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Single-cell transcriptomics reveals a multiphasic Wolbachia host infection trajectory

Jacobs, Jodie1; Lum, Alexandra1; Nykamp, Jonas1; Lagousis, Cayla R. M.1; Russell, Shelbi L.1

Research facility: UCSC Chemical Screening Center
Published Jul 17, 2026 on Dryad. https://doi.org/10.5061/dryad.612jm64md

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Abstract

To establish persistent infections, intracellular bacterial symbionts must navigate complex host cellular environments, co-opt host biology, and evade immune clearance, yet the molecular mechanisms underlying infection remain poorly characterized. The alphaproteobacterial endosymbiont Wolbachia pipientis, prevalent in diverse arthropods and nematodes, exemplifies this challenge. The wMel strain, natively associated with Drosophila melanogaster, has proven particularly effective in biocontrol applications for blocking viral transmission and suppressing reproduction when transinfected into mosquito populations. Despite these successful applications, the molecular mechanisms underlying initial infection establishment remain poorly understood in wMel’s native host. Importantly, Wolbachia demonstrates host cell-type preferences and induces tissue-specific phenotypes, yet bulk RNA-sequencing studies have yielded inconsistent signals of differential expression, likely due to signal averaging across cells with heterogeneous infection titers, states, and developmental stages. Here, we employed single-cell RNA sequencing to investigate how wMel colonization influences the host transcriptome during the establishment of stable infection of D. melanogaster JW18 somatic cell lines. First, we use 10X Genomics Chromium 3’ single cell RNA-seq to validate the lower-cost Illumina 3’ single cell RNA-seq (PIPseq) platform and show that mis-priming of symbiont and host ribosomal RNAs can be leveraged as a proxy to measure intracellular bacterial titer.Using the PIPseq platform to characterize six timepoints across the three months required to establish stable infection, we demonstrate that nascent wMel infections induce distinct transcriptional changes that create novel cellular states diverged from uninfected controls. Through unsupervised clustering, pseudotime analysis, and marker gene identification, we reveal how Wolbachia reprograms host somatic cells to evade immune responses, establish infection, and facilitate nutrient acquisition. These findings uncover the hidden role Wolbachia plays in eukaryotic cellular biology and provide mechanistic insights from the native host cell system that will enable future efforts to develop advanced control measures leveraging cell-type specific phenotypes of Wolbachia strains.