Dual transcriptional analysis provides insights into the replicative niche of <i>P. salmonis</i> and the host response during infection

<i>Piscirickettsia salmonis</i> is a facultative intracellular pathogen and the causative agent of piscirickettsiosis, a systemic disease affecting salmonid fish. Successful replication within host phagocytes is central to disease progression, yet the intracellular niche supporting bacterial growth remains incompletely defined. Here, we employed dual RNA sequencing to characterize the coordinated host and bacterial transcriptional responses during active intracellular replication of <i>P. salmonis</i> in SHK-1 cells, a macrophage-like cell line derived from the head kidney of <i>Salmo salar</i>. Infection was associated with the induction of lysosome-related pathways in host cells, including increased expression of genes involved in lysosomal biogenesis and proteolytic activity. Concomitantly, <i>P. salmonis</i> upregulated genes associated with intracellular adaptation, including components of the Dot/Icm type IVB secretion system, stress-response pathways, and iron-acquisition systems. Confocal microscopy revealed that bacteria reside within an acidified, Lamp-1-positive vacuole resembling a late endosomal/lysosomal compartment. Pharmacological disruption of vacuolar acidification significantly reduced intracellular <i>P. salmonis</i> replication, and iron supplementation partially restored growth under conditions of impaired acidification. In line with this observation, experimental manipulation of host iron availability modulated intracellular bacterial growth. Finally, bacteria recovered from infected cells exhibit enhanced replication during subsequent infections, indicating that intracellular passage influences bacterial physiological state. Together, these findings delineate key physiological features of the intracellular niche that shape <i>P. salmonis</i> replication and provide a framework for understanding host-pathogen interactions during infection.<h4>Importance</h4>Successful intracellular replication is a defining feature of many bacterial pathogens and directly influences disease outcome. For the salmonid pathogen <i>Piscirickettsia salmonis</i>, the intracellular environment that supports bacterial growth has remained incompletely characterized. Here, we show that <i>P. salmonis</i> replicates within an acidified, Lamp-1-positive vacuole and that intracellular growth is influenced by host iron availability. Infection is accompanied by activation of lysosomal pathways in host cells and coordinated induction of bacterial stress-response mechanisms, secretion systems, iron-acquisition pathways, and numerous genes of previously unknown function. Intracellular passage also alters bacterial behavior during subsequent infection cycles, suggesting a physiological adaptation associated with host-cell residence. By defining the intracellular context in which <i>P. salmonis</i> proliferates and situating these features within the broader landscape of intracellular bacterial strategies, this work advances understanding of host-pathogen interactions in non-mammalian systems and provides a foundation for future functional studies relevant to aquaculture and intracellular microbiology.