scholarly journals The Francisella tularensis FabI Enoyl-Acyl Carrier Protein Reductase Gene Is Essential to Bacterial Viability and Is Expressed during Infection

2012 ◽  
Vol 195 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Luke C. Kingry ◽  
Jason E. Cummings ◽  
Kerry W. Brookman ◽  
Gopal R. Bommineni ◽  
Peter J. Tonge ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Francisella Tularensis ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Transcriptional Profiling ◽  
Acid Synthesis ◽  
Content Type ◽  
Enoyl Acp Reductase ◽  
Long Chain

ABSTRACTFrancisella tularensisis classified as a category A priority pathogen and causes fatal disseminated disease in humans upon inhalation of less than 50 bacteria. Although drugs are available for treatment, they are not ideal because of toxicity and route of delivery, and in some cases patients relapse upon withdrawal. We have an ongoing program to develop novel FAS-II FabI enoyl-ACP reductase enzyme inhibitors forFrancisellaand other select agents. To establishF. tularensisFabI (FtFabI) as a clinically relevant drug target, we demonstrated that fatty acid biosynthesis and FabI activity are essential for growth even in the presence of exogenous long-chain lipids and that FtfabIis not transcriptionally altered in the presence of exogenous long-chain lipids. Inhibition of FtFabI or fatty acid synthesis results in loss of viability that is not rescued by exogenous long-chain lipid supplementation. Importantly, whole-genome transcriptional profiling ofF. tularensiswith DNA microarrays from infected tissues revealed that FtfabIandde novofatty acid biosynthetic genes are transcriptionally active during infection. This is the first demonstration that the FabI enoyl-ACP-reductase enzyme encoded byF. tularensisis essential and not bypassed by exogenous fatty acids and thatde novofatty acid biosynthetic components encoded inF. tularensisare transcriptionally active during infection in the mouse model of tularemia.

scholarly journals Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

2011 ◽  
Vol 78 (5) ◽  
pp. 1563-1573 ◽  
Author(s):  
Juanli Cheng ◽  
Jincheng Ma ◽  
Jinshui Lin ◽  
Zhen-Chuan Fan ◽  
John E. Cronan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Ralstonia Solanacearum ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Phytopathogenic Bacterium ◽  
Content Type ◽  
Long Chain

ABSTRACTRalstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes ofR. solanacearumshowed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) andcis-vaccenic (C18:1) acids, little is known regardingR. solanacearumfatty acid synthesis. TheR. solanacearumGMI1000 genome is unusual in that it contains four genes (fabF1,fabF2,fabF3, andfabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes,fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles inR. solanacearumfatty acid synthesis. Mutant strains lackingfabF1are nonviable, and thus, FabF1 is essential forR. solanacearumfatty acid biosynthesis. Moreover,R. solanacearumFabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.

scholarly journals The Two Functional Enoyl-Acyl Carrier Protein Reductases of Enterococcus faecalis Do Not Mediate Triclosan Resistance

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lei Zhu ◽  
Hongkai Bi ◽  
Jincheng Ma ◽  
Zhe Hu ◽  
Wenbin Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enterococcus Faecalis ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Content Type ◽  
Enoyl Acp Reductase ◽  
Triclosan Resistance

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.

Triclosan Resistance in Francisella tularensis: A Site-Directed Mutagenesis Study of the FabI Enzyme

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
N. Silas ◽  
R. Demissie ◽  
L.W.M. Fung
Keyword(s):  
Fatty Acid ◽  
Francisella Tularensis ◽  
Bacterial Resistance ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Enzymatic Reaction ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Bacterial Fatty Acid ◽  
Triclosan Resistance

An NADH-dependent enoyl-acyl carrier protein reductase, FabI, catalyzes the final step of bacterial fatty acid biosynthesis, reducing the double bond of trans-2-enoyl-ACP to a single bond forming acyl-ACP. Given its importance in bacterial fatty acid synthesis, FabI has become a recognized drug target. Triclosan, a diphenyl ether, targets the FabI, inhibits its activity, and stops bacterial growth. However, as a consequence of triclosan's popularity, and thus its overuse, bacterial resistance to triclosan has been reported. The mutation G93V in Escherichia coli (E. coli) FabI allows E. coli to resist the action of triclosan. We have identified the equivalent residue of G93 in Francisella tularensis FabI (ftFabI) as A92, and prepared a mutant A92V. E. coli cells, transformed with a plasmid containing the ftFabI-A92V gene, were grown, and the gene was overexpressed. From two growths (6 G of cells), 62 mG of protein, with a histidine tag, at a purity of 85% were obtained. Enzymatic activity was assayed by monitoring the absorbance of NADH at 340 nm. In the presence of triclosan, the wild-type protein was almost completely inhibited after NADH was converted to NAD$^+$ in the enzymatic reaction; however the A92V mutant exhibited similar activity with and without triclosan, demonstrating that triclosan resistance may also develop in Francisella tularensis.

scholarly journals The Burkholderia pseudomallei Enoyl-Acyl Carrier Protein Reductase FabI1 Is Essential forIn VivoGrowth and Is the Target of a Novel Chemotherapeutic with Efficacy

2013 ◽  
Vol 58 (2) ◽  
pp. 931-935 ◽  
Author(s):  
Jason E. Cummings ◽  
Luke C. Kingry ◽  
Drew A. Rholl ◽  
Herbert P. Schweizer ◽  
Peter J. Tonge ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Specific Inhibitor ◽  
Carrier Protein ◽  
Wild Type ◽  
Content Type ◽  
Fatty Acid Biosynthesis Pathway ◽  
Enoyl Acp Reductase

ABSTRACTThe bacterial fatty acid biosynthesis pathway is a validated target for the development of novel chemotherapeutics. However, sinceBurkholderia pseudomalleicarries genes that encode both FabI and FabV enoyl-acyl carrier protein (ACP) reductase homologues, the enoyl-ACP reductase that is essential forin vivogrowth needs to be defined so that the correct drug target can be chosen for development. Accordingly, ΔfabI1, ΔfabI2, and ΔfabVknockout strains were constructed and tested in a mouse model of infection. Mice infected with a ΔfabI1strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ΔfabI2and ΔfabVmutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ΔfabI2and ΔfabVstrains were not significantly different from those of the wild-type strain, a slight delay was observed. A FabI1-specific inhibitor was used to confirm that inhibition of FabI1 results in reduced bacterial burden and efficacy in an acuteB. pseudomalleimurine model of infection. This work establishes that FabI1 is required for growth ofBurkholderia pseudomalleiin vivoand is a potential molecular target for drug development.

scholarly journals Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2020 ◽  
Author(s):  
Michael Burkart ◽  
Thomas Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D Lee ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Acid Biosynthesis ◽  
E Coli

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.

scholarly journals Taurine-Mediated IDOL Contributes to Resolution of Streptococcus uberis Infection

2021 ◽  
Vol 89 (5) ◽  
Author(s):  
Zhixin Wan ◽  
Riguo Lan ◽  
Yilin Zhou ◽  
Yuanyuan Xu ◽  
Zhenglei Wang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Bacterial Load ◽  
Critical Role ◽  
Acid Synthesis ◽  
Content Type ◽  
Streptococcus Uberis

ABSTRACT Metabolic alterations occur in pathogenic infections, but the role of lipid metabolism in the progression of bacterial mastitis is unclear. Cross talk between lipid droplets (LDs) and invading bacteria occurs, and targeting of de novo lipogenesis inhibits pathogen reproduction. In this study, we investigate the role(s) of lipid metabolism in mammary cells during Streptococcus uberis infection. Our results indicate that S. uberis induces the synthesis of fatty acids and production of LDs. Importantly, taurine reduces fatty acid synthesis, the abundance of LDs and the in vitro bacterial load of S. uberis. These changes are mediated, at least partly, by the E3 ubiquitin ligase IDOL, which is associated with the degradation of low-density lipoprotein receptors (LDLRs). We have identified a critical role for IDOL-mediated fatty acid synthesis in bacterial infection, and we suggest that taurine may be an effective prophylactic or therapeutic strategy for preventing S. uberis mastitis.

scholarly journals NovelXanthomonas campestrisLong-Chain-Specific 3-Oxoacyl-Acyl Carrier Protein Reductase Involved in Diffusible Signal Factor Synthesis

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Zhe Hu ◽  
Huijuan Dong ◽  
Jin-Cheng Ma ◽  
Yonghong Yu ◽  
Kai-Hui Li ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Xanthomonas Campestris ◽  
Octanoic Acid ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Content Type ◽  
Diffusible Signal Factor ◽  
Acyl Chains

ABSTRACTThe precursors of the diffusible signal factor (DSF) family signals ofXanthomonas campestrispv.campestrisare 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by theX. campestrispv.campestrisXCC0416 gene (fabG2), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of anEscherichia colifabGtemperature-sensitive strain. However, when transformed into theE. colistrain together with a plasmid bearing theVibrio harveyiacyl-ACP synthetase gene (aasS), growth proceeded, but only when the medium contained octanoic acid.In vitroassays showed that FabG2 catalyzes the reduction of long-chain (≥C8) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C4, C6) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a strain that overexpressedfabG2but only in octanoic acid-supplemented media. Growth of theX. campestrispv.campestrisΔfabG1strain overexpressingfabG2requiredfabHfor growth with octanoic acid, indicating that octanoyl coenzyme A is elongated byX. campestrispv.campestrisfabH. Deletion offabG2reduced DSF family signal production, whereas overproduction of either FabG1 or FabG2 in the ΔfabG2strain restored DSF family signal levels.IMPORTANCEQuorum sensing mediated by DSF signaling molecules regulates pathogenesis in several different phytopathogenic bacteria, includingXanthomonas campestrispv.campestris. DSF signaling also plays a key role in infection by the human pathogenBurkholderia cepacia. The acyl chains of the DSF molecules are diverted and remodeled from a key intermediate of the fatty acid synthesis pathway. We report aXanthomonas campestrispv.campestrisfatty acid synthesis enzyme, FabG2, of novel specificity that seems tailored to provide DSF signaling molecule precursors.

Sign in / Sign up

Export Citation Format

Share Document