The use of molecular genetic tools to generate and complement mutations in pathogenic bacteria is central to our understanding of how microorganisms cause disease. Unfortunately, many clinically important pathogens, such as Chlamydia trachomatis -the causative agent of blinding trachoma and many sexually transmitted diseases- are not amenable to such analysis. Here we propose to establish a comprehensive system for forward and reverse genetic analysis in Chlamydia based on the use of chemical mutagens, genetic recombination and """"""""deep"""""""" DNA sequencing technologies. Our specific goals are to 1) identify factors important in C. trachomatis pathogenesis by implementing forward genetic screens, including a genetic analysis of Type II secretion and 2) develop a method for systematic reverse genetic analysis in C. trachomatis. By the completion of these experiments we will have established methods to perform routine forward and genetic screens - the lynchpin of microbial pathogenesis research. The tools and reagents generated through the proposed work will constitute an important resource for the Chlamydia research community.
Chlamydia trachomatis is the leading cause of infectious blindness (trachoma) and many sexually transmitted infections. Despite their clinical and public health importance, these obligate intracellular bacterial pathogens remain understudied because of their intractability to molecular genetic manipulation. Here we describe methods to perform genetic analysis in these pathogens, which will significantly accelerate our understanding of virulence gene function and the potential development of attenuated strains for vaccine applications.
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