Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: The JE2 strain was grown in SerFA (BHI containing 10% serum and an equimolar mixture of 3 FAs) or in SerFA supplemented with the anti-FASII (AFN-1252, 0.5 µg/ml; SerFA-AFN). A. Transmission electron microscopy: left, exponential (3 h) growth of non-treated S. aureus in SerFA to OD 600 = ∼3; middle, 6 h growth post anti-FASII-treatment; right, 10 h post-anti-FASII-treatment, OD 600 = ∼3. White bar, 200 nm. A zoom of the surface within the dotted box highlights morphological differences in septal regions. Envelope thickness in the three conditions are shown in the graph at right. P-values were determined using a two-sided T-test based on 73, 89, and 60 measurements respectively on at least 15 individual bacteria ( Source data). Additional images are in Supplementary Fig. S2 . B. Ethidium bromide (EtBr) retention was compared in non-treated (NT) and anti-FASII-adapted (AD) S. aureus in exponential (exp, N=3) and stationary (stat; N=6) phase cultures. EtBr retention is presented at 20 minutes; see Source data for the full data set. P-values were determined using a two-sided T-test on biological triplicates for exponential and six replicates for stationary cultures. NS, non-significant.

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Transmission Assay, Electron Microscopy, Bacteria

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: S. aureus and numerous streptococci compensate FASII inhibition, deletion, or repression, by incorporating exogenous FAs in membranes [ , , , ] . A. Antibiotic- or fabD- mutation- generated FASII inhibition leads to LTA depletion in S. aureus . WT JE2 was grown in SerFA without (NT, non-treated) or with FabI inhibitor AFN-1252 (AD, anti-FASII-adapted) . Δ fabD is a FASII auxotroph ([ , ]), and was grown in SerFA (N=3). B. Both anti-FabI AFN-1252 (AFN) and anti-FabF platensimycin (PTM, ) lead to LTA depletion in S. aureus (N=3). C. Inhibition of FASII by exogenous FAs in the streptococcus species S. agalactiae leads to LTA depletion. The S. agalactiae FASII pathway is repressed by FabT when bound to exogenous FAs . Here, S. agalactiae strain NEM316 was grown in BHI plus 0.025% FA-free bovine serum albumin without or with C17:1 (100 µM); exogenous FA addition represses FASII but allows bacterial growth. LTA was monitored by immunoblotting using anti-LTA antibody (N=5). Samples were taken from exponential phase OD 600 = ∼2-3 ( A and B ) and OD 600 = ∼1 ( C ).

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Inhibition, Mutagenesis, Generated, Western Blot

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: A. Congo red inhibits LtaS activity . Non-treated (NT) and anti-FASII-adapted (AD) S. aureus JE2 overnight cultures were adjusted to the same OD 600 and dilutions were plated on solid SerFA medium without and with Congo Red (N=3). AD cultures show greater resistance than NT to Congo Red, possibly suggesting less reliance on LtaS. Black zones surrounding AD colonies on Congo Red indicates exopolysaccharide production [ , ]. B , C . LtaS is required for growth but does not restore LTA in anti-FASII-adapted S. aureus . B. The IPTG inducible locus iltaS established in the USA300 LAC derivative strain ANG2505 was grown overnight with 1 mM IPTG. Growth of ANG2505 NT (orange) and AD (treated by AFN-1252; green) cultures was followed without (dashed line) or with 1 mM IPTG (solid line). Results show the mean ± SD (standard deviation) of 4 biological replicates. C , D , E . LTA was detected by immunoblotting using anti-LTA antibody. C . Increasing LtaS expression does not restore LTA synthesis in FASII bypass conditions. The WT parental LAC strain and ANG2505 iltaS were grown in NT and AD conditions in the presence of 1 mM IPTG. N=3. D . PK150 stimulates SpsB, which enhances LtaS cleavage and reportedly increases LTA [ , ]. PK150 was added as indicated in NT and AD conditions and LTA production was assessed. E . Mutations in mspA derepress SpsB protease, leading to increased LTA . LTA levels in JE2 WT and mspA mutants (USA300_FPR3757 mutant positions 2379899 [ mspA 1 ] or 2380097 [ mspA 2 ]) were examined in exponential phase SerFA (NT) or SerFA-AFN (AD) cultures. Samples at right were also treated with 1 µM PK150 during growth (N=3).

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Activity Assay, Standard Deviation, Western Blot, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: S. aureus non-treated (NT), anti-FASII-adapted (AD), and Δ fabD (disabled for FASII) cultures were grown in A , 250 µM “Natural Mix”, which mimics endogenously produced FAs. B. The same strains and conditions as in A , plus a Δ plsX strain (deleted for plsX , see Supplementary Fig. 1 ; were grown in 250 µM ai 15, the major FA synthesized by S. aureus ; ai 15 is elongated to ai 17 and ai 19 only in the non-treated WT strain. A , B , upper: FA composition of membrane extracts determined by gas chromatography. Peak heights correspond to relative responses (mV) of each FA in a sample. Predominant FAs are indicated; N=2. A , B , lower: Cell extracts were prepared and submitted to immunoblotting using anti-LTA antibody N=3. Samples in B were run on the same gel and subjected to the same exposure time.

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Produced, Synthesized, Membrane, Gas Chromatography, Western Blot

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: A . Multiple biosynthetic pathways underly synthesis of LTA, and carry an energy cost. LTA production relies on 3 major biosynthetic pathways (FASII and FASII bypass , ①a and ①b; phosphatic acid synthesis, ②; and phosphatidylglycerol synthesis, ③) before branching to LTA synthesis. FAs are in green; lipoprotein sometimes comprises a third FA (dashed line) . ATP and GroP or Gro expenses (boxed in red), gains (in green), or neutral changes (in grey) during production of the 4 possible PG products, including LTA . For a single LTA molecule, one PG is used to produce the anchor, while ∼25 PGs donate GroP to produce the polymer. The GroP donors are then recycled to regenerate PGs, which requires ATP. Synthesis of a single LTA molecule costs about 150 ATPs , 25 GroPs, and 26 PGs. In contrast, production of the three other lipid products leads to positive or neutral ATP and GroP footprints. PG product distribution in the inner and outer membrane leaflets is asymmetric: cardiolipin, but not the other lipid products, is preferentially enriched in the membrane inner leaflet . B and C , bacteria were grown in SerFA (non-treated) or in SerFA-AFN (FASII bypass ). B. ATP pools are greater in FASII bypass conditions, as FASII bypass uses ∼7-10 times less ATP per FA molecule compared to FASII (see A ). N=5. C . FASII bypass causes depletion of GroP pools. These measurements do not include GroP net loss associated with depletion of the GroP polymer attached to LTA. N=6. Data in B and C are shown as mean values ± SD normalized to cognate measurements in non-treated samples; P-values were determined using the two-sided T-test. Full data sets for B and C is in and Source data.

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Polymer, Membrane, Bacteria

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: A . Lipid extractions were performed on non-treated (NT) and anti-FASII-adapted (AD) S. aureus JE2, and the Δ plsX derivative where indicated, from OD 600 = ∼3 cultures prepared in BHI plus 10% delipidated serum, supplemented by ‘Natural Mix’ (FA Mix), ai 15 and C18:0 (125 µM each), or ai 15 (250 µM). Relative mass proportions (% lipid (mass)) of monoglucosyl diacylglycerol (MG-DAG), diglucosyl diacylglycerol (DG-DAG; the LTA lipid anchor), phosphatidylglycerol (PG), and cardiolipin (CL) are shown, with data points (black dots), ranges, and average of biological duplicates. Of note, the Δ plsX strain produces LTA and its lipid distribution is similar to that of the WT NT strain. See Source data for original data readouts. B and C . WT S. aureus JE2 and LAC strains, and the Δ cls1 Δ cls2 (Δ cls12 ) LAC derivative were grown in SerFA without and with anti-FASII (AFN-1252; AFN). B . Growth of WT LAC and Δ cls12 strains was compared in NT and AD cultures, and was comparable in each condition (N=3). C . LTA detection by immunoblotting was performed on WT JE2 and LAC, and Δ cls12 cultures using anti-LTA antibody (N=3).

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The fatty acid synthesis pathway is a checkpoint for lipoteichoic acid synthesis in Staphylococcus aureus

doi: 10.64898/2026.04.06.715823

Figure Lengend Snippet: A and B . Detection of WTA by Alcian blue staining ( A ) and LTA by immunodetection ( B ) in non-treated (NT) and anti-FASII-adapted (AD) S. aureus JE2 (N=6 and N=5 respectively). Representative gels, and means ± SDs of the independent samples are presented; P-values were determined using the two-sided T-test. C . Growth and survival of S. aureus upon bitherapy treatment with anti-FASII (AFN-1252, 0.5 µg/ml) and anti-WTA (Targocil, 15 µg/ml for growth, and 5-15 µg/ml for survival). Bacteria were precultured in SerFA prior to simultaneous antibiotic addition. Growth was monitored on a Tecan plate reader. Survival after 15 h growth was determined by serial dilution plating of cultures (5 µL per spot) that were treated with one or two antibiotics as indicated above each column.

Article Snippet: They were then diluted in SerFA to OD 600 = 0.1 without or with antibiotics, and growth was followed for at least 10 h. FASII inhibitors were AFN-1252 (anti-FabI; Bioaustralis, Australia) or platensimycin (anti-FabF, MedChemExpress, France), both used at 0.5 μg/ml.

Techniques: Staining, Immunodetection, Bacteria, Serial Dilution