Abstract
BackgroundThe Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis.ResultsHere we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates.ConclusionThis first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.
Highlights
The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis
We report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified open reading frames (ORFs) of Cpn (Chlamydia pneumonia strain CWL029) in GatewayW ‘entry’ vectors
The ORFeome of C. pneumoniae represents 98.5% of the predicted ORFs For construction and characterization of the Cpn ORFeome, we employed the same strategy as we used in developing the first Staphylococcus aureus ORFeome [10]
Summary
The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is a prominent human pathogen responsible for respiratory infections like pneumoniae and bronchitis [1,2]. The involvement of Cpn in other human diseases such as atherosclerosis [3], reactive arthritis [4], and myocarditis [5], or the association between Cpn infection and lung cancer [6], makes this pathogen an important risk factor in human disease. About half of the 1052 open reading frames (ORFs) of Cpn encode proteins without any known function [9]. A flexible gene collection that contains all Cpn genes is a precondition to identify new or additional protein functions, e.g. through the discovery of possible interaction partners or the production of recombinant proteins for enzymatic assays or to test their influence on gene expression
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