Analysis of Spatial Gene Expression in Chlamydia Using Fluorescent In Situ Hybridization

The obligate intracellular bacterium Chlamydia employs a unique, asynchronous biphasic developmental cycle characterized by distinct morphological forms: the replicative reticulate body (RB), the infectious elementary body (EB), and an intermediate body (IB). The simultaneous presence of these phenotypically distinct cell types throughout infection complicates the study of gene expression regulation during development. Conventional population-level assays are inadequate for dissecting the regulatory mechanisms within this mixed population. This chapter describes a robust method utilizing hybridization chain reaction (HCR) RNA fluorescence in situ hybridization (FISH) in conjunction with dual-promoter reporter Chlamydia strains to determine cell-form-specific transcript expression. We leverage dual-reporter strains to provide visual identification of RBs and EBs. Probes targeting genes of interest are designed to interact with spectrally distinct fluorescent HCR amplifiers. This versatile, multiplexing system allows for precise spatial and temporal localization of specific mRNAs within identified Chlamydial cell forms, offering a powerful tool to overcome current limitations in Chlamydia research and elucidate the regulatory underpinnings of its complex life cycle.