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Characterization of Pseudomonas aeruginosa Enoyl-Acyl Carrier Protein Reductase (FabI): a Target for the Antimicrobial Triclosan and Its Role in Acylated Homoserine Lactone Synthesis

Published on Sep 1, 1999in Journal of Bacteriology 3.23
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
Abstract
The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-l-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-l-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
  • References (45)
  • Citations (159)
References45
Newest
Published on Sep 1, 1999in Journal of Bacteriology 3.23
Alecksandr J. Kutchma2
Estimated H-index: 2
(CSU: Colorado State University),
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally c...
Published on Apr 16, 1999in Journal of Biological Chemistry
Richard J. Heath34
Estimated H-index: 34
(St. Jude Children's Research Hospital),
J. Ronald Rubin3
Estimated H-index: 3
(St. Jude Children's Research Hospital)
+ 3 AuthorsCharles O. Rock26
Estimated H-index: 26
(St. Jude Children's Research Hospital)
Abstract Triclosan is a broad-spectrum antibacterial agent that inhibits bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. Resistance to triclosan inEscherichia coli is acquired through a missense mutation in the fabI gene that leads to the expression of FabI[G93V]. The specific activity and substrate affinities of FabI[G93V] are similar to FabI. Two different binding assays establish that triclosan dramatically increases the affinity of FabI for NAD+. In co...
Published on Apr 1, 1999in Nature 43.07
Colin Levy25
Estimated H-index: 25
(University of Sheffield),
Anna Roujeinikova18
Estimated H-index: 18
(University of Sheffield)
+ 5 AuthorsJohn B. Rafferty27
Estimated H-index: 27
(University of Sheffield)
Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) has been used for more than 30 years as a general antibacterial and antifungal agent, and is found in formulations as diverse as toothpastes, cosmetics, antiseptic soaps, carpets, plastic kitchenware and toys. It has recently been suggested that triclosan blocks lipid biosynthesis by specifically inhibiting the enzyme enoyl-acyl carrier protein reductase (ENR)1. We have carried out a structural analysis and inhibition experiments on a complex o...
Published on Mar 1, 1999in Antimicrobial Agents and Chemotherapy 4.71
Laura M. McMurry24
Estimated H-index: 24
,
Patrick F. McDermott52
Estimated H-index: 52
,
Stuart B. Levy77
Estimated H-index: 77
(Tufts University)
Three Mycobacterium smegmatis mutants selected for resistance to triclosan each had a different mutation in InhA, an enoyl reductase involved in fatty acid synthesis. Two expressed some isoniazid resistance. A mutation originally selected on isoniazid also mediated triclosan resistance, as did the wild-type inhA gene on a multicopy plasmid. Replacement of the mutant chromosomal inhA genes with wild-type inhA eliminated resistance. These results suggest that M. smegmatis InhA, like its Escherichi...
Published on Mar 1, 1999in Journal of Bacteriology 3.23
Alex Glessner1
Estimated H-index: 1
(UD: University of Dayton),
Roger S. Smith8
Estimated H-index: 8
(UR: University of Rochester)
+ 1 AuthorsJayne B. Robinson15
Estimated H-index: 15
(UD: University of Dayton)
Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were sign...
Published on Nov 13, 1998in Journal of Biological Chemistry
Richard J. Heath34
Estimated H-index: 34
(St. Jude Children's Research Hospital),
Yuen-Tsu Yu1
Estimated H-index: 1
(St. Jude Children's Research Hospital)
+ 2 AuthorsCharles O. Rock26
Estimated H-index: 26
(St. Jude Children's Research Hospital)
Abstract The broad spectrum antibacterial properties of 2-hydroxydiphenyl ethers have been appreciated for decades, and their use in consumer products is rapidly increasing. We identify the enoyl-acyl carrier protein reductase (fabI) component of the type II fatty acid synthase system as the specific cellular target for these antibacterials. Biologically active 2-hydroxydiphenyl ethers effectively inhibit fatty acid synthesis in vivo and FabI activity in vitro. Resistant mechanisms include up-re...
Published on Sep 1, 1998in Journal of Bacteriology 3.23
Jesús Campos-García18
Estimated H-index: 18
(UNAM: National Autonomous University of Mexico),
Alma Delia Caro3
Estimated H-index: 3
(UNAM: National Autonomous University of Mexico)
+ 3 AuthorsGloria Sobero27
Estimated H-index: 27
(UNAM: National Autonomous University of Mexico)
A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnol...
Published on Aug 1, 1998in Nature 43.07
Laura M. McMurry24
Estimated H-index: 24
(Tufts University),
Margret Oethinger11
Estimated H-index: 11
(Tufts University),
Stuart B. Levy77
Estimated H-index: 77
(Tufts University)
Triclosan is a broad-spectrum antibacterial and antifungal agent1,2, which acts by previously undetermined mechanisms, that is used in products such as antiseptic soaps, toothpastes, fabrics and plastics. Here we show that triclosan blocks lipid synthesis in Escherichia coli, and that mutations in, or overexpression of, the gene fabI (which encodes enoyl reductase, involved in fatty acid synthesis) prevents this blockage. This is, to our knowledge, the first evidence that triclosan acts on a spe...
Published on May 1, 1998in Gene 2.64
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
RoxAnn R. Karkhoff-Schweizer10
Estimated H-index: 10
(CSU: Colorado State University)
+ 1 AuthorsHerbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
Abstract An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZα -allele for blue–white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains ...
Published on May 1, 1998in Journal of Bacteriology 3.23
Dale L. Val6
Estimated H-index: 6
,
John E. Cronan73
Estimated H-index: 73
(UIUC: University of Illinois at Urbana–Champaign)
Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditi...
Cited By159
Newest
Published on Aug 1, 2019in Bioresource Technology 6.67
Jin Du , Jin Du + 2 AuthorsJunrui Cao
Abstract A comparative study of rhamnolipid congener synthesis was performed using the neotype Pseudomonas aeruginosa ATCC 10145 and two marine isolates (1A01151 and 1A00364). Compared with the neotype, 1A01151 and 1A00364 showed increased production of rhamnolipids containing higher proportion of congeners with longer fatty acid chains (C 10 -C 12 and C 10 -C 12:1 ) and/or di-rhamnose moiety. These could reduce surface tension of water to lower minimum values of 21.3 mN m −1 and 25.7 mN m −1 . ...
Published on Jul 2, 2019in Microbiology 1.03
K. C. Gupta7
Estimated H-index: 7
(Panjab University, Chandigarh),
Priya Daroch (Panjab University, Chandigarh)+ 1 AuthorsSanjay Chhibber23
Estimated H-index: 23
(Panjab University, Chandigarh)
Quorum sensing (QS) refers to chemical signalling between micro-organisms and defines a social concord among them. Once a threshold of signal is accumulated, certain virulent traits are regulated within bacteria in response to the surrounding environment. These virulence traits are known to contribute in the pathogenicity of bacterial diseases. To prevent the activation of virulence factors, QS is inhibited in different ways through a strategy known as quorum quenching. Various types of quorum-q...
Published on Feb 5, 2019in Frontiers in Cellular and Infection Microbiology 3.52
Osmel Fleitas Martínez (UCB: Universidade Católica de Brasília), Pietra Orlandi Rigueiras (UCB: Universidade Católica de Brasília)+ 4 AuthorsOctávio L. Franco29
Estimated H-index: 29
(UCB: Universidade Católica de Brasília)
Faced with the global health threat of increasing resistance to antibiotics, researchers are exploring interventions that target bacterial virulence factors. Quorum sensing is a particularly attractive target because several bacterial virulence factors are controlled by this mechanism. Furthermore, attacking the quorum-sensing signaling network is less likely to select for resistant strains than using conventional antibiotics. Strategies that focus on the inhibition of quorum-sensing signal prod...
Published on Feb 1, 2019in Microbial Pathogenesis 2.58
Mona I. Shaaban14
Estimated H-index: 14
(Technology College),
Abdelaziz Elgaml4
Estimated H-index: 4
(Mansoura University)
+ 0 AuthorsElsayed E. Habib11
Estimated H-index: 11
(Technology College)
Abstract High incidence of antibiotic resistance among bacterial clinical isolates necessitates the discovery of new targets for inhibition of microbial pathogenicity, without stimulation of microbial resistance. This could be achieved by targeting virulence determinants, which cause host damage and disease. Many pathogenic bacteria elaborate signaling molecules for cellular communication. This signaling system is named quorum sensing system (QS), and it is contingent on the bacterial population...
Published on Feb 1, 2019
Manmohit Kalia3
Estimated H-index: 3
(MNNIT: Motilal Nehru National Institute of Technology Allahabad),
Vivek Kumar Yadav3
Estimated H-index: 3
(MNNIT: Motilal Nehru National Institute of Technology Allahabad)
+ 4 AuthorsVishnu Agarwal8
Estimated H-index: 8
(MNNIT: Motilal Nehru National Institute of Technology Allahabad)
Pseudomonas aeruginosa infections are a leading cause of death in patients suffering from respiratory diseases. The multidrug-resistant nature of Pseudomonas is potentiated by a process known as quorum sensing. The aim of this study was to reveal new inhibitors of a well-validated but quite unexplored target, enoyl-ACP reductase, which contributes acyl chain lengths of N-acyl homoserine lactones that are major signaling molecules in gram-negative bacteria. In the present study, the crystal struc...
Published on Jan 1, 2019in European Journal of Medicinal Chemistry 4.83
Gemma Howse1
Estimated H-index: 1
(University of Reading),
Richard Bovill1
Estimated H-index: 1
(Thermo Fisher Scientific)
+ 1 AuthorsHelen M. I. Osborn22
Estimated H-index: 22
(University of Reading)
Abstract There is an ongoing urgent need for new targeted antibacterial compounds with novel mechanisms of action for the treatment of infections caused by bacteria that are resistant to currently available materials. Since the expression of glycosidase enzymes within bacteria is unequally distributed, glycoside derivatives of antibacterial agents offer potential as targeted prodrugs for bacterial infections. Herein we report the synthesis and characterisation of four α-D-glycopyranosides and th...
Published on Sep 1, 2018in Fibers and Polymers 1.44
Khaldi Zineb1
Estimated H-index: 1
(University of Limoges),
Nzambe Ta Keki Jean Kerim1
Estimated H-index: 1
(University of Limoges)
+ 3 AuthorsZerrouki Rachida1
Estimated H-index: 1
(Université du Québec)
With the increased risk of disease transmissions and cross-infection caused by microorganisms, the control of microbial infections becomes a very important issue in modern societies. Moreover, with the emergence of antibio-resistant bacterial strains, it is necessary to control the bacterial growth. One of ways to limit the bacterial proliferation is to develop antimicrobial surfaces. The present work describes the synthesis process of a direct linking of propargyled Triclosan to a modified Kraf...
Published on May 7, 2018in Frontiers in Cellular and Infection Microbiology 3.52
Jianfei Liu2
Estimated H-index: 2
,
Kaifei Fu3
Estimated H-index: 3
+ 3 AuthorsLijun Zhou3
Estimated H-index: 3
N-Acyl Homoserine Lactones (N-AHLs) are an important group of small quorum-sensing molecules generated and released into the surroundings by Gram-negative bacteria. N-AHLs play a crucial role in various infection-related biological processes of marine Vibrio species, including survival, colonization, invasion and pathogenesis. With the increasing problem of antibiotic abuse and subsequently the emergence of drug-resistant bacteria, studies on AHLs are therefore expected to bring potential new br...
Published on Mar 1, 2018in Archives of Microbiology 1.64
Abhirup Mookherjee2
Estimated H-index: 2
(IIT-KGP: Indian Institute of Technology Kharagpur),
Shivangi Singh1
Estimated H-index: 1
(IIT-KGP: Indian Institute of Technology Kharagpur),
Mrinal K. Maiti17
Estimated H-index: 17
(IIT-KGP: Indian Institute of Technology Kharagpur)
Endophytes are microbes which reside inside the plant tissues asymptomatically or causing pathogenicity to the host plant for a brief period. Owing to their presence in a specialized niche, endophytes are capable of synthesizing diverse types of bioactive molecules. Continuous development of resistance mechanism by pathogens to the currently available health treatments and pharmaceuticals has led researchers to explore new therapeutic agents. Quorum sensing has a role in the development of micro...
Published on Feb 8, 2018in PLOS ONE 2.78
Raees Khan4
Estimated H-index: 4
,
Nazish Roy2
Estimated H-index: 2
+ 1 AuthorsSeon-Woo Lee11
Estimated H-index: 11