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Project 2

Assigning function of unknown genes of Acinetobacter baumannii by cell envelope analysis

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Principal Investigator: Samuel Miller

Goals and Objectives

The overall goal of Project 2 is to identify genes of unknown function (GUF) that are essential in maintaining the cell envelope barrier of A. baumannii. Thus, we propose to undertake genome-wide transposon and chemical mutagenic screens to discover mutants that have altered cell envelope structure and function. The screen will be based on phenotypes that occur due to changes in envelope functions such as increased cell permeability, alteration in phospholipid composition, defects in cell division, increased cell density and alteration in capsule production. To conduct these screens, we have developed a series of FACS based cell sorting techniques. Next, using biochemical, phenotypical and microscopic methods developed in the lab, we will determine how these genes contribute to maintaining a healthy Gram-negative envelope. Additionally, using animal models, we will determine how the products from these genes promote resistance to and alter sensing by the host innate immune system. Lastly, we will integrate gene functions discovered in these aims with genomic, proteomics and gene expression data to build gene regulatory circuits using bioinformatics annotation and functional protein-protein interactions.


The Gram-negative envelope consists of: inner and outer membranes, a periplasmic space, a peptidoglycan cell wall and extracellular capsules. It is an integral structural component of Gram-negative bacteria that serves as a first line of defense by maintaining a barrier against toxic compounds, innate immune effector molecules, and antibiotics. Therefore, we hypothesize that complete identification of genes needed for normal envelope function in Acinetobacter will aid in understanding pathogenesis and developing new treatment/eradication programs.