Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

Download Full-text

Lipid Acyl Chain Remodeling in Yeast

Lipid Insights ◽

10.4137/lpi.s31780 ◽

2015 ◽

Vol 8s1 ◽

pp. LPI.S31780 ◽

Cited By ~ 1

Author(s):

Mike F. Renne ◽

Xue Bao ◽

Cedric H. De Smet ◽

Anton I. P. M. De Kroon

Keyword(s):

Intracellular Transport ◽

De Novo ◽

Acyl Chain ◽

Model Organism ◽

Lipid Synthesis ◽

Membrane Lipid ◽

Lipid Homeostasis ◽

Synthetic Process ◽

Acyl Chains ◽

Phospholipid Acyl Chain

Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation. Experimental evidence for the occurrence of phospholipid acyl chain exchange in cardiolipin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine is summarized, including methods and tools that have been used for detecting remodeling. Progress in the identification of the enzymes involved is reported, and putative functions of acyl chain remodeling in yeast are discussed.

Download Full-text

Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽

10.1128/mbio.00395-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 14

Author(s):

Matthew J. Belousoff ◽

Zohar Eyal ◽

Mazdak Radjainia ◽

Tofayel Ahmed ◽

Rebecca S. Bamert ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Structural Information ◽

Structural Basis ◽

Common Mechanism ◽

Resistance Mutations ◽

Antimicrobial Drugs ◽

Linezolid Resistance ◽

Drug Modification ◽

Antibiotic Resistance Mutations ◽

Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

Download Full-text

First person – Wei Sheng Yap

Journal of Cell Science ◽

10.1242/jcs.256016 ◽

2020 ◽

Vol 133 (21) ◽

pp. jcs256016

Keyword(s):

Biological Sciences ◽

Membrane Lipid ◽

Early Career ◽

Lipid Homeostasis ◽

First Person ◽

Early Career Researchers ◽

Technological University ◽

Selection Of

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Wei Sheng Yap is first author on ‘The yeast FIT2 homologs are necessary to maintain cellular proteostasis and membrane lipid homeostasis’, published in JCS. Wei Sheng works in the lab of Guillaume Thibault in the School of Biological Sciences, Nanyang Technological University, Singapore, studying the interplay between the proteostasis network and lipid homeostasis.

Download Full-text

Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel

Science ◽

10.1126/science.aav9334 ◽

2019 ◽

Vol 363 (6430) ◽

pp. eaav9334 ◽

Cited By ~ 66

Author(s):

Ying Yin ◽

Son C. Le ◽

Allen L. Hsu ◽

Mario J. Borgnia ◽

Huanghe Yang ◽

...

Keyword(s):

Transient Receptor Potential ◽

Structural Information ◽

Receptor Potential ◽

Membrane Lipid ◽

Structural Basis ◽

Trpm8 Channel ◽

Allosteric Coupling ◽

Transient Receptor Potential Melastatin ◽

Lipid Sensing ◽

Transient Receptor

Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca2+)–permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo–electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with the menthol analog WS-12 and PIP2. Our structures reveal the binding sites for cooling agonists and PIP2in TRPM8. Notably, PIP2binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP2and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.

Download Full-text

Proteomic and Membrane Lipid Correlates of Re-duced Host Defense Peptide Susceptibility in a snoD Mutant of Staphylococcus aureus

Antibiotics ◽

10.3390/antibiotics8040169 ◽

2019 ◽

Vol 8 (4) ◽

pp. 169

Author(s):

Christian Kohler ◽

Richard Proctor ◽

Arnold Bayer ◽

Michael Yeaman ◽

Michael Lalk ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Compositional Data ◽

Insertion Site ◽

Growth Conditions ◽

Membrane Lipid ◽

Anaerobic Growth ◽

Short Chain ◽

Aureus Strain ◽

Branched Chain ◽

The Impact

We previously described a transposon mutant in Staphylococcus aureus strain SH1000 that exhibited reduced susceptibility to cationic thrombin-induced platelet microbicidal proteins (tPMPs). The transposon insertion site was mapped to the gene snoD, the staphylococcal nuo orthologue. Hence, further studies have been performed to understand how this mutation impacts susceptibility to tPMP, by comparing proteomics profiling and membrane lipid analyses of the parent vs. mutant strains. Surprisingly, the mutant showed differential regulation of only a single protein when cultivated aerobically (FadB), and only a small number of proteins under anaerobic growth conditions (AdhE, DapE, Ddh, Ald1, IlvA1, AgrA, Rot, SA2366, and SA2367). Corresponding to FadB impact on lipid remodeling, membrane fatty acid analyses showed that the snoD mutant contained more short chain anteiso-, but fewer short chain iso-branched chain fatty acids under both aerobic and anaerobic conditions vs. the parental strain. Based upon these proteomic and membrane compositional data, a hypothetical “network” model was developed to explain the impact of the snoD mutation upon tPMP susceptibility.

Download Full-text