Study of secretory and membrane proteins of Pseudomonas aeruginosa
EMSL Project ID
2394
Abstract
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium isolated from soil, water, as well as host systems including plants, insects and vertebrates. It is one of the top three causes of opportunistic human infections, due in part to its intrinsic resistance to antibiotics and disinfectants. Over 80% of patients with cystic fibrosis acquire a P. aeruginosa infection that results in progressive loss of lung function and early death. It is also the most common causative organism of sepsis in burn patients, as well as of many other life-threatening infections in HIV, and cancer patients. The pathophysiology of infections due to P. aeruginosa is complex, as shown by the clinical diversity of the diseases associated with this organism and by the multiplicity of virulence factors it produces. The Objective of the proposed research is to elucidate the strategies employed by the pathogen for the success of infection and proliferation, and used by infected hosts for self-defense in the form of immune response. These understandings will contribute to the reduction in the incidence and complications of bacterial infections associated with P. aeruginosa and other pathogens sharing similar mechanisms. To achieve this important and challenging goal, an interdisciplinary approach has been adopted that takes advantage of expertise from leading groups in the field of genomics and molecular biology (Dr. R.W.Davis? lab at Stanford Medical School), bacterial genetics (Dr. L.G.Rahme's lab at Harvard Medical School), proteomics (Dr. R.D.Smith?s lab at EMSL/PNL), pathology and burn wound infections (Dr. R.G.Tompkins' lab at Massachusetts General Hospital), and bioinformatics (Dr. A.P.Arkins? lab at University of California at Berkeley). In this project, we propose to sequence the genome of a common virulent strain of P. aeruginosa, PA14. To determine virulent factors involved in pathogenic infection, research is underway to make systematic mutations in P. aeruginosa and to perform infection assays. Expression analysis will then be performed on virulent and non-virulent strains to elucidate infection related pathways. To study the host defense system, we are doing expression and mutational analysis on infected Drosophila and Arabidopsis. The results will be verified using mouse system. The study on P. aeruginosa proteins is essential for the success of this project. Specific Aim 1: The advanced high throughput proteome analysis using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry pioneered by Dr.Smith?s lab will be applied to identify accurate mass tags for the membrane and secretory proteins of P. aeruginosa. Currently, about five hundred or so genes are implicated in the infection process, but the location of the majority of these genes is not known. However, most of these virulent factors are expected to be secretory proteins, membrane proteins, or transcription factors in the cytosol. Therefore identifying the location of these genes shall provide us significant clues about their functions in pathogenicity, which will be further studied by gene knock-out experiments and infection assays. Specific Aim 2: Comparative analysis of the virulent wild type and non-virulent strains due to deletions of virulence-related transcription factors. By identifying the differences in secretory and membrane proteins, together with data from gene expression analysis using DNA microarray, we shall gain thorough knowledge on the regulatory circuits of the infection machinery. Understandings acquired from these and other studies in this project shall allow us to formulate detailed model for the mechanism of pathogen infection as well as propose novel antibiotic drug targets.
Project Details
Project type
Exploratory Research
Start Date
2002-02-28
End Date
2003-09-29
Status
Closed
Released Data Link
Team
Principal Investigator