Tick cells as a tractable arthropod model for studying <i>Orientia tsutsugamushi</i>

ABSTRACT Orientia tsutsugamushi ( Ot ), the causative agent of scrub typhus, is an obligate intracellular bacterium naturally maintained in Leptotrombidium mites, yet its interactions within arthropod hosts remain poorly understood. Here, we employed two tick cell lines, Ixodes scapularis ISE6 and Rhipicephalus microplus BME/CTVM23, as arthropod models to investigate the intracellular lifecycle of Ot strains TA686 and Karp. Both strains efficiently infected tick cells and replicated within them, with ISE6 supporting more robust growth. Electron microscopy images revealed that Ot maintains its characteristic cytoplasmic, non-vacuolar location in tick cells and exits infected cells by budding off the surface in a membrane-encased structure. Time-course immunofluorescence imaging demonstrated progressive intracellular replication and dynamic expression of Ot outer membrane autotransporters ScaA and ScaC, with ScaC enriched early in infection and ScaA at later stages. A microscopy-based analysis of metabolic activity using a clickable methionine analog L-homopropargylglycine showed that high ScaA abundance correlated with reduced translational activity, suggesting a link between ScaA abundance and late-stage or extracellular-like developmental states. The subcellular location of Ot in tick cells differs from the characteristic dynein-driven perinuclear clustering observed in mammalian cells and was not sensitive to disruption of microtubules, suggesting a distinct mode of distribution. Together, these findings identify tick cell lines as tractable and biologically relevant arthropod models for studying Ot and dissecting host-microbe interactions. IMPORTANCE Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium naturally maintained in trombiculid mites, yet its interactions with arthropod host cells remain poorly understood. This study establishes, for the first time, a tractable in vitro arthropod model for O. tsutsugamushi using tick cell lines from Ixodes scapularis and Rhipicephalus microplus . We demonstrate that both virulent and avirulent Orientia strains efficiently infect and replicate in these cells while maintaining their characteristic cytoplasmic location and budding mode of exit. Distinct temporal expression of the bacterial autotransporters ScaA and ScaC and their relationship to metabolic activity reveal stage-specific adaptations during infection. These findings provide essential mechanistic insight into how O. tsutsugamushi persists and develops within arthropod hosts. The tick cell model enables comparative studies of host-pathogen interactions across vector species and offers a powerful tool to investigate transmission biology and potential targets for intervention.