Primary transcript enrichment reveals a density-dependent Wolbachia transcriptome and active mosquito RNA viruses.

Wolbachia pipientis is a widespread obligate intracellular bacterium and an effective biocontrol agent being deployed in the field against mosquito-borne viral diseases. Investigating the transcriptomic dynamics of Wolbachia within host cells is highly desired but is fundamentally challenged by the overwhelming abundance of eukaryotic host RNA and bacterial ribosomal RNA. Here, we demonstrate that Cappable-seq specifically captures the active, primary transcriptome of Wolbachia while drastically reducing host background. We applied this methodology to monitor Wolbachia gene expression changes over a single host cell passage cycle, tracking the transition from early infection to a high-titer maintenance phase. Differential gene expression analysis identified the upregulation of rpoH transcription factor, stress-response chaperones and WO prophage elements. This was coupled with a continuous downregulation of key host-manipulation effectors, including Ankyrin-repeat domain-containing proteins and secretion systems, alongside the broad suppression of metabolic transporters over the time-course. In addition, we discovered that Cappable-seq concurrently co-enriches the transcripts and genomes from various single-stranded RNA viruses that are known to be present in these mosquito cells. In summary, Cappable-seq technology provides a robust framework to investigate complex, tripartite host-microbe-virus interactions, demonstrating that Wolbachia actively senses and adapts to the dynamic constraints of its intracellular environment by regulating its transcriptome.