Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.

A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype. That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion. These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.     

Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA

SdiA is an Escherichia coli protein that regulates cell division in a cell density-dependent, or quorum-sensing, manner. We report that SdiA also controls multidrug resistance by positively regulating the multidrug resistance pump AcrAB. Overproduction of SdiA confers multidrug resistance and increased levels of AcrAB. Conversely, sdiA null mutants are hypersensitive to drugs and have decreased levels of AcrB protein. Our findings provide a link between quorum sensing and multidrug efflux. Combined with previously published reports, our data support a model in which a role of drug efflux pumps is to mediate cell-cell communication in response to cell density. Xenobiotics expelled by pumps may resemble the communication molecules that they normally efflux.     

Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein

AcrAB of Escherichia coli, an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low-molecular-weight inducers specifically bind to the C-terminal, non-DNA-binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre-existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407-1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli, were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob-mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.     

Isolation and characterization of VceC gain-of-function mutants that can function with the AcrAB multiple-drug-resistant efflux pump of Escherichia coli

VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.     

Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes

Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.     

Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae

In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB(HI)). Similar to other RND transporters AcrB(HI) associates with AcrA(HI), the periplasmic membrane fusion protein, and the outer membrane channel TolC(HI). Here, we report that AcrAB(HI) confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC(EC)) protein. To investigate the intracellular dynamics of AcrAB(HI), single cysteine mutations were constructed in AcrB(HI) in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB(HI) and in the presence or absence of AcrA(HI) and TolC(EC). We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB(HI).     

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.     

Active efflux,a common mechanism for biocide and antibiotic resistance

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.     

RobA-lnduced Multiple Antibiotic Resistance Largely Depends on the Activation of the AcrAB Efflux

RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a ΔompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1^? mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.     

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.     

Active efflux of bile salts by Escherichia coli.

Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming delta pH without affecting delta psi. The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with Vmax and Km values in the neighborhood of 0.3 nmol min(-1) mg of protein(-1) and 50 microM, respectively.     

Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics

In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.     

marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.     

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.