Developmental Progression and Single-Cell Heterogeneity of Orientia tsutsugamushi during Intracellular Infection

Orientia tsutsugamushi , the causative agent of human scrub typhus disease, is an obligate intracellular bacterium transmitted by mites. It replicates exclusively within eukaryotic cells, inhabiting a range of host cell types in mammals and mites. Previously, we demonstrated that its extracellular, non-replicative form is developmentally distinct from the intracellular replicative form. In this study, we investigated spatial and temporal heterogeneity within the intracellular bacterial population. By quantifying bacterial DNA replication, protein synthesis, cell division, and subcellular localization, we defined four distinct intracellular growth stages: pre-active, lag, log, and maturation. Using microscopy-based single-cell analysis of selected transcripts and proteins, we further revealed transcriptional and translational heterogeneity within microcolonies. These findings highlight the complex, asynchronous nature of intracellular O. tsutsugamushi growth and underscore the importance of single-cell analysis in understanding its pathobiology. Importance Scrub typhus is a serious, sometimes life-threatening disease caused by the bacterium Orientia tsutsugamushi , which is spread by the bites of tiny mites and is common in many rural parts of Asia. This bacterium can only survive and grow inside living cells, where hundreds of individual bacteria may infect a single host cell. Until now, it was unclear whether all the bacteria inside a cell behaved the same way or if they existed in different stages of growth. In this study, we show that O. tsutsugamushi goes through several distinct stages while growing inside host cells and that not all bacteria are at the same stage at the same time. Even within a single infected cell, some bacteria are actively dividing, while others are preparing to leave the cell. Understanding these stages provides a new framework for exploring how the bacterium develops, spreads, and causes disease — insights that could help guide future research and improve ways to control or treat scrub typhus.