This proposal will define genes of unknown function (GUF) of the Gram-negative bacteria Acinetobacter baumannii (Ab) that contribute to the structure and barrier function of the cell envelope. The Gram-negative bacterial envelope is an ordered complex of macromolecules that is unique in being comprised of two membranes. The outer membrane (OM), a unique structure composed of an asymmetrical bilayer consisting of lipopolysaccharide (LPS) (outer leaflet) and glycerophospholipids (inner leaflet), forms the first line of defense against antiseptics and antimicrobials by functioning as a permeability barrier. Ab has a novel cell envelope that is relatively impermeable to antibiotics, and colistin resistant Ab have been isolated that cannot synthesize LPS from patients treated with colistin. These novel properties suggest that the study of the Ab envelope should reveal important new knowledge of clinical relevance. Our preliminary data and published studies of transcriptional profiles of the Ab LPS null mutant have led to bioinformatics analysis and selection o f a set of Ab GUF that are candidates for participation in maintenance o f a regulated permeability barrier. These GUF will be characterized by construction of mutant strains that will be analyzed for altered permeability to chemical probes. Strains with altered envelope permeability will be subjected to susceptibility testing to antimicrobial peptides and antibiotics, chemical analysis of phospholipids and lipid A, and, when appropriate, testing in animal models of disease. In an iterative process to define new GUF for characterization in further years, we will employ specific genetic screens using FACS-based cell sorting, drug, and temperature sensitivity screens to identify GUF that contribute to cell envelope structure and function. Lastly, we will integrate data from the projects of Drs. Manoil and Harwood and utilize the technological core of Dr. Bruce to define additional GUF for characterization. This proposal will test the hypothesis that regulated undiscovered structural alterations of the Gram-negative envelope encoded by GUF are a component of pathogen defense against host innate immune, antimicrobial, and disinfectant attack.

Public Health Relevance

Opportunistic infections with antibiotic resistant organisms are an important medical problem. Many antibiotics in current use target the bacterial cell envelope. More information about the bacterial cell surface and its role in resistance to antimicrobials is essential to develop new antibiotics to treat antibiotic resistant infections. This proposal should lead to new scientific knowledge of the Gram-negative bacerial cell envelope that can be used to develop new treatments for antibiotic resistant opportunistic infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZAI1-FDS-M (M1))
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University of Washington
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Schweppe, Devin K; Chavez, Juan D; Lee, Chi Fung et al. (2017) Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry. Proc Natl Acad Sci U S A 114:1732-1737
Zhong, Xuefei; Navare, Arti T; Chavez, Juan D et al. (2017) Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers. J Proteome Res 16:720-727
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Miller, Samuel I (2016) Antibiotic Resistance and Regulation of the Gram-Negative Bacterial Outer Membrane Barrier by Host Innate Immune Molecules. MBio 7:
Schweppe, Devin K; Chavez, Juan D; Navare, Arti T et al. (2016) Spectral Library Searching To Identify Cross-Linked Peptides. J Proteome Res 15:1725-31
Wu, Xia; Chavez, Juan D; Schweppe, Devin K et al. (2016) In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075. Nat Commun 7:13414
Schweppe, Devin K; Chavez, Juan D; Bruce, James E (2016) XLmap: an R package to visualize and score protein structure models based on sites of protein cross-linking. Bioinformatics 32:306-8
Chavez, Juan D; Eng, Jimmy K; Schweppe, Devin K et al. (2016) A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry. PLoS One 11:e0167547
Schweppe, Devin K; Zheng, Chunxiang; Chavez, Juan D et al. (2016) XLinkDB 2.0: integrated, large-scale structural analysis of protein crosslinking data. Bioinformatics 32:2716-8

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