Hopanoids as functional analogues of cholesterol in bacterial membranes

Sáenz, J. P.; Grosser, D.; Bradley, A. S.; Lagny, T. J.; Lavrynenko, O.; Broda, M.; Simons, K. (2015) Proceedings of the National Academy of Sciences, 112(38), 11971–11976. DOI: 10.1073/pnas.1515607112

Summary

Having established in model membranes that Diplopterol can form liquid-ordered phases analogously to cholesterol (Sáenz et al., 2012, PNAS), we asked whether hopanoids actually perform this function in living bacteria. Working with [[methylobacterium-extorquens|Methylobacterium extorquens]], a gram-negative plant-associated bacterium, we showed that hopanoids are localized to the outer membrane, where they interact with lipid A to determine membrane order — analogous to sterol-sphingolipid interactions in eukaryotic plasma membranes. Deletion of hopanoid synthesis (ΔSHC mutant) reduced outer membrane order and impaired energy-dependent multidrug efflux, revealing a link between membrane order and active transport in prokaryotes.

Key Findings

• Hopanoid localization: Diplopterol and 2-methyl-diplopterol are highly enriched in the outer membrane (OM) of M. extorquens, comprising ~19 mol% of total lipids (phospholipids, LPS, and diplopterols), as shown by TLC of purified membrane fractions.
• Hopanoids determine OM order: The ΔSHC mutant (squalene-hopene cyclase deletion) has significantly lower OM order than wild-type, as measured by C-laurdan GP. Depleting hopanoids from WT OM decreases order; loading diplopterol into ΔSHC OM increases order. Loading cholesterol into ΔSHC OM also restores order, demonstrating functional interchangeability.
• Preferential interaction with lipid A: Gibbs excess free energy of mixing (ΔGex) measurements on a Langmuir trough showed that diplopterol interacts favorably (negative ΔGex) only with saturated lipids — lipid A and sphingomyelin — and repulsively with unsaturated phospholipids. This contrasts with cholesterol, which interacts favorably with phospholipids of varying unsaturation.
• Detergent sensitivity: The lethal concentration of Triton X-100 is >1,000-fold lower in ΔSHC than in wild-type, a phenotype consistent with impaired multidrug transport.
• Impaired multidrug efflux: Using NPN uptake and Hoechst 33342 efflux assays, we demonstrated that the ΔSHC mutant accumulates dyes that wild-type cells actively pump out. The 2.2-fold higher NPN accumulation slope indicates increased OM permeability, but the >1,000-fold reduction in detergent tolerance cannot be explained by permeability alone — impaired energy-dependent efflux is the primary cause.
• M. extorquens also produces extended-side-chain polar hopanoids (BHT-CE and BHT-GCE), though diplopterol and 2-methyl-diplopterol are the major hopanoids.

Methods

• Membrane fractionation: gradient centrifugation to separate OM and inner membrane fractions, analyzed by TLC with MS confirmation of hopanoid identity.
• C-laurdan and Di-4-ANEPPDHQ spectroscopy: fluorescent probes responsive to membrane packing/order applied to purified OM fractions.
• Methyl-β-cyclodextrin: used to deplete hopanoids from WT OM or load diplopterol/cholesterol into ΔSHC OM, demonstrating reversibility of order changes.
• Langmuir trough ΔGex measurements: quantified the favorability of diplopterol and cholesterol interactions with different lipid classes.
• NPN uptake assay and Hoechst 33342 efflux assay: standard methods to measure membrane permeability and energy-dependent multidrug transport, respectively. CCCP used to inhibit ATP synthesis; succinate addition to restore it.

Significance

This paper moved the hopanoid-sterol analogy from model membranes into living cells. The convergence between hopanoid-lipid A ordering in bacteria and sterol-sphingolipid ordering in eukaryotes reveals a deeply conserved principle of membrane organization. The link to multidrug efflux has practical implications: hopanoid biosynthesis pathways could be targets to combat bacterial multidrug resistance. The finding that diplopterol interacts selectively with saturated lipids (unlike cholesterol) adds nuance to the analogy and reflects the distinct lipid composition of bacterial outer membranes versus eukaryotic plasma membranes.