The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

A short-chain acyl-CoA synthetase that supports branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.

Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii.

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus.

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C5-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C5-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria.

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the ß-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI).

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1-3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix-helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

Fatty acid activation and utilization by Alistipes finegoldii, a representative Bacteroidetes resident of the human gut microbiome.

Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram-negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium-chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long-chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2-position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long-chain acyl-ACP. The ability to assimilate a broad-spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl-acyl carrier protein (ACP) synthetases. Acyl-ACP synthetase 1 had a substrate preference for medium-chain fatty acids and synthetase 2 had a substrate preference for long-chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium- and long-chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.

Cardiac PANK1 deletion exacerbates ventricular dysfunction during pressure overload.

Coenzyme A (CoA) is an essential cofactor required for intermediary metabolism. Perturbations in homeostasis of CoA have been implicated in various pathologies; however, whether CoA homeostasis is changed and the extent to which CoA levels contribute to ventricular function and remodeling during pressure overload has not been explored. In this study, we sought to assess changes in CoA biosynthetic pathway during pressure overload and determine the impact of limiting CoA on cardiac function. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, pantothenate kinase 1 (Pank1). We found that constitutive, cardiomyocyte-specific Pank1 deletion (cmPank1-/-) significantly reduced PANK1 mRNA, PANK1 protein, and CoA levels compared with Pank1-sufficient littermates (cmPank1+/+) but exerted no obvious deleterious impact on the mice at baseline. We then subjected both groups of mice to pressure overload-induced heart failure. Interestingly, there was more ventricular dilation in cmPank1-/- during the pressure overload. To explore potential mechanisms contributing to this phenotype, we performed transcriptomic profiling, which suggested a role for Pank1 in regulating fibrotic and metabolic processes during the pressure overload. Indeed, Pank1 deletion exacerbated cardiac fibrosis following pressure overload. Because we were interested in the possibility of early metabolic impacts in response to pressure overload, we performed untargeted metabolomics, which indicated significant changes to metabolites involved in fatty acid and ketone metabolism, among other pathways. Collectively, our study underscores the role of elevated CoA levels in supporting fatty acid and ketone body oxidation, which may be more important than CoA-driven, enzyme-independent acetylation in the failing heart.NEW & NOTEWORTHY Changes in CoA homeostasis have been implicated in a variety of metabolic diseases; however, the extent to which changes in CoA homeostasis impacts remodeling has not been explored. We show that limiting cardiac CoA levels via PANK deletion exacerbated ventricular remodeling during pressure overload. Our results suggest that metabolic alterations, rather than structural alterations, associated with Pank1 deletion may underlie the exacerbated cardiac phenotype during pressure overload.

A fatty acid-binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids.

Streptococcus pneumoniae is responsible for the majority of pneumonia, motivating ongoing searches for insights into its physiology that could enable new treatments. S. pneumoniae responds to exogenous fatty acids by suppressing its de novo biosynthetic pathway and exclusively utilizing extracellular fatty acids for membrane phospholipid synthesis. The first step in exogenous fatty acid assimilation is phosphorylation by fatty acid kinase (FakA), whereas bound by a fatty acid-binding protein (FakB). Staphylococcus aureus has two binding proteins, whereas S. pneumoniae expresses three. The functions of these binding proteins were not clear. We determined the SpFakB1- and SpFakB2-binding proteins were bioinformatically related to the two binding proteins of Staphylococcus aureus, and biochemical and X-ray crystallographic analysis showed that SpFakB1 selectively bound saturates, whereas SpFakB2 allows the activation of monounsaturates akin to their S. aureus counterparts. The distinct SpFakB3 enables the utilization of polyunsaturates. The SpFakB3 crystal structure in complex with linoleic acid reveals an expanded fatty acid-binding pocket within the hydrophobic interior of SpFakB3 that explains its ability to accommodate multiple cis double bonds. SpFakB3 also utilizes a different hydrogen bond network than other FakBs to anchor the fatty acid carbonyl and stabilize the protein. S. pneumoniae strain JMG1 (ΔfakB3) was deficient in incorporation of linoleate from human serum verifying the role of FakB3 in this process. Thus, the multiple FakBs of S. pneumoniae permit the utilization of the entire spectrum of mammalian fatty acid structures to construct its membrane.

Oleate hydratase from Staphylococcus aureus protects against palmitoleic acid, the major antimicrobial fatty acid produced by mammalian skin.

Oleate hydratases (OhyAs) belong to a large family of bacterial proteins catalyzing the hydration or isomerization of double bonds in unsaturated fatty acids. A Staphylococcus aureus gene (Sa0102) is predicted to encode an OhyA. Here, we recombinantly expressed and purified SaOhyA and found that it forms a homodimer that requires FAD for activity. SaOhyA hydrates only unsaturated fatty acids containing cis-9 double bonds, but not fatty acids with trans-9 double bonds or cis double bonds at other positions. SaOhyA products were not detected in S. aureus phospholipids and were released into the growth medium. S. aureus does not synthesize unsaturated fatty acids, and the SaOhyA substrates are derived from infection sites. Palmitoleate (16:1(9Z)) is a major mammalian skin-produced antimicrobial fatty acid that protects against S. aureus infection, and we observed that it is an SaOhyA substrate and that its hydroxylated derivative is not antimicrobial. Treatment of S. aureus with 24 µm 16:1(9Z) immediately arrested growth, followed by growth resumption after a lag period of 2 h. The ΔohyA mutant strain did not recover from the 16:1(9Z) challenge, and increasing SaOhyA expression using a plasmid system prevented the initial growth arrest. Challenging S. aureus with sapienic acid (16:1(6Z)), an antimicrobial fatty acid produced only by human skin, arrested growth without recovery in WT, ΔohyA, and SaOhyA-overexpressing strains. We conclude that SaOhyA protects S. aureus from palmitoleic acid, the antimicrobial unsaturated fatty acid produced by most mammals, and that sapienic acid, uniquely produced by humans, counters the OhyA-dependent bacterial defense mechanism.

Acyl-chain selectivity and physiological roles of Staphylococcus aureus fatty acid-binding proteins.

Fatty acid (FA) kinase produces acyl-phosphate for the synthesis of membrane phospholipids in Gram-positive bacterial pathogens. FA kinase consists of a kinase protein (FakA) that phosphorylates an FA substrate bound to a second module, an FA-binding protein (FakB). Staphylococcus aureus expresses two distinct, but related, FakBs with different FA selectivities. Here, we report the structures of FakB1 bound to four saturated FAs at 1.6-1.93 Å resolution. We observed that the different FA structures are accommodated within a slightly curved hydrophobic cavity whose length is governed by the conformation of an isoleucine side chain at the end of the tunnel. The hydrophobic tunnel in FakB1 prevents the binding of cis-unsaturated FAs, which are instead accommodated by the kinked tunnel within the FakB2 protein. The differences in the FakB interiors are not propagated to the proteins' surfaces, preserving the protein-protein interactions with their three common partners, FakA, PlsX, and PlsY. Using cellular thermal shift analyses, we found that FakB1 binds FA in vivo, whereas a significant proportion of FakB2 does not. Incorporation of exogenous FA into phospholipid in ΔfakB1 and ΔfakB2 S. aureus knockout strains revealed that FakB1 does not efficiently activate unsaturated FAs. FakB2 preferred unsaturated FAs, but also allowed the incorporation of saturated FAs. These results are consistent with a model in which FakB1 primarily functions in the recycling of the saturated FAs produced by S. aureus metabolism, whereas FakB2 activates host-derived oleate, which S. aureus does not produce but is abundant at infection sites.

Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae.

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria.

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase.

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis.

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine.

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 pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain 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 of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless 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 of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. 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 of L. monocytogenes.

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae.

PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.