Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

Download Full-text

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

mBio ◽

10.1128/mbio.00613-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 26

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.

Download Full-text

Suppression offabBMutation byfabF1Is Mediated by Transcription Read-through in Shewanella oneidensis

Journal of Bacteriology ◽

10.1128/jb.00463-16 ◽

2016 ◽

Vol 198 (22) ◽

pp. 3060-3069 ◽

Cited By ~ 7

Author(s):

Meng Li ◽

Qiu Meng ◽

Huihui Fu ◽

Qixia Luo ◽

Haichun Gao

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Tandem Repeats ◽

Unsaturated Fatty Acids ◽

Acyl Carrier Protein ◽

Shewanella Oneidensis ◽

Acid Synthesis ◽

Carrier Protein ◽

Type Ii ◽

Content Type

ABSTRACTAs type II fatty acid synthesis is essential for the growth ofEscherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of thecis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case inShewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB inS. oneidensis. InfabB+ordesA+(encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of bothfabBanddesAgenes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of thefabF1gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator foracpP, an acyl carrier protein gene immediately upstream offabF1. There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.IMPORTANCEIt has been firmly established that FabB for UFA synthesis via type II fatty acid synthesis in FabA-containing bacteria such asE. coliis essential. However,S. oneidensisappears to be an exception. In this bacterium, FabF1, when sufficiently expressed, is able to fully complement the FabB loss. Importantly, such a capability can be obtained by spontaneous mutations, which lead to transcription read-through. Therefore, our data, by identifying the functional overlap between FabB and FabFs, provide new insights into the current understanding of KAS and help reveal novel ways to block UFA synthesis for therapeutic purposes.

Download Full-text

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

mBio ◽

10.1128/mbio.00596-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 7

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.

Download Full-text

Staphylococcus aureusUtilizes Host-Derived Lipoprotein Particles as Sources of Fatty Acids

Journal of Bacteriology ◽

10.1128/jb.00728-17 ◽

2018 ◽

Vol 200 (11) ◽

Cited By ~ 18

Author(s):

Phillip C. Delekta ◽

John C. Shook ◽

Todd A. Lydic ◽

Martha H. Mulks ◽

Neal D. Hammer

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Acid Synthesis ◽

Low Density ◽

Content Type ◽

Lipoprotein Particles ◽

Bacterial Fatty Acid

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects ofS. aureusphysiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids forS. aureusduring infection. We sought to test the ability of LDLs to serve as a fatty acid source forS. aureusand show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth ofS. aureusfatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids forS. aureusduring infection.IMPORTANCEInhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused byS. aureusand other human pathogens. However,S. aureusincorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited byS. aureusis not well understood. Human low-density lipoprotein particles represent a particularly abundantin vivosource of fatty acids and are present in tissues thatS. aureuscolonizes. Herein, we establish thatS. aureusis capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply thatS. aureustargets LDLs as a source of fatty acids during pathogenesis.

Download Full-text

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02515-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 14

Author(s):

Karine Gloux ◽

Mélanie Guillemet ◽

Charles Soler ◽

Claire Morvan ◽

David Halpern ◽

...

Keyword(s):

Fatty Acids ◽

Staphylococcus Aureus ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Bacterial Infections ◽

Impact Resistance ◽

Acid Synthesis ◽

Type Ii ◽

Content Type ◽

Triclosan Resistance

ABSTRACT The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.07335-11 ◽

2011 ◽

Vol 78 (5) ◽

pp. 1563-1573 ◽

Cited By ~ 8

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.

Download Full-text