High levels of antisense transcription from numerous genes in the obligate intracellular bacterium Orientia tsutsugamushi

Orientia tsutsugamushi (Ot) is a cytoplasmic, Gram-negative, obligate intracellular bacterium that causes the human disease scrub typhus. It has a highly repetitive genome whereby approximately 50% is composed of multiple copies of a proliferated integrative and conjugative element, the Rickettsial amplified Genetic Element (RAGE). Previous RNA sequencing analysis revealed a high level of antisense transcription in Ot, particularly from RAGE-encoded genes, with 18% of all genes having a sense:antisense ratio <1 and 5% with a ratio <0.1. In our current study, we have confirmed the earlier RNA sequencing findings via PCR-based methods and established that this antisense transcription is a consistent feature across eight distinct Ot strains. Furthermore, we have utilized PCR to monitor differences in sense and antisense expression between intracellular and extracellular bacteria. Our findings lend weight to the hypothesis that antisense transcription in Ot is a regulated mechanism, possibly playing a crucial role in the pathogen’s lifecycle during infection.