Research into the pathogenesis of microorganisms adapted to humans is difficult because the small animal models commonly used are often unable to replicate human physiology. However, elucidating the molecular infection mechanisms of bacterial pathogens such as Shigella and the microorganism-host interactions is crucial for combating them. The bacterium Shigella flexneri causes severe intestinal inflammation in humans and is responsible for numerous deaths each year, especially in young children.

The research team has now created a genome-wide map of Shigella genes that are used by the bacterium to colonize a species-specific intestinal epithelium. For their investigations, the researchers generated organotypic tissue cultures from three-dimensional intestinal epithelium organoids developed from human stem cells. The source tissue for this came from surplus surgical tissue. They performed high-throughput infection tests of the cells and tissues with Shigella and used the method of transposon-driven insertion site sequencing (TraDIS), functional genomics, proteomics, and computer-aided modeling. ⁽¹⁾

In the TraDIS method, mutants of the bacterium are generated with the help of “jumping genes” (transposons), thereby creating a mutant library.⁽²⁾ If the transposon inserts into an essential gene or an essential region of the bacterium, the mutant is not viable and does not survive selection after transformation. However, if the transposon inserts into a non-essential gene or non-essential region, the mutant forms a colony and is represented in the library. The transposon genomic DNA linkages of the mutant pool can then be sequenced to identify the regions of the chromosome that can be disrupted. With sufficient mutant density, this method can be used to identify all genetic loci that are essential for the growth and survival of the bacteria.

Bioinformatic analyses can then trace this sequence back to the reference genome to determine the exact position of the transposon insertion. This can then be visualized and quantified to identify essential genes, non-essential genes, and essential regions of genes or genomic sequences.⁽³⁾
The researchers succeeded in identifying the virulence effectors of Shigella that are essential for the colonization of epithelial cells in different geometries and intestinal segments. They identified over 100 chromosomal genes involved in this process. ⁽¹⁾

Original publication:
⁽¹⁾ Di Martino ML, Jenniches L, Bhetwal A, Eriksson J, Lopes ACC, Ntokaki A, Pasqua M, Sundbom M, Skogar M, Graf W, Webb DL, Hellström PM, Mateus A, Barquist L, Sellin ME: A scalable gut epithelial organoid model reveals the genome-wide colonization landscape of a human-adapted pathogen. Nature Genetics, 2025, DOI: 10.1038/s41588-025-02218-x

Further information:
https://www.helmholtz-hzi.de/media-center/newsroom/news-detailseite/mini-organe-aus-dem-labor-enthuellen-strategien-aggressiver-bakterien/

Additional information:
⁽²⁾ Warner IA, Kok WJ, Martinelli N, Yang Z, Goodall ECA, Henderson I. Microbial Primer: Transposon directed insertion site sequencing (TraDIS): A high throughput method for linking genotype to phenotype. Microbiology (Reading). 2023 Nov;169(11):001385. doi: 10.1099/mic.0.001385. PMID: 37909267; PMCID: PMC10710833.
⁽³⁾ Endres JL, Yajjala VK, Fey PD, Bayles KW. Construction of a Sequence-Defined Transposon Mutant Library in Staphylococcus aureus. Methods Mol Biol. 2019;2016:29-37. doi: 10.1007/978-1-4939-9570-7_3. PMID: 31197706.