scholarly journals Erwinia amylovoraAuxotrophic Mutant Exometabolomics and Virulence on Apples

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Sara M. Klee ◽  
Judith P. Sinn ◽  
Melissa Finley ◽  
Erik L. Allman ◽  
Philip B. Smith ◽  
...  
Keyword(s):  
Fire Blight ◽  
De Novo ◽  
Virulent Strain ◽  
Auxotrophic Mutant ◽  
Management Tool ◽  
Wild Type ◽  
Management Options ◽  
Substantial Impact ◽  
Content Type ◽  
Arginine Auxotroph

ABSTRACTThe Gram-negative bacteriumErwinia amylovoracauses fire blight disease of apples and pears. While the virulence systems ofE. amylovorahave been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophicE. amylovoramutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-typeE. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenicE. amylovoraauxotrophs could have utility as fire blight biocontrol agents.IMPORTANCEThis study has revealed the availability of a range of host metabolites toE. amylovoracells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterialde novosynthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophicE. amylovoramutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulentE. amylovoraon flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenicE. amylovoraauxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

scholarly journals Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Elizabeth A. Walker ◽  
Gary C. Port ◽  
Michael G. Caparon ◽  
Blythe E. Janowiak
Keyword(s):  
Streptococcus Agalactiae ◽  
De Novo ◽  
Single Gene ◽  
Neonatal Meningitis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Glutathione Synthesis ◽  
Content Type ◽  
Resistance Pathway

ABSTRACT Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

scholarly journals Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

2013 ◽  
Vol 79 (15) ◽  
pp. 4586-4594 ◽  
Author(s):  
Masato Ikeda ◽  
Aya Miyamoto ◽  
Sumire Mutoh ◽  
Yuko Kitano ◽  
Mei Tajima ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Pimelic Acid ◽  
Wild Type ◽  
Biotin Synthase ◽  
Content Type ◽  
Carbon Carbon Bond

ABSTRACTTo develop the infrastructure for biotin production through naturally biotin-auxotrophicCorynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribedbioBFgenes ofEscherichia coliwere introduced into theC. glutamicumgenome, which originally lacked thebioFgene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), thebioIgene ofBacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of thede novosynthesis of biotin. On the other hand, thebioYgene responsible for biotin uptake was disrupted in wild-typeC. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, thebioYdisruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioYstrain showed a similar high requirement for the precursor dethiobiotin, a substrate forbioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, thebioBgene was further disrupted in both the wild-type strain and the ΔbioYstrain. By selectively using the resulting two strains (ΔbioBand ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph ofC. glutamicumproduced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).

scholarly journals Enterococcus faecalisEncodes an Atypical Auxiliary Acyl Carrier Protein Required for Efficient Regulation of Fatty Acid Synthesis by Exogenous Fatty Acids

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Lei Zhu ◽  
Qi Zou ◽  
Xinyun Cao ◽  
John E. Cronan
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Acid Synthesis ◽  
Wild Type ◽  
Content Type

ABSTRACTAcyl carrier proteins (ACPs) play essential roles in the synthesis of fatty acids and transfer of long fatty acyl chains into complex lipids. TheEnterococcus faecalisgenome contains two annotatedacpgenes, calledacpAandacpB. AcpA is encoded within the fatty acid synthesis (fab) operon and appears essential. In contrast, AcpB is an atypical ACP, having only 30% residue identity with AcpA, and is not essential. Deletion ofacpBhas no effect onE. faecalisgrowth orde novofatty acid synthesis in media lacking fatty acids. However, unlike the wild-type strain, where growth with oleic acid resulted in almost complete blockage ofde novofatty acid synthesis, theΔacpBstrain largely continuedde novofatty acid synthesis under these conditions. Blockage in the wild-type strain is due to repression offaboperon transcription, leading to levels of fatty acid synthetic proteins (including AcpA) that are insufficient to supportde novosynthesis. Transcription of thefaboperon is regulated by FabT, a repressor protein that binds DNA only when it is bound to an acyl-ACP ligand. Since AcpA is encoded in thefaboperon, its synthesis is blocked when the operon is repressed andacpAthus cannot provide a stable supply of ACP for synthesis of the acyl-ACP ligand required for DNA binding by FabT. In contrast to AcpA,acpBtranscription is unaffected by growth with exogenous fatty acids and thus provides a stable supply of ACP for conversion to the acyl-ACP ligand required for repression by FabT. Indeed,ΔacpBandΔfabTstrains have essentially the samede novofatty acid synthesis phenotype in oleic acid-grown cultures, which argues that neither strain can form the FabT-acyl-ACP repression complex. Finally, acylated derivatives of both AcpB and AcpA were substrates for theE. faecalisenoyl-ACP reductases and forE. faecalisPlsX (acyl-ACP; phosphate acyltransferase).IMPORTANCEAcpB homologs are encoded by many, but not all, lactic acid bacteria (Lactobacillales), including many members of the human microbiome. The mechanisms regulating fatty acid synthesis by exogenous fatty acids play a key role in resistance of these bacteria to those antimicrobials targeted at fatty acid synthesis enzymes. Defective regulation can increase resistance to such inhibitors and also reduce pathogenesis.

scholarly journals Differential Role of the Interleukin-17 Axis and Neutrophils in Resolution of Inhalational Anthrax

2011 ◽  
Vol 80 (1) ◽  
pp. 131-142 ◽  
Author(s):  
Kévin Garraud ◽  
Aurélie Cleret ◽  
Jacques Mathieu ◽  
Daniel Fiole ◽  
Yves Gauthier ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Crucial Role ◽  
Pulmonary Infection ◽  
Virulent Strain ◽  
The Self ◽  
Interleukin 17 ◽  
Wild Type ◽  
Content Type ◽  
Inhalation Anthrax

ABSTRACTThe roles of interleukin-17 (IL-17) and neutrophils in the lung have been described as those of two intricate but independent players. Here we identify neutrophils as the primary IL-17-secreting subset of cells in a model of inhalation anthrax using A/J and C57BL/6 mice. With IL-17 receptor A knockout (IL-17RA−/−) mice, we confirmed that IL-17A/F signaling is instrumental in the self-recruitment of this population. We also show that the IL-17A/F axis is critical for surviving pulmonary infection, as IL-17RA−/−mice become susceptible to intranasal infection byBacillus anthracisSterne spores. Strikingly, infection with a fully virulent strain did not affect IL-17RA−/−mouse survival. Eventually, by depleting neutrophils in wild-type and IL-17RA−/−mice, we demonstrated the crucial role of IL-17-secreting neutrophils in mouse survival of infection by fully virulentB. anthracis. This work demonstrates the important roles of both IL-17 signaling and neutrophils in clearing this pathogen and surviving pulmonaryB. anthracisinfection.

scholarly journals Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant

2011 ◽  
Vol 77 (17) ◽  
pp. 6133-6140 ◽  
Author(s):  
Yongjin Zhou ◽  
Lei Wang ◽  
Fan Yang ◽  
Xinping Lin ◽  
Sufang Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Essential Gene ◽  
Auxotrophic Mutant ◽  
Genetically Engineered ◽  
Reduced Form ◽  
Live Cells ◽  
Regulation Mechanism ◽  
Content Type ◽  
High Concentrations

ABSTRACTNAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels inEscherichia colicells that were genetically engineered to be NAD+auxotrophic. First, we expressed thentt4gene encoding the NAD(H) transporter in theE. colimutant YJE001, which had a deletion of thenadCgene responsible for NAD+de novobiosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential genenadE, which is responsible for the last step of NAD+biosynthesis in cells harboring thentt4gene. The minimal NAD+level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.

scholarly journals Protective Role of the Capsule and Impact of Serotype 4 Switching on Streptococcus mitis

2014 ◽  
Vol 82 (9) ◽  
pp. 3790-3801 ◽  
Author(s):  
Håkon V. Rukke ◽  
Raja Sab Kalluru ◽  
Urska Repnik ◽  
Alice Gerlini ◽  
Ricardo J. José ◽  
...  
Keyword(s):  
Mouse Model ◽  
Type Strain ◽  
Virulent Strain ◽  
Protective Role ◽  
Lung Model ◽  
Close Relative ◽  
Wild Type ◽  
Streptococcus Mitis ◽  
Protective Function ◽  
Content Type

ABSTRACTThe polysaccharide capsule surroundingStreptococcus pneumoniaeis essential for virulence. Recently,Streptococcus mitis, a human commensal and a close relative ofS. pneumoniae, was also shown to have a capsule. In this study, theS. mitistype strain switched capsule by acquisition of the serotype 4 capsule locus ofS. pneumoniaeTIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protectedS. mitisagainst phagocytosis by RAW 264.7 macrophages. This effect was enhanced in theS. mitisstrain expressing theS. pneumoniaecapsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant.S. mitissurvival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models,S. pneumoniaeTIGR4 showed higher rates of survival than theS. mitistype strain or the capsule-switching mutant, except in the lung model, in which significant differences betweenS. pneumoniaeTIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule inS. mitis. Under such conditions,S. mitisresistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strainS. pneumoniaeTIGR4.

scholarly journals Mutation of theErwinia amylovora argDGene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

2014 ◽  
Vol 80 (21) ◽  
pp. 6739-6749 ◽  
Author(s):  
Laura S. Ramos ◽  
Brian L. Lehman ◽  
Kari A. Peter ◽  
Timothy W. McNellis
Keyword(s):  
Fire Blight ◽  
Virulent Strain ◽  
Apple Fruit ◽  
Infection Process ◽  
Bacterial Disease ◽  
Primary Metabolism ◽  
In Planta ◽  
Coding Region ◽  
Content Type ◽  
Expression Of Genes

ABSTRACTFire blight is caused byErwinia amylovoraand is the most destructive bacterial disease of apples and pears worldwide. In this study, we found thatE. amylovoraargD(1000)::Tn5, anargDTn5transposon mutant that has the Tn5transposon inserted after nucleotide 999 in theargDgene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. TheE. amylovoraargDgene encodes a predictedN-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-typeargDgene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of theargD(1000)::Tn5mutant. However, even when mixed with virulentE. amylovoracells and inoculated onto immature apple fruit, theargD(1000)::Tn5mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argDcomplementation plasmid was stably maintained in theargD(1000)::Tn5mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argDcomplementation plasmid could be useful for the expression of genes, markers, and reporters inE. amylovoragrowingin planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered anE. amylovoravirulence factor because theargD(1000)::Tn5mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate thatE. amylovoracannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.

scholarly journals Vaccination of BALB/c Mice with an Avirulent Mycoplasma pneumoniae P30 Mutant Results in Disease Exacerbation upon Challenge with a Virulent Strain

2012 ◽  
Vol 80 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
S. M. Szczepanek ◽  
S. Majumder ◽  
E. S. Sheppard ◽  
X. Liao ◽  
D. Rood ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Virulent Strain ◽  
Gliding Motility ◽  
Respiratory Pathogen ◽  
High Morbidity ◽  
Wild Type ◽  
Disease Exacerbation ◽  
Live Attenuated Vaccine ◽  
Th17 Response ◽  
Content Type

Mycoplasma pneumoniaeis a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to preventM. pneumoniaeinfection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-typeM. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms ofM. pneumoniaepathogenesis.

scholarly journals Host Fatty Acid Utilization by Staphylococcus aureus at the Infection Site

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Matthew W. Frank ◽  
Jiangwei Yao ◽  
Justin L. Batte ◽  
Jessica M. Gullett ◽  
Chitra Subramanian ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Molecular Species ◽  
De Novo ◽  
New Drugs ◽  
Pentadecanoic Acid ◽  
Infection Site ◽  
Wild Type ◽  
Content Type ◽  
Branched Chain

ABSTRACT Staphylococcus aureus utilizes the fatty acid (FA) kinase system to activate exogenous FAs for membrane synthesis. We developed a lipidomics workflow to determine the membrane phosphatidylglycerol (PG) molecular species synthesized by S. aureus at the thigh infection site. Wild-type S. aureus utilizes both host palmitate and oleate to acylate the 1 position of PG, and the 2 position is occupied by pentadecanoic acid arising from de novo biosynthesis. Inactivation of FakB2 eliminates the ability to assimilate oleate and inactivation of FakB1 reduces the content of saturated FAs and enhances oleate utilization. Elimination of FA activation in either ΔfakA or ΔfakB1 ΔfakB2 mutants does not impact growth. All S. aureus strains recovered from the thigh have significantly reduced branched-chain FAs and increased even-chain FAs compared to that with growth in rich laboratory medium. The molecular species pattern observed in the thigh was reproduced in the laboratory by growth in isoleucine-deficient medium containing exogenous FAs. S. aureus utilizes specific host FAs for membrane biosynthesis but also requires de novo FA biosynthesis initiated by isoleucine (or leucine) to produce pentadecanoic acid. IMPORTANCE The shortage of antibiotics against drug-resistant Staphylococcus aureus has led to the development of new drugs targeting the elongation cycle of fatty acid (FA) synthesis that are progressing toward the clinic. An objection to the use of FA synthesis inhibitors is that S. aureus can utilize exogenous FAs to construct its membrane, suggesting that the bacterium would bypass these therapeutics by utilizing host FAs instead. We developed a mass spectrometry workflow to determine the composition of the S. aureus membrane at the infection site to directly address how S. aureus uses host FAs. S. aureus strains that cannot acquire host FAs are as effective in establishing an infection as the wild type, but strains that require the utilization of host FAs for growth were attenuated in the mouse thigh infection model. We find that S. aureus does utilize host FAs to construct its membrane, but host FAs do not replace the requirement for pentadecanoic acid, a branched-chain FA derived from isoleucine (or leucine) that predominantly occupies the 2 position of S. aureus phospholipids. The membrane phospholipid structure of S. aureus mutants that cannot utilize host FAs indicates the isoleucine is a scarce resource at the infection site. This reliance on the de novo synthesis of predominantly pentadecanoic acid that cannot be obtained from the host is one reason why drugs that target fatty acid synthesis are effective in treating S. aureus infections.

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