Validation of chlamydia virulence determinants by complementation

Federal Grant Award Summary

The National Institute of Allergy and Infectious Diseases (NIAID) awarded a $431,750 Project Grant to the University of California, Irvine under the Allergy and Infectious Diseases Research program (CFDA 93.855) for the period August 1, 2025 through July 31, 2027. The research project focuses on developing and validating new complementation tools and conditional expression methods to enhance genetic approaches for studying Chlamydia trachomatis virulence determinants. This addresses a critical gap in current research capabilities, as existing complementation methods utilizing tetracycline (tet) promoters cannot independently control expression levels for gene knockdown and complementing alleles simultaneously, and may introduce confounding gene dosage effects when expressed from the chlamydial plasmid.

The project deliverables encompass two primary objectives: (1) development of new conditional expression systems for C. trachomatis using alternative induction mechanisms such as cumate, IPTG, and arabinose to enable independent control of multiple exogenous genes; and (2) identification of neutral genomic insertion sites within C. trachomatis where genes can be inserted without disrupting other genes or intracellular infection capabilities. These research outputs will provide the scientific community with enhanced tools to confirm that genetic knockout or knockdown phenotypes result from targeted genes rather than secondary effects, and will facilitate the creation of backbone strains expressing additional genetic factors—with broad applicability to chlamydial gene function studies and host-pathogen interaction research.

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.