Oxidative stress is intrinsic to staphylococcal adaptation to fatty acid synthesis antibiotics

Abstract

Antibiotics inhibiting the fatty acid synthesis pathway (FASII) of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed the consequences and effectors of FASII-antibiotic (anti-FASII) adaptation. Anti-FASII induced lasting expression changes without genomic rearrangements. Several identified regulators affected the timing of adaptation outgrowth. Adaptation resulted in decreased expression of major virulence factors. Conversely, stress responses were globally increased and adapted bacteria were more resistant to peroxide killing. Importantly, pre-exposure to peroxide led to faster anti-FASII-adaptation by stimulating eFA incorporation. This adaptation differs from reports of peroxide-stimulated antibiotic efflux, which leads to tolerance. In vivo, anti-FASII-adapted S. aureus killed the insect host more slowly but continued multiplying. We conclude that staphylococcal adaptation to FASII antibiotics involves reprogramming, which decreases virulence and increases stress resistance. Peroxide, produced by the host to combat infection, favors anti-FASII adaptation.

Keywords: Microbial metabolism; Microbiology.

Graphical abstract

Figure 1

Proteomic kinetics and global expression changes related to S. aureus USA300 strain adaptation to anti-FASII (A) Schematics of sample preparation. Samples (4 biological replicates) were grown and prepared in conditions and harvesting times (or OD₆₀₀ for controls) as indicated below flasks. (B) Sample growth. Means and standard deviations are indicated. Points correspond to sampling times. See Table S1 for details. Dashed blue line represents typical growth in SerFA medium determined separately. S. aureus adapts to anti-FASII within 10h on SerFA-Tric, but not on FA-Tric, after a latency period (double arrow). (C) Heatmap of proteins showing altered levels in at least 1 condition relative to other samples. All sample conditions are shown. Sampling times (h) previous steps correspond to 2, 4, 6, 8, and 10 h for FA-Tric and SerFA-Tric. The heatmap shows global protein changes, and is determined relative to weighted value for each protein, as on scale at left (navy, down-represented; yellow, up-represented; see STAR Methods for analyses).

Figure 2

Anti-FASII affects pools and phosphorylation status of regulatory proteins (A) Expression of known or putative regulators that differ in at least one time point during anti-FASII adaptation (SerFA-Tric) compared to non-treated (NT) SerFA sample. Changes in putative regulatory protein levels are shown in anti-FASII adaptation SerFA medium (see Table S1 for all test conditions). Gene loci and names are at right. Times (h) of sampling are indicated above green steps. Heatmap (scale at left) is determined relative to weighted value for each protein (navy, down-represented; yellow, up-represented; see STAR Methods for analyses). (B) Phosphorylated regulatory proteins with altered expression during anti-FASII adaptation. Peptides showing differential phosphorylation at 10 h post-anti-FASII adaptation are indicated (see Table S2 for complete information). ^a Peptide positions in respective protein sequences are indicated. Phosphorylated amino acids are in bold red or in bold black when there is an ambiguity. (C) Effects of regulator gene inactivation on anti-FASII adaptation. Kinetics of anti-FASII-adaptation of USA300 (WT) and insertional mutants, based on regulators with altered protein and/or phosphorylation levels (A and/or B; selected loci in bold), were compared to that of the parental strain. Cultures were grown without (squares) and with anti-FASII AFN-1252 (circles). Growth curves and standard deviations are based on biological triplicates.

Figure 3

Expression changes during anti-FASII treatment (A) Heat maps of known or putative virulence factors. Samples shown are in anti-FASII adaptation medium (SerFA; see Table S1 for results in all test conditions). Kinetics (in h) of sampling is indicated above green steps. Gene loci, protein names, and functional categories are at right. Correspondence between color and expression in heatmap (scale at left) is determined relative to weighted value for each protein (navy, down-represented; yellow, up-represented see STAR Methods for analyses). (B) Differential NT and anti-FASII-adapted S. aureus adhesion to macrophage. Non-treated (NT) or AFN-1252-treated S. aureus USA300 (3x10⁵) were added to of THP-1 macrophage monolayers (3x10⁵ cells per well), and incubated for 1 h at 4°C. Colony forming units (CFU) of bacteria that adhered to macrophage were determined on five independent bacterial samples; means and standard errors are shown (p = 0.003; see STAR Methods). (C) Secreted virulence factor activities. Non-treated (NT) and anti-FASII-adapted cultures treated with triclosan (Tric-ad) or AFN-1252 (AFN-ad) were grown overnight, and reached similar OD₆₀₀ values (=13, 9 and 9 respectively for NT, Tric-ad, and AFN-ad). Cultures (for protease detection) and culture supernatants (lipase, nuclease, and hemolysin detection) were prepared (see STAR Methods) and spotted on appropriate detection medium. Representative results of 3 biologically independent replicates are shown (Figure S6 for replicate results).

Figure 4

Anti-FASII adaptation confers oxidative stress resistance, and is accelerated by prior exposure to peroxide stress (A) Stress response heatmap. Results are shown for anti-FASII adaptation medium (SerFA; see Table S1 for all test conditions). Sampling times (h) are indicated above green steps. Gene names and functional categories are at right. Heatmap (scale at left) is determined relative to weighted value for each protein (navy, down-represented; yellow, up-represented; see STAR Methods for analyses). (B) S. aureus anti-FASII adaptation confers increased H₂O₂ resistance. Upper: USA300 non-treated (NT, orange bar) and AFN-1252-adapted overnight cultures (AD, green bar) were challenged with 0.5 mM H₂O₂ for 5 h, and CFUs were determined; means and standard errors are shown. ∗∗, p < 0.01. Lower: Lawns of NT and AD cultures (100 μL of dilutions adjusted to OD₆₀₀ = 0.1) were prepared on SerFA solid medium, and plates were spotted with 1.5 mm H₂O₂ and 4 nm (1.5 μg) AFN-1252, and photographed after 48 h incubation at 37°C. Representative of 3 independent assays. (C) Priming S. aureus with H₂O₂ accelerates anti-FASII adaptation and requires PerR. Upper: USA300 (WT) and perR (SAUSA300_1842) mutant SerFA cultures were grown overnight without or with 0.5 mM H₂O₂. Cultures were diluted (OD₆₀₀ = 0.1) in SerFA without or with 0.5 μg/mL AFN-1252, and growth was monitored. Results are shown for biological triplicates. H₂O₂ –primed samples, without or with AFN addition at T0 are as indicated. Lower: FA profiles of indicated strains harvested at 6 h post-anti-FASII treatment (arrow in “C”). In green, FA profile of fully adapted H₂O₂-pretreated cultures at 10 h, shown here for perR and non-distinguishable from WT. 1, 2, and 3, eFAs present in SerFA medium (respectively C14, C16, and C18:1). At left of each profile, proportions of incorporated eFAs (%) are the average of two independent measurements (<3% difference between replicates).

Figure 5

Comparison of untreated and anti-FASII-treated S. aureus in a G. mellonella infection model Insects were injected with 10⁶ CFU S aureus USA300 that were either non-treated (NT) or preadapted to anti-FASII AFN-1252 (AD). (A) Insect mortality in NT and AD S. aureus. Survival was plotted using Kaplan-Meier with pooled values of biologically independent triplicates (60 insects per condition). Survival curves between treatment groups were analyzed by log rank (Mantel-Cox) tests, which showed that both NT and AD survival kinetics were significantly different from the PBS control group and from each other p values <0.001 (∗∗∗). The Cox proportional hazard model, conducted between NT and AD infected larvae, confirms that NT-infected larvae had 8.77-fold higher hazard than AD-infected larvae infected (p value = 0.001). (B) legend: “Left, bacterial CFUs in surviving larvae. CFUs were determined on insects infected as described in “A” and at the same time intervals. For NT, standard deviation (std dev) values were based on 9 insects at both 0 and 24 h points. For AD, the means and standard deviation were based on 9, 9, 9, and 8 for respective consecutive time points. †, no surviving insects. As all NT-infected larvae died by 48h, significance between NT and AD was only determined at 0 h and 24 h. This was performed by non-parametric Mann-Whitney test. Further experimentation is needed to analyze the significance of declining CFUs in the AD-treated insects. Right, bacterial CFUs in dead infected insects (9 NT- and 8 AD-infected larvae) at 48 h. Analyses were done using the non-parametric Mann-Whitney test (GraphPad Prism 9.5.1 software). ∗, p = 0.02; ns, non-significant.

Figure 6

Summary model of anti-FASII adaptation and S. aureus fitness S. aureus synthesizes fatty acids (FAs) to produce membrane phospholipids (upper left, FAs in orange). Anti-FASII treatment in SerFA medium promotes FASII bypass, during which exogenous FAs (eFAs, in green) are incorporated and constitute the membrane phospholipid FAs. FASII bypass is accelerated by H₂O₂ priming, which requires PerR, but is lower if KatA is present. Anti-FASII adaptation is accompanied by massive changes in protein expression. Membrane perturbation in the new phospholipid environment is proposed as the primary signal for protein reprogramming; as reported, membrane FAs or phospholipids may also shed internally and bind to regulatory proteins to modulate their function.^,,, Decreased virulence factor production may help bacteria escape host immune surveillance. Up-regulation of stress response protein levels confers greater ROS tolerance, and could facilitate survival during infection. Peroxide priming accelerates FA incorporation and anti-FASII adaptation by a novel PerR-dependent process. Anti-FASII treatment would favor emergence of S. aureus populations that are transiently less infectious, but that may persist in the host.