Hybrid capture RNA-seq defines temporal gene expression in Rickettsia.

Area of Science:

• Microbiology and Molecular Biology
• Genomics and Transcriptomics
• Infectious Diseases and Pathogenesis

Background:

• Pathogenic Rickettsia are obligate intracellular bacteria with poorly understood gene regulatory networks.
• Standard RNA sequencing (RNA-seq) struggles with host RNA contamination, hindering transcriptional analysis of Rickettsia during infection.
• Dynamic gene expression changes during Rickettsia infection have been difficult to profile comprehensively.

Purpose of the Study:

• To adapt and apply the Pathogen Hybrid Capture (PatH-Cap) method for sensitive transcriptional profiling of Rickettsia species.
• To investigate temporal gene expression trends during the first 24 hours of Rickettsia infection, including infection initiation.
• To identify and analyze antisense RNA molecules and predict operon structures in Rickettsia parkeri.

Main Methods:

• Adaptation of the PatH-Cap technique for Rickettsia species to enrich bacterial transcripts and deplete host/rRNA.
• Application of PatH-Cap RNA-sequencing to analyze transcriptional changes during the initial 24 hours of infection.
• Clustering of genes based on temporal expression trends and identification of antisense RNA molecules.

Main Results:

• PatH-Cap successfully enriched bacterial transcripts, enabling profiling of gene expression during infection initiation.
• Identified distinct cohorts of genes with up- or down-regulated expression at different infection stages.
• Discovered 310 antisense RNA molecules, many exhibiting strong temporal expression trends, and highlighted diverse expression patterns of virulence-related genes.

Conclusions:

• PatH-Cap is a powerful tool for sensitive transcriptional profiling of intracellular pathogens like Rickettsia.
• This study provides the most comprehensive analysis of temporal Rickettsia gene expression to date.
• The findings lay the groundwork for future investigations into Rickettsia gene regulation under various conditions and disease models.