A triclosan‐resistance protein from the soil metagenome is a novel enoyl‐acyl carrier protein reductase: Structure‐guided functional analysis

ABSTRACTEnoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. TheEnterococcus faecalisgenome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of anEscherichia colifabItemperature-sensitive mutant strain under nonpermissive conditions.In vitroassays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression ofE. faecalis fabKconfers toE. colihigh levels of resistance to the antimicrobial triclosan, deletion offabKfrom theE. faecalisgenome showed that FabK does not play a detectable role in the inherent triclosan resistance ofE. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion offabKgrow normally without fatty acid supplementation, whereasfabIdeletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs.IMPORTANCEThe finding that exogenous fatty acids support growth ofE. faecalisstrains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in counteringE. faecalisinfections because host serum fatty acids support growth of the bacterium.

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Defining and Combating the Mechanisms of Triclosan Resistance in Clinical Isolates of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3343-3347.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3343-3347 ◽

Cited By ~ 61

Author(s):

Frank Fan ◽

Kang Yan ◽

Nicola G. Wallis ◽

Shannon Reed ◽

Terrance D. Moore ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Acyl Carrier Protein ◽

Clinical Isolates ◽

Stable Complex ◽

Carrier Protein ◽

Wild Type ◽

Binding Studies ◽

Additional Increase ◽

Triclosan Resistance

ABSTRACT The MICs of triclosan for 31 clinical isolates of Staphylococcus aureus were 0.016 μg/ml (24 strains), 1 to 2 μg/ml (6 strains), and 0.25 μg/ml (1 strain). All the strains for which triclosan MICs were elevated (>0.016 μg/ml) showed three- to fivefold increases in their levels of enoyl-acyl carrier protein (ACP) reductase (FabI) production. Furthermore, strains for which triclosan MICs were 1 to 2 μg/ml overexpressed FabI with an F204C alteration. Binding studies with radiolabeled NAD+ demonstrated that this change prevents the formation of the stable triclosan-NAD+-FabI complex, and both this alteration and its overexpression contributed to achieving MICs of 1 to 2 μg/ml for these strains. Three novel, potent inhibitors of FabI (50% inhibitory concentrations, ≤64 nM) demonstrated up to 1,000-fold better activity than triclosan against the strains for which triclosan MICs were elevated. None of the compounds tested from this series formed a stable complex with NAD+-FabI. Consequently, although the overexpression of wild-type FabI gave rise to an increase in the MICs, as expected, overexpression of FabI with an F204C alteration did not cause an additional increase in resistance. Therefore, this work identifies the mechanisms of triclosan resistance in S. aureus, and we present three compounds from a novel chemical series of FabI inhibitors which have excellent activities against both triclosan-resistant and -sensitive clinical isolates of S. aureus.

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Cloning and Functional Analysis of Peanut Δ9-Stearoyl-Acyl Carrier Protein Desaturase Promoter

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2016.01629 ◽

2016 ◽

Vol 42 (11) ◽

pp. 1629

Author(s):

Lei SHI ◽

Li-Juan MIAO ◽

Fei-Yan QI ◽

Zhong-Xin ZHANG ◽

Wei GAO ◽

...

Keyword(s):

Functional Analysis ◽

Acyl Carrier Protein ◽

Carrier Protein

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Triclosan Resistance of Pseudomonas aeruginosa PAO1 Is Due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01152-09 ◽

2009 ◽

Vol 54 (2) ◽

pp. 689-698 ◽

Cited By ~ 107

Author(s):

Lei Zhu ◽

Jinshui Lin ◽

Jincheng Ma ◽

John E. Cronan ◽

Haihong Wang

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutant Strain ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Carrier Protein ◽

Active Efflux ◽

E Coli ◽

Genes Encoding ◽

Enoyl Acp Reductase ◽

Triclosan Resistance

ABSTRACT Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active efflux of the compound (R. Chuanchuen, R. R. Karkhoff-Schweizer, and H. P. Schweizer, Am. J. Infect. Control 31:124-127, 2003). We report that P. aeruginosa contains two enoyl-ACP reductase isozymes, the previously characterized FabI homologue plus a homologue of FabV, a triclosan-resistant enoyl-ACP reductase recently demonstrated in Vibrio cholerae. By deletion of the genes encoding P. aeruginosa FabI and FabV, we demonstrated that FabV confers triclosan resistance on P. aeruginosa. Upon deletion of the fabV gene, the mutant strain became extremely sensitive to triclosan (>2,000-fold more sensitive than the wild-type strain), whereas the mutant strain lacking FabI remained completely resistant to the biocide.

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Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

Scientific Reports ◽

10.1038/s41598-019-51895-2 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Raees Khan ◽

Amir Zeb ◽

Kihyuck Choi ◽

Gihwan Lee ◽

Keun Woo Lee ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Fatty Acid Biosynthesis ◽

Catalytic Domain ◽

Acyl Carrier Protein ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Carrier Protein ◽

The Novel ◽

Triclosan Resistance ◽

Structural Insights

Abstract Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

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Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00648-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 3

Author(s):

Raees Khan ◽

Amir Zeb ◽

Nazish Roy ◽

Roniya Thapa Magar ◽

Hyo Jeong Kim ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Carrier Protein ◽

Content Type ◽

Triclosan Resistance ◽

Bacterial Type

ABSTRACTEnoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found inEpsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the generaHelicobacterandCampylobacter. Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenicEpsilonproteobacteria.

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