Single-Cell Sequencing Reveals Ehrlichia Reprogramming Across Diverse Tick Cell Subpopulations

A new investigation into how the bacterial pathogen Ehrlichia muris eauclairensis interacts with host tick cells has revealed that the Ixodes scapularis embryonic cell line ISE6 possesses profound structural and transcriptional heterogeneity while lacking commitment to any single tick tissue lineage. Published in Frontiers in Cellular and Infection Microbiology, the study demonstrated that this embryonic line comprises fifteen distinct transcriptional cell clusters rather than a uniform population. Ehrlichia muris eauclairensis, an obligate intracellular bacterium causing human ehrlichiosis, established active intracellular infection across these diverse morphological and transcriptional states without displaying preference for a specific subpopulation. Rather than being restricted by the absence of tissue-specific differentiation, the pathogen initiated coordinated, time-dependent transcriptional reprogramming within the host cells. This finding provides researchers with a detailed cellular atlas of an essential laboratory model, clarifying its functional complexity while establishing an empirical framework for evaluating intracellular bacterial propagation in tick vectors.

Vector-borne infections transmitted by the blacklegged tick Ixodes scapularis continue to expand in geographical range, highlighting the need to understand how pathogens survive within vectors and alter host physiological systems. Ehrlichia muris eauclairensis persists across all tick developmental stages, migrating through tissues during blood feeding to enable transmission to mammalian hosts. Earlier research revealed that the bacterium manipulates nymphal tick behavior by accelerating host-seeking velocity and extending feeding duration, phenotypic changes linked to synganglion colonization and altered expression of synganglion neuropeptides including CCHamide-1 and nicotinic acetylcholine receptors. Dissecting the molecular mechanisms underlying such behavioral changes requires tractable experimental platforms. Although the embryonic cell line ISE6 has been utilized for decades to culture tick-borne organisms and study tick-pathogen relationships, its baseline cellular identities remained uncharacterized. Researchers hypothesized that the pathogen drives time-dependent transcriptional shifts across distinct tick cell populations, offering an in vitro approach to examine neuroendocrine dysregulation.

To establish baseline cellular characteristics before infection, the research team conducted brightfield microscopy, transmission electron microscopy, and scanning electron microscopy on uninfected ISE6 cultures. Brightfield imaging uncovered prominent diversity in cellular dimensions, shapes, and internal granularity. Ultrastructural examination through transmission electron microscopy confirmed marked heterogeneity, showing substantial differences in nuclear contours and numbers across individual cells. While certain cells exhibited high nuclear-to-cytoplasmic ratios where enlarged nuclei occupied most of the cell space, others contained centrally positioned nuclei surrounded by abundant cytoplasm. Electron densities within nuclei and cytoplasm varied independently of nuclear counts, with some cells displaying multi-lobed nuclei and others irregular boundaries. Following inoculation, transmission electron microscopy identified intracytoplasmic bacterial morulae across cells possessing single or multiple nuclei, confirming active infection without cell-type selectivity. Scanning electron microscopy corroborated these observations, revealing that infected populations retained diverse surface features, sizes, and geometries, establishing that bacterial colonization proceeds broadly across heterogeneous tick cell states.

The researchers next tracked bacterial replication and host survival across multiple culture timepoints to establish optimal parameters for single-cell sequencing. Using flow cytometry with amine-reactive dyes, the authors observed that host viability remained high during early infection, averaging 93.7 percent at six hours, 89.4 percent at twenty-four hours, and 93.3 percent at forty-eight hours post-inoculation. Viability progressively declined during subsequent intervals, falling to 81.2 percent at seventy-two hours, 77.6 percent at ninety-six hours, 62.7 percent at one hundred twenty hours, and 28.3 percent by one hundred sixty-eight hours. Quantitative polymerase chain reaction assays measuring the ehrlichial dsb gene confirmed that bacterial DNA copy numbers climbed steadily from 3.05 million at forty-eight hours to 6.07 million at ninety-six hours and reached 13.1 million at one hundred twenty hours. Based on these kinetics, investigators selected forty-eight and ninety-six hours post-infection for transcriptomic profiling, capturing an early adaptive stage and a later high-burden phase while maintaining host viability above eighty percent.

Single-cell transcriptomic libraries were prepared from uninfected and infected cultures across two independent experiments conducted thirty days apart, targeting roughly five thousand cells per sample on droplet microfluidic systems. Following paired-end sequencing, reads were mapped against the Ixodes scapularis reference genome, ambient RNA was removed, and graph-based clustering in Seurat resolved fifteen distinct clusters at a resolution of 0.2. Cluster zero formed the predominant subpopulation across all conditions, followed by cluster one. In uninfected cultures, composition remained relatively stable between forty-eight and ninety-six hours, with cluster two increasing from 2.90 percent to 5.49 percent. In infected cultures, however, bacterial proliferation induced substantial shifts: cluster zero declined from 36.62 percent to 29.50 percent, cluster one expanded from 21.18 percent to 26.05 percent, and cluster two surged from 9.34 percent at forty-eight hours to 15.14 percent at ninety-six hours. This 5.80 percent expansion in cluster two surpassed the uninfected increase, demonstrating that infection selectively enriches specific transcriptional states.

To delineate the functional roles of these subpopulations, the authors applied stringent criteria to identify cluster markers, requiring expression in seventy percent of cells within a cluster and no more than twenty percent in other clusters. Putative markers were identified for ten of the fifteen clusters. Cluster six and cluster eleven showed strong enrichment for transcriptional regulators and cell-cycle factors, while cluster eleven uniquely expressed neuropeptide receptors including cardioacceleratory peptide receptor, pyrokinin-1 receptor, and diuretic hormone receptor. Cluster thirteen exhibited high levels of heat shock proteins, and cluster fourteen possessed a muscle-like profile featuring myosin isoforms, troponin I, and myocyte-specific enhancer factor 2. To test whether clusters corresponded to specific organs, the team generated a reference atlas from seven dissected tick tissues and hemocyte profiles. Despite individual markers aligning with organ transcripts, cluster profiles showed no correspondence to any single tissue, confirming that the embryonic cell line retains developmental plasticity.

Evaluation of infection-responsive gene regulation uncovered forty cluster marker genes that changed significantly during bacterial propagation, with suppressive effects peaking at ninety-six hours post-infection. The most pronounced response appeared in cluster four, where LOC8030973, encoding the pyrokinin-1 receptor, exhibited sharp downregulation across infection progression, dropping with a log2 fold-change of negative 2.80 between forty-eight and ninety-six hours and negative 3.62 relative to uninfected controls. In cluster zero, LOC8042156, encoding cAMP-dependent protein kinase catalytic subunit 3, was similarly repressed, decreasing by a log2 fold-change of negative 1.68 over infection time and negative 1.42 compared with uninfected cells. In cluster eleven, LOC8030997, encoding a diuretic hormone receptor, was reduced relative to uninfected controls by a log2 fold-change of negative 1.17, despite minor upregulation between early and late infection. Because these neuropeptides participate in neuroendocrine signaling, visceral regulation, and fluid homeostasis, their downregulation provides direct evidence that intracellular bacterial infection disrupts neuronal-associated signaling programs within tick host cells.

In addition to neuroendocrine receptors, bacterial progression altered cluster markers involved in membrane transport, metabolic activity, and cytoskeletal structure. In cluster four, downregulation of the pyrokinin-1 receptor was accompanied by reduced expression of monocarboxylate transporter 12, reflecting coupled suppression of signaling and nutrient transport. Cluster seven exhibited significant downregulation of LOC8052878, encoding the organic anion transporter Oatp74D, which mediates ecdysone uptake and has been linked to vector survival during arboviral infection. In cluster eleven, late infection decreased expression of a lysosomal acid glucosylceramidase-like gene and a doublesex-related transcription factor, suggesting impaired autophagic processes that might otherwise clear intracellular bacteria. Conversely, certain stress and structural genes showed selective induction. Cluster thirteen increased expression of heat shock protein beta-6-like by a log2 fold-change of 1.75 during late infection, representing an activated cellular stress program. Cluster eight upregulated unconventional myosin-XV by a log2 fold-change of 1.06, suggesting that the pathogen induces targeted cytoskeletal remodeling to facilitate intracellular maintenance.

Global differential expression profiling identified a distinct two-phase transcriptional transition in host cells as bacterial burden escalated. While uninfected cultures showed minimal drift, altering only two genes between forty-eight and ninety-six hours, infected cultures differed from controls by 399 differentially expressed genes at forty-eight hours and 640 genes at ninety-six hours, with 121 genes shifting between infection stages. At forty-eight hours, host cells mounted an early adaptive response dominated by gene activation, with Gene Ontology analysis highlighting pathways for unfolded protein binding, response to chemical stimuli, and mitochondrial organization. Upregulation of endoplasmic reticulum chaperones, IRE1 signaling, peroxiredoxins, and catalases indicated robust antioxidant and proteostatic defenses, counterbalanced by early downregulation of DNA mismatch repair components. By ninety-six hours, this adaptive activation was replaced by widespread transcriptional silencing. Enriched pathways among downregulated transcripts included mitotic division, DNA replication, minichromosome maintenance complexes, cyclin-dependent kinases, fatty acid biosynthesis, and microtubule organization, accompanied by reductions in host transcription factor activity scores.

The authors concluded that while ISE6 cells do not mirror mature tick organs, their diverse transcriptional states and expression of neuroendocrine receptors provide a valuable system for dissecting pathogen-induced cellular dysregulation. The absence of specific tissue commitment likely stems from the embryonic origin of the cell line and three decades of continuous culture adaptation, an evolutionary divergence documented across other long-maintained arthropod lines. Crucially, in vitro downregulation of protein kinase A catalytic subunit 3, pyrokinin-1 receptor, and diuretic hormone receptor aligns with previous in vivo observations in nymphal ticks, where infection colonized the synganglion, altered CREB transcription factor levels, and modified host-seeking locomotion. Although cell culture platforms cannot fully replicate whole-organism complexity, these findings supply critical candidate pathways for ongoing in vivo validation in live ticks. By resolving how intracellular pathogens reshape vector transcription and manipulate neuronal signaling, the research advances fundamental understanding of tick-pathogen relationships while highlighting targets for disrupting disease transmission cycles.

Subject of Research: Single-cell transcriptomic dynamics of Ixodes scapularis ISE6 cells during Ehrlichia muris eauclairensis infection

Article Title: Ehrlichia infection progression drives tissue-independent transcriptional heterogeneity in tick cells

Article References: Adegoke, A., Aspinwall, J., McNinch, C., Ho, M. C. W., Miranda, A. X., Hoyt, F., Nair, V., Lack, J., & Saito, T. B. (2026). Ehrlichia infection progression drives tissue-independent transcriptional heterogeneity in tick cells. Frontiers in Cellular and Infection Microbiology, 16, Article 1961724. https://doi.org/10.3389/fcimb.2026.1961724

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1961724

Keywords: Ehrlichia muris eauclairensis, Ixodes scapularis, ISE6 cell line, single-cell RNA sequencing, tick-borne pathogens, transcriptional heterogeneity, synganglion signaling, pyrokinin receptor, vector biology, cell cycle regulation, Ehrlichia, infection