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PROJECT SUMMARYDuring an infection, the bacterial envelope mediates interactions between bacteria and their hosts throughstructures such as adhesins and polysaccharides. The structure and composition of the cell envelope are alsocrucial determinants of whether a bacterium will be defeated by or resistant to antibiotics. For example, Gram-negative bacteria are naturally resistant to many antibiotics and detergents because their outermost envelopelayer, the outer membrane, excludes hydrophobic small molecules that otherwise can cross typical lipid bilayers.Therefore, understanding how Gram-negative bacteria build their cell envelope and maintain envelope integrityand impermeability is crucial for the development of new antibacterial strategies to combat infections.Our long-term goal is to understand at the molecular level how Gram-negative bacteria build their cell envelope.The Gram-negative envelope is delimited by two lipid bilayers (the inner and outer membranes), which areseparated by an aqueous compartment (the periplasm) where a thin layer of peptidoglycan cell wall resides.Although many factors involved in the biogenesis and maintenance of the cell envelope have been identified inthe last few decades, there are still many envelope proteins whose function remains unknown. This proposalseeks to investigate the AsmA-like protein clan, which includes proteins that have been reported to be importantfor bacterial-host interactions, as well as for maintaining the impermeability to antibiotics and the integrity of thecell envelope of some Gram-negative bacteria. Furthermore, we have found that members of the AsmA-likeprotein clan are ubiquitously present in diderm bacteria, even Gram-negative endosymbionts that haveundergone massive genome reduction. From these lines of evidence, we hypothesize that AsmA-like proteinsperform a yet-to-be-identified fundamental function in the envelope of double membrane bacteria. We also arguethat this function has remained elusive in organisms used as models to study envelope biogenesis and functionbecause of redundancy among AsmA-like paralogs. Indeed, our preliminary data has uncovered functionalredundancy among AsmA-like paralogs in the Gram-negative bacterium Escherichia coli.Here, we propose to take advantage of the genetics system, and the extensive base of knowledge andexperimental tools available to study the cell envelope of E. coli in order to conduct the first in-depth functionalcharacterization of multiple AsmA-like paralogs. We expect that the proposed studies will identify: 1) thecontribution of each AsmA-like paralog to envelope homeostasis; 2) the functional relationship between AsmA-like paralogs; 3) which pathways are affected by the loss of AsmA-like proteins; and 4) potential functionalpartners of AsmA-like proteins. To achieve these goals, we will combine genetic and biochemical approachestogether with detailed phenotypic characterization, a combination that has been proven effective for theidentification and characterization of envelope biogenesis factors. The knowledge gained from the proposedstudies will contribute to the development of novel therapies to combat infections by Gram-negative pathogens.

Ruiz, Natividad
Ohio State University
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