were the less diverse within all M. bovis and mbxA were the less diverse within all M. bovis and M. bovoculi strains. PCR amplifications suggest the occurrence of differences between both Moraxella species, related to evaluated genes within Moraxella spp. strains and suggests that both species may have different pathogenic attributes. MbxA and the outer membrane protein TolC might be considered for future studies to develop new vaccines against IBK.

Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations

Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt-bridge R367-D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate.

Free energy profiles of ion permeation and doxorubicin translocation in TolC

The outer membrane protein TolC of Escherichia coli forms a channel-tunnel pore spanning the periplasmic space and outer membrane, serving as the main exit duct for bacteria multidrug resistance and protein export. Many aspects of the transport mechanism of TolC are still unclear. Here, we have investigated the substrate permeability and gating mechanism of TolC by calculating the potential of mean forces (PMFs) for transporting sodium ion and doxorubicin through TolC using the adaptive biasing force (ABF) method. The transport mechanism is turned out to be substrate dependent. It is found that the periplasmic gate is required to open for the passage of both Na+and doxorubicin, but the conformational gating does not lead to permeation barrier for Na+at this region. The extracellular loops and K283 residues cause permeation barriers for Na+at the extracellular entrance, but not for doxorubicin due to the extensive interactions between the drug molecule and the protein. TolC exhibits high conformational flexibility during the transport of Na+, while doxorubicin seems to be able to stabilize TolC in the resting state with the periplasmic gate closed. The association of the TolC docking domain of AcrB does not lower the permeation barrier for doxorubicin at the periplasmic gate, while the gate opening induces the dissociation of the TolC–AcrB complex.