Biogenesis and maintenance of the outer membrane of Gram-negative bacteria

革兰氏阴性菌外膜的生物发生和维持

• 批准号: 10693911
• 负责人: Thomas J. Silhavy
• 金额: $ 80.33万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10693911
• 关键词: Antibiotics; Architecture; Biogenesis; Biological Models; Cell surface; Cells; Collection; Combined Modality Therapy; Cytoplasm; Defect; DegP protease; Environment; Enzymes; Escherichia coli; Face; Funding; Genes; Goals; Gram-Negative Bacteria; Island; Knowledge; Ligase; Lipid Bilayers; Lipids; Lipopolysaccharides; Lipoproteins; Maintenance; Membrane; Modeling; Molecular; Molecular Chaperones; Mutation; Nature; O Antigens; Pathway interactions; Peptides; Periodicity; Phenotype; Phospholipids; Physiology; Process; Proteins; Role; Surface; Testing; Work; aqueous; beta barrel; biological adaptation to stress; cell envelope; cell killing; enterobacterial common antigen; insight; membrane biogenesis; mutant; new therapeutic target; novel; novel antibiotic class; periplasm; protein protein interaction; response; sortase; tool; trafficking; translational study

PROJECT SUMMARY The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and β-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins required to assemble LPS (LptABCDEFG) and OMPs (BamABCDE) in the OM and we have provided evidence of a diffusive mechanism of phospholipid transport between the IM and the OM. In the current funding period, we have shown that the conditional lethal phenotype of bamB bamE double mutants can be suppressed simply by deleting a surface-exposed lipoprotein, we revealed the existence of an alternate lipoprotein trafficking pathway, we uncovered a role for the cyclic form of Enterobacterial Common Antigen in maintaining the OM barrier, and we identified mutations that activate or prime the σE stress response that suppress a variety of OMP and Bam defects. In translational studies, we used our knowledge of OM biogenesis to discover a new class of antibiotics that work to inhibit BamA at the cell surface. We propose to use our large collection of mutations that alter the Bam components or various OMP substrates together with our collection of suppressors as tools to probe the OMP assembly process. In particular, we will probe the function of the non-essential BamBCE lipoproteins and test our hypothesis that BamD does not perform a truly essential mechanistic role, but rather functions as a regulator to control the activity of BamA. We will test the role of the chaperone Skp as a specific adaptor for the periplasmic protease DegP. We also posit that the trimeric nature of the major OMPs functions as a global organizer of OM architecture by providing multiple interacting faces to allow the protein-protein interactions necessary for the formation of protein islands. Our studies on LPS assembly will utilize a mutant O-antigen ligase and the enzyme sortase to attach peptides or proteins to LPS to challenge the capabilities of the LptDE translocon. We will also test our model that three essential IM proteins, YejM, YciM, and FtsH comprise a novel pathway that regulates LPS synthesis in response to the lipid status of the OM. The mlaA* mutation destabilizes the OM by increasing LPS levels. This causes membrane loss by OM vesiculation and IM PLs flow into the OM to replace the loss. We have identified a mutation that slows this lipid flow and we believe that continued study of this gene may provide insights into the poorly understood process of anterograde PL transport.

项目总结

项目成果

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen.

• DOI: 10.1128/mbio.02846-21
• 发表时间: 2021-12-21
• 期刊: mBio
• 影响因子: 6.4
• 作者: Rai AK;Carr JF;Bautista DE;Wang W;Mitchell AM
• 通讯作者: Mitchell AM

Trade-offs constrain adaptive pathways to the type VI secretion system survival.

• DOI: 10.1016/j.isci.2023.108332
• 发表时间: 2023-12-15
• 期刊: ISCIENCE
• 影响因子: 5.8
• 作者: Macgillivray, Kathryn A.;Ng, Siu Lung;Wiesenfeld, Sophia;Guest, Randi L.;Jubery, Tahrima;Silhavy, Thomas J.;Ratcliff, William C.;Hammer, Brian K.
• 通讯作者: Hammer, Brian K.

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine.

• DOI: 10.1073/pnas.2114997119
• 发表时间: 2022-01-04
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Combs AN;Silhavy TJ
• 通讯作者: Silhavy TJ

Physical properties of the bacterial outer membrane.

• DOI: 10.1038/s41579-021-00638-0
• 发表时间: 2022-04
• 期刊: Nature reviews. Microbiology
• 作者: Sun J;Rutherford ST;Silhavy TJ;Huang KC
• 通讯作者: Huang KC

A periplasmic phospholipase that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa.

• DOI: 10.1073/pnas.2302546120
• 发表时间: 2023-07-25
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Guest, Randi L.;Lee, Michael J.;Wang, Wei;Silhavy, Thomas J.
• 通讯作者: Silhavy, Thomas J.