A genomic approach towards understanding of bacterial two-component signal...


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Title A genomic approach towards understanding of bacterial two-component signal transduction systems
Period 01 / 2001 - unknown
Status Completed
Dissertation Yes
Research number OND1278486


Prokaryotes have evolved complex adaptation machineries to survive at various ecological niches. The most prevalent are bacterial signal transduction systems that consist of a sensor protein ('receptor' ) and a cytoplasmic effector protein (i.e. two-component systems). This regulator protein interacts with the DNA and influences the transcription of distinct subsets of genes. Bacteria may display up to 30 two-component systems, many of which are critical for bacterial virulence. Despite their global presence in the prokaryotic world and the often detailed knowledge about the
intracellular signaling cascade, surprisingly little is known about the external stimuli that activate the various systems and the subsets of genes that are affected. The aim of this project is to fill this gap in knowledge for the bacterial pathogen Campylobacter jejuni using a functional genomics and proteomics approach. Analysis of the recently completed Campylobacter genome sequence indicates the presence of at least seven putative protein kinase sensor proteins and of twelve cytoplasmic response regulators. The proposed work involves (a) mutagenesis of each of the
response regulator genes to establish their role in virulence and fusion of a reporter gene to the response regulator operons to assess their activation state and putative regulatory functions under various environmental conditions, (b) gene expression profiling via DNA microarrays and, if necessary, 2-D electrophoresis in conjunction with MALDI-TOF biospectrometry to determine the complete repertoire of genes affected by the individual regulators, (c) coupling of individual sensors and regulators via comparative gene expression profiling using regulator and sensor mutants and via protein phosphorylation experiments, (d) identification of the external stimuli and corresponding sensor recognition domains by biochemical methods including protein digestions, tryptophane intrinsic fluorescence spectrometry, radioactivity or radical cleavage, and targeted mutagenesis of putative recognition domains, and (e) functional analysis of environmentally regulated genes that affect key steps in bacterial pathogenesis. This work should greatly extend our
fundamental knowledge on prokaryotic signal transduction systems and may lead to the construction of recombinant strains that have limited adaptive capabilities. These attenuated strains may have vaccine potential. In addition, identification of the signals and recognition domains of two-component systems that impact bacterial virulence may facilitate the design of inhibitors that can be utilized for prophylactic and/or therapeutic purposes. This project nicely complements ongoing pathogenesis and functional genomics research at the Institute of Infectious Diseases and
This project will utilize the bacterial pathogen Campylobacter jejuni as a model system. This bacterium is the leading cause of human food-borne diarrheal disease and is capable to survive under highly diverse environmental conditions. Additional advantages are that the complete bacterial genome sequence is available and a series of putative two-component systems have been identified. This provides a firm basis for the proposed approach (see above). The project will start with monitoring of the activation state of the various systems under various conditions including
the bacterial survival inside eukaryotic cells. Hereto, two-component regulator and sensor gene reporter constructs will be developed. The genes that are regulated by the individual two-component systems will be identified via DNA micro-array based expressing profiling. DNA microarrays covering the complete genome of C. jejuni have been constructed and will be made available to us by Dr. J. Wagenaar (ID-Lelystad) in collaboration with Dr. B. Wren (Londen, UK),
the initiator of the C. jejuni genome project. Specific DNA binding of the response regulators will be verified via gel shift assays using isolated DNA fragments and purified His-tagged response regulator proteins. When appropriate, gene regulation will be monitored at the level of the mature protein by 2D electrophoresis in combination with MALDI-TOF (in collaboration with Dr. P. Hermans, EUR, Rotterdam). This part of the project is expected to continue well into the second year. In the second and part of the third year, identified response regulators will be coupled to their respective sensors proteins by comparison of mutant expression profiles mutants (using DNA micro-arrays) and via phosphorylation experiments using affinity purified recombinant sensor and regulator proteins. In the third year, biochemical methods and a site-specific mutagenesis will be applied to identify the specific ligands and to map the signal recognition domains on distinct sensor proteins. This will be limited to two-component systems that are relevant for bacterial virulence. The function of the regulated genes in bacterial pathogenesis, the behavior of the constructed mutants and, when available, the effects of potential inhibitors of distinct two-component systems will be evaluated in association with complementary research projects running at the institute.

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