Microsporidia are single-celled, obligate intracellular parasites that are closely related to fungi. One of the common microsporidian species that infect humans, Encephalitozoon intestinalis, is an enteric parasite that invades and replicates within the epithelial lining of the small intestine. Understanding host and parasite transcriptional programs during infection can illuminate mechanisms of host detection, immune signaling, and parasite development.
This study used single-cell transcriptomics to profile both host and parasite RNA during infection of two epithelial cell lines routinely employed as in vitro models of E. intestinalis infection: Caco-2 and Vero cells. The aim was to resolve heterogeneity in host responses and to characterize parasite developmental transcriptional dynamics at single-cell resolution.
The authors applied single-cell RNA sequencing (scRNA-seq) to infected cultures of Caco-2 and Vero cells to measure host and parasite transcripts in individual cells. Using scRNA-seq enables detection of cell-to-cell variability in transcriptional responses, identification of rare responsive cells, and characterization of parasite gene expression programs across developmental stages.
The dataset and analytic approach were presented as a resource for further study; detailed methods, sequencing depth, cell counts, and bioinformatic pipelines are described in the original preprint and supplementary materials. These specific quantitative and procedural details were not included in the provided source excerpt.
A principal observation is that the majority of host cells showed no direct transcriptional response to parasite invasion or intracellular replication. This suggests that many infected epithelial cells either fail to detect E. intestinalis intracellular presence or fail to mount a measurable transcriptional response upon direct infection in these in vitro models. The pattern seen in these epithelial lines mirrors prior single-cell transcriptomic findings in infected macrophages, supporting a recurrent biological theme.
Only a small subset of host cells exhibited detectable transcriptional changes attributable to sensing intracellular parasites. Those responsive cells were observed late in the parasite life cycle rather than immediately following invasion.
The data indicate that the small fraction of cells that detect intracellular parasites subsequently signal to neighboring cells. This intercellular signaling produces a broader activation of a TNF-ɑ response across the culture, implying that the inflammatory signature observed at the population level may arise from paracrine amplification rather than from uniform direct detection by each infected cell.
This mechanism—limited direct detection by most infected cells coupled with signaling from a minority of sensors—provides a plausible explanation for how tissue-level inflammatory responses can develop despite a muted per-cell response in most infected epithelial cells.
Analysis of parasite-derived transcripts recovered by scRNA-seq revealed a developmental transcriptional program that correlates with the morphological stages of the E. intestinalis life cycle. In other words, parasite gene expression profiles change in concert with known parasite developmental transitions, and these patterns are observable at single-cell resolution within infected host cultures.
These parasite transcriptional dynamics provide an internally consistent map linking gene expression to morphological stage and life-cycle progression in vitro.
The observed limited direct host transcriptional response and the late paracrine-driven TNF-ɑ activation are consistent with previous single-cell transcriptomic studies of E. intestinalis infection in macrophages. That consistency suggests a possibly shared host response pattern across different host cell types and experimental systems.
By profiling both host and parasite transcripts at single-cell resolution, the study contributes insights into (1) how epithelial cells detect and respond to intracellular microsporidia, (2) how small numbers of sensing cells can drive tissue-level inflammatory signaling, and (3) how parasite transcriptional programs unfold during development. The authors present the datasets as a valuable resource for follow-up mechanistic work.
This work is reported as a preprint and has not been peer-reviewed. The preprint lists authors affiliated with Johns Hopkins University. Funding sources declared include a Johns Hopkins Catalyst Award and a National Science Foundation award (OAC1920103). The authors declared no competing interests. The preprint and supplementary materials contain additional dataset and methodological information.
Specific experimental parameters (for example, multiplicity of infection, time points sampled, number of cells analyzed, sequencing depth, and the precise bioinformatic workflows) were not included in the supplied source excerpt. Therefore, details required to fully evaluate reproducibility and quantitative strength of the findings are available in the full preprint and supplementary files but were not reproduced here.
Single-cell transcriptomics of E. intestinalis infection in Caco-2 and Vero cells reveals that most host epithelial cells do not mount a detectable direct transcriptional response to intracellular infection, while a minority of late-detecting cells trigger a paracrine TNF-ɑ response that spreads within the culture. Concurrently, parasite transcripts delineate a developmental program aligned with morphological life-cycle stages. The datasets are offered as a resource to enable further mechanistic and comparative investigations.