ORCID Profile
0000-0001-7672-5153
Current Organisation
Princeton University
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Publisher: American Society for Microbiology
Date: 06-2019
DOI: 10.1128/JB.00745-18
Abstract: Gram-negative bacteria, such as Escherichia coli , inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σ E stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.
Publisher: American Society for Microbiology
Date: 11-01-2013
DOI: 10.1128/JB.02142-12
Publisher: Proceedings of the National Academy of Sciences
Date: 17-06-2014
Abstract: The outermost membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), and its proper placement on the cell surface is required to establish an effective permeability barrier. The presence of LPS prevents small hydrophobic molecules from entering the cell making it difficult to develop antibiotics. LPS is synthesized inside the cell and must move across three compartments to the cell surface. The final step of LPS transport (Lpt), translocation of LPS across the outer membrane, is accomplished by a two-protein complex (LptDE) that must insert LPS into its outer leaflet. We have identified a binding site within LptE critical for the proper function of the translocon. LptE binds LPS and changes its physical state to facilitate its translocation through the outer membrane.
Publisher: American Society for Microbiology
Date: 02-05-2018
Abstract: The outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM’s barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified an Escherichia coli mutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle. IMPORTANCE The outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these mislocalized phospholipids has a second, equally important function as a sensor. The fatty acids produced by hydrolysis of the phospholipids act as second messengers to signal the cell that more lipopolysaccharide is needed. These fatty acids diffuse across the periplasm and are transported into the cytoplasm by a process that attaches coenzyme A. The acyl-CoA molecule produces signals to inhibit the degradation of the critical enzyme LpxC by the ATP-dependent protease FtsH, increasing lipopolysaccharide production.
Publisher: Proceedings of the National Academy of Sciences
Date: 29-02-2016
Abstract: The cell envelope of Gram-negative bacteria contains two membranes and a cell wall located in the aqueous compartment between them. The outer membrane (OM) functions as a barrier that contributes to antibiotic resistance. We describe a dominant mutation in a gene for an OM lipoprotein that leads to cell death under starvation conditions in medium with limited cation concentrations. We show that death occurs not by rapid cell lysis but by a previously uncharacterized mechanism involving flow of material from the inner membrane to the OM that results in rupture of the inner membrane and the slow leakage of cytoplasmic contents. Our study highlights the vital need for balanced synthesis across the Gram-negative envelope and may empower the development of new therapeutics.
Publisher: eLife Sciences Publications, Ltd
Date: 31-12-2014
DOI: 10.7554/ELIFE.05334
Abstract: The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs).
Publisher: American Society for Microbiology
Date: 15-12-2014
DOI: 10.1128/JB.02298-14
Publisher: Elsevier BV
Date: 11-2017
Publisher: American Society for Microbiology
Date: 04-05-2016
Abstract: The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.
No related grants have been discovered for Thomas Silhavy.