Comparative study on nutrient depletion-induced lipidome adaptations in Staphylococcus haemolyticus and Staphylococcus epidermidis

Staphylococcus species are emerging opportunistic pathogens that cause outbreaks of hospital and community-acquired infections. Some of these bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to multiple antibiotics. We carried out a comparative study on the lipidome adaptations in response to starvation in the two most common coagulase-negative Staphylococcus species: a S. epidermidis strain sensitive to ampicillin and erythromycin and a S. haemolyticus strain resistant to both. The predominant fatty acid composition in glycerolipids was (17:0–15:0) in both bacteria. During the exponential phase, the two bacterial lipidomes were similar. Both were dominated by diacylglycerol (DAG), phosphatidylglycerol (PG), lysyl-phosphatidylglycerol (Lysyl-PG) and Diglucosyl-diacylglycerol (DGDG). Alanyl-PG was detected in small amounts in both bacterial lipids. N-succinyl-lysyl-PG was detected only in S. haemolyticus, while lysyl-DAG only in S. epidermidis. As the two bacteria entered stationary phase, both lipidomes became essentially nitrogen-free. Both bacteria accumulated large amounts of free fatty acids. Strikingly, the lipidome of S. epidermidis became dominated by cardiolipin (CL), while that of S. haemolyticus was simplified to DGDG and PG. The S. epidermidis strain also produced acyl-phosphatidylglycerol (APG) in the stationary phase.


Supplementary data
Introduction: Here we present the microbial typing results, and the manually assigned fatty acid compositions based on tandem mass spectrometry acquired with the 4000 QTRAP system. The fatty acyl assignment was mainly based on dissociation patterns described in the following two articles: Murphy, R.C. and P.H. Axelsen, Mass spectrometric analysis of long-chain lipids.
Coulon, D. and Bure, C. Acylphosphatidylglycerol (acyl-PG) or Nacylphosphatidylethanolamine (NAPE). J. Mass Spectrom, 2015Spectrom, . 50. 1318 In negative mode, the [FA-H]ion dissociated from the sn-2 position is generally 2-3 fold more abundant than that from the sn-1 position. We did a rather complete survey of PG and lysyl-PG. The compositions of DAG and DGDG were based on the assumption they were produced from the same pool of DAG for synthesizing phospholipids. Their MS/MS spectra in positive mode, especially their [DAG-OH] + and [MAG-OH] + fragments, were used to confirm the two fatty acyl composition.  Since PG anions were the most abundant, we were able to acquire meaningful MS/MS spectra of several of such anions and assigned their fatty acid compositions, as listed in table S1. These compositions serve as a near complete survey of fatty acid compositions in glycerophospholipids found in the two staphylococcus strains.
Many of these PG anions were mixtures of multiple fatty acid compositions, which are listed in the order of descending abundance of corresponding FA anions in the MS/MS spectra. As we follow the general rule that fatty acid anion dissociated from the sn-2 position is more abundant by 2-3 fold, it appeared that fatty acyls at the sn-1 position were always equal to or larger that those at the sn-2 position. The minor compositions of FA pairs were assigned based on their equal overall mass to that of the major composition and that the ion counts of each pair of FA anions differ by 2-3 fold.  Since lysyl-PG anions were also abundant, we were able to acquire meaningful MS/MS spectra of several of such anions and assigned their fatty acid compositions, as listed in table S2. As in PG anions, it appeared that fatty acyls at the sn-1 position were always equal to or larger that those at the sn-2 position.  DGDG anions were not abundant in negative mode, and they overlap with lysyl-PG anions with larger fatty acyl groups. Sodiated DGDG ion s were chosen as the target species to survey this type of lipids by mass spectrometry. These cations produced strong signals due to neutral loss of FAs, which were used to confirm the identity of the two fatty acyl groups. We also acquired tandem MS spectra of several [DGDG+NH 4 ] + ions, which produced extremely strong signals of [DAG-OH] + as well as [MAG-OH] + fragment ions. The assigned fatty acyl compositions are listed in table S4.  Tri-acylated lipid APG was only observed in lipids extracted from S. epidermidis in the stationary phase. After purification by TLC, we were able to acquire meaningful MS/MS spectra of four such anions and assigned their fatty acid compositions, as listed in table S5. The two fatty acyl compositions of the DAG moiety and that of the head group glycerol is separated by a slash. We noticed that all these four MS/MS spectra shared one feature that the 241 m/z fragment ion was at least 4-fold as intense as other fatty acid anions. This intensity difference could not be explained by the difference at the sn-1 and sn-2 positions in PG or lysyl-PG, which is normally 2.5 fold favoring the sn-2 fatty acid anion. We tentatively interpreted this unusual intensity of the 241 anion as suggesting that all APG is likely formed by acylation of the PG head group by a (15:0) fatty acid (242 amu). In addition to the strong signals at 241 m/z and 269 m/z corresponding to (15:0) and (17:0) FA anions, this MS/MS spectrum also revealed much weaker signals of (14:0) and (18:0) FA anions at 227 m/z and 283 m/z, respectively. Remarkably, intensity ratio of the two weaker signals was 2.2, indicating a (18:0-14:0) minor composition of the phosphatidyl group, and leading to the assignment of a common (15:0) fatty acyl to the second glycerol moiety. Both CL and LCL were observed in noticeable amounts only in lipids extracted from S. epidermidis in the stationary phase. After purification by TLC, we were able to acquire meaningful MS/MS spectra of several CL double anions and assigned their fatty acid compositions, as listed in table S6. The two fatty acyl compositions of the two phosphatidyl moieties were based on FA anions and cyclic PG anions. For instance, the above spectrum suggested a cyclic PG anion at 703 m/z, matching a fatty acid composition of (32:0). The FA anions at 241 and 269 m/z suggest the fatty acids are (17:0) and (15:0) at the sn-1 and sn-2 positions, respectively.

Table S5. [APG -H] -(m/z) fatty acid composition
There was a rather wide spectrum of CL produced by S. epidermidis during the stationary phase. Their fatty acid compositions were assigned manually and listed in Table S6.  Both CL and LCL were observed in noticeable amounts only in lipids extracted from S. epidermidis in the stationary phase. After purification by TLC, we were able to acquire meaningful MS/MS spectra of several LCL double anions and assigned their fatty acid compositions, as listed in table S7. The two fatty acyl compositions of the two phosphatidyl moieties were based on FA anions and cyclic PG anions. For instance, the above spectrum suggested a cyclic PG anion at 703 m/z, matching a fatty acid composition of (32:0). The FA anions at 241 and 269 m/z suggest the fatty acids are (17:0) and (15:0) at the sn-1 and sn-2 positions, respectively. An extra (17:0) FA must be assigned to the other glycerol moiety.

PG-H 3 O
There was much less LCL than CL anions that were observed at adequate intensity for MS/MS analysis. The longer FA on the sn-1 position of the monoacylated glycerol moiety appeared to be preserved in LCL, implying activity of a phospholipase A2.  Lysyl-DAG was observed in lipids extracted from S. epidermidis in the exponential phase. It did not form a visible band on thin-layer chromatogram. We could not rule out the possibility that this lipid species may be the product of spontaneous chemical reaction between two major lipids of DAG and lysyl-PG in the extract.  The peaks were clustered arounf 721 m/z separated by 14 m/z, indicating lack of cardiolipin or lysocardiolipin double anions which would be separated by 7 m/z. Cardiolipin double anions was expected to cluster around a major peak at 675 m/z, which was not observed. As shown in Figure S1 and Table S1, lack of any monolysoor dilyso-cardiolipin fragments in the MS/MS spectra of the major precursors further indicates that they are PG anions with varied fatty acid composition. In comparison with the precursor scans of lipids from S. haemolyticus in exponential phase shown in Figure S10, the profile of the spectra shown above are very similar except for noticeable decrease in intensity by nearly an order of magnitude. There was no indication cardiolipin or lysocardiolipin accumulated in the bacterial