Characterization of the Mechanisms of Daptomycin Resistance among Gram-Positive Bacterial Pathogens by Multidimensional Lipidomics

Abstract

This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E. faecalis, S. aureus, and C. striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S. aureus and C. striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

Characterisation ofStaphylococcus aureuslipids by nanoelectrospray ionisation tandem mass spectrometry (nESI-MS/MS)

“…The presence of unsaturated lipids in S. aureus is somewhat controversial, with a common assumption in the community that the biophysical role of unsaturated fatty acids is substituted by the abundant branched-chain fatty acids in Gram-positive bacteria [25]. Indeed, in two recent LC-MS studies on S. aureus lipids, one reported only saturated lipids to exist in Col and Newman strains [20], while the other on the N315 strain detected lipids containing unsaturated fatty acid chains (specifically C29:1 to C35:1 PG species) [21], though no comment was made on the existence of these molecular species. However, it is common to have minor variations in fatty acid contents and thus the profiles of bacterial lipidomes due to variations in growth phase, choice of strain and culture media, and indeed these earlier studies utilised brain-heart infusion media [20,21].…”

“…Nevertheless, a need exists for a rapid, sensitive and accurate quantitative method to characterise in detail the lipid composition of the S. aureus cell membrane. The recent applications of two distinct liquid chromatographic (LC) MS approaches, reversed-phase LC-MS [20] and hydrophilic interaction LC-ion mobility-MS [21], have addressed some of the limitations in earlier methodologies. Both of these studies describe similar, but not identical S. aureus lipidomes, but these remain incomplete, as the authors did not obtain positional assignments for the individual fatty acid chains to enable a full characterisation of each lipid species.…”

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

“…For example, more cationic antibiotic resistant bacteria have been shown to change their overall membrane surface charge from negative to neutral, and in the most resistant strains, to positive (48). The membranes of S. aureus have been shown to be composed primarily of PG and LPG phospholipids while E. faecalis has been shown to contain PG, CL, DGDG and some LPG phospholipids (5,49). In order to further support the difference in surface charge expected between the bacterial species, we examined whether there were overall differences in the abilities of S. aureus and E. faecalis to bind the positively charged (cationic) protein cytochrome c using a previously described method by Peschel et al (39).…”

“…S. aureus is known to have a greater percentage of branched acyl chain structures in its lipidome in comparison to E. faecalis (4,48,49,53,54). In addition, the CL headgroup that is more prevalent in E. faecalis attaches four chains, as opposed to two chains for the other headgroups mentioned, which could also contribute to altered fluidity (5,49,55). While it is well known that bacteria do change their lipid tail composition in response to environmental temperature (2), the potentially more fluid membrane environment of S. aureus due to branched acyl chains may lead to the more dynamic behavior of the probes in these membranes over longer time periods.…”

“…The underlying mechanism behind this contrasting activity is currently unclear. Multiple studies have investigated the composition of the membranes of these two bacteria and their differences (3)(4)(5)(6)(7)(8), but there is significantly less work in the field examining the broader implications of how these differences impact small molecule interactions. Here, we measure second harmonic generation (SHG) simultaneously with two-photon fluorescence (TPF) to examine differences between these bacterial species in vivo as well as their sensitivity to small structural changes to the adsorbing molecules.…”