Validation of Chlamydia virulence determinants by complementation

Project Summary/Abstract More than 1.6 million cases of Chlamydia trachomatis infections are reported to the CDC each year, making it the most commonly reported infectious disease in the country. This bacterial pathogen infects human and other eukaryotic cells and only reproduces in a host cell. Chlamydia research has been revolutionized by the recent development of genetic methods to knockout or knockdown specific genes. However, a roadblock in this genetic approach is the limited availability and utility of complementation methods to express an exogenous copy of the gene. Current conditional expression in Chlamydia relies on the Tet promoter, and it is not possible to independently control expression levels for gene knockdown and also for a complementing allele. A further limitation is that expression of the complementing allele from the chlamydial plasmid introduces gene dosage effects. This project will develop new complementation tools that will enhance the rigor of genetic approaches used to study chlamydial gene function. In Aim 1, we will develop new conditional expression methods for C. trachomatis. These studies will test induction systems, including cumate and IPTG, and arabinose, that have been used in other bacteria. In Aim 2, we will identify sites in the C. trachomatis genome where a gene can be inserted without affecting other genes or the intracellular infection. These studies have high potential impact because they will enhance the use of complementation to confirm that a knockout or knockdown phenotype is due to a targeted gene. The ability to independently control the expression of more than one exogenous gene will also be broadly useful for studies of chlamydial gene function and host- Chlamydia interactions. In addition, the identification of neutral sites for gene insertion will be useful for creating C. trachomatis backbone strains that express additional factors, such as Cre-Lox recombination systems or antibiotic resistance markers.