Toxin import through the antibiotic efflux channel TolC.
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ABSTRACT:
SUBMITTER: Housden NG
PROVIDER: S-EPMC8324772 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug-resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria (Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, et al. 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 61(10); Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. 2018. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018(1):60-66; Petrovic Fabijan A, Lin RCY, Ho J, Maddocks S, Ben Zakour NL, Iredell JR, Westmead Bacteriophage Therapy Team. 2020. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat Microbiol. 5(3):465-472). One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS phage (Pagie L, Hogeweg P. 1999. Colicin diversity: a result of eco-evolutionary dynamics. J Theor Biol. 196(2):251-261; Andersen C, Hughes C, Koronakis V. 2000. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep. 1(4):313-318; Koronakis V, Andersen C, Hughes C. 2001. Channel-tunnels. Curr Opin Struct Biol. 11(4):403-407; Czaran TL, Hoekstra RF, Pagie L. 2002. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. 99(2):786-790; Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev. 71(1):158-229). By systematically assessing the distribution of fitness effects of ∼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.
| S-EPMC8476145 | biostudies-literature
Project description:The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.
| S-EPMC5366289 | biostudies-literature
Project description:There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections.
| S-EPMC9781404 | biostudies-literature
Project description:Bacterial resistance to biocides used as antiseptics, dyes, and disinfectants is a growing concern in food preparation, agricultural, consumer manufacturing, and health care industries, particularly among Gram-negative Enterobacteriaceae, some of the most common community and healthcare-acquired bacterial pathogens. Biocide resistance is frequently associated with antimicrobial cross-resistance leading to reduced activity and efficacy of both antimicrobials and antiseptics. Multidrug resistant efflux pumps represent an important biocide resistance mechanism in Enterobacteriaceae. An assortment of structurally diverse efflux pumps frequently co-exist in these species and confer both unique and overlapping biocide and antimicrobial selectivity. TolC-dependent multicomponent systems that span both the plasma and outer membranes have been shown to confer clinically significant resistance to most antimicrobials including many biocides, however, a growing number of single component TolC-independent multidrug resistant efflux pumps are specifically associated with biocide resistance: small multidrug resistance (SMR), major facilitator superfamily (MFS), multidrug and toxin extruder (MATE), cation diffusion facilitator (CDF), and proteobacterial antimicrobial compound efflux (PACE) families. These efflux systems are a growing concern as they are rapidly spread between members of Enterobacteriaceae on conjugative plasmids and mobile genetic elements, emphasizing their importance to antimicrobial resistance. In this review, we will summarize the known biocide substrates of these efflux pumps, compare their structural relatedness, Enterobacteriaceae distribution, and significance. Knowledge gaps will be highlighted in an effort to unravel the role that these apparent "lone wolves" of the efflux-mediated resistome may offer.
| S-EPMC5840245 | biostudies-literature
Project description:The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.
| S-EPMC4361902 | biostudies-literature
Project description:Two TolC homologs, PM0527 and PM1980, were identified for Pasteurella multocida. A pm0527 mutant displayed increased susceptibility to a range of chemicals, including rifampin (512-fold) and acridine orange (128-fold). A pm1980 mutant showed increased susceptibility to rifampin, ceftazidime, and vancomycin.
| S-EPMC2573115 | biostudies-literature
Project description:In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.
| S-EPMC2752513 | biostudies-literature
Project description:Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity/plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na+/Cl- -dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.
| S-EPMC549289 | biostudies-literature
Project description:Efflux-mediated β-lactam resistance is a major public health concern, reducing the effectiveness of β-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines β-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under β-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.
| S-EPMC11347637 | biostudies-literature
Project description:In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.
| S-EPMC1068630 | biostudies-literature