Work

Automated high-throughput microscopy screening unveiled new Listeria monocytogenes genes involved in cell infection

Year: 2026

Type: article

Abstract: To uncover novel genetic factors required for Listeria monocytogenes cell infection, we developed an automated high-throughput microscopy screening pipeline that integrates GFP-expressing bacteria with machine learning-based image analysis. Using this approach, we screened a mariner transposon library comprising 4,224 L. monocytogenes EGDe mutants and identified 58 with significantly reduced numbers of intracellular bacteria. Sequencing revealed 24 unique insertion sites corresponding to 14 genes, including previously known virulence factors and nine novel candidates not previously implicated in cell infection. These genes encode the protease chaperone ClpX, the ferric uptake regulator Fur, the sensor histidine kinase LisK, the peptide chain release factor 2 PrfB, proteins involved in proline and purine biosynthesis (ProAB, PurAB), and Lmo2217, a protein of unknown function. Among these, the targeted deletion of the adenylosuccinate synthetase gene, purA, resulted in impaired growth in minimal medium, severely reduced proliferation in epithelial and macrophage cell lines, and attenuated virulence in mice. Unexpectedly, PurA was also essential for bacterial internalization into cells. Supplementation with AMP or adenine, but not ATP, rescued the invasion capacity of the ΔpurA mutant. Mechanistically, purA deletion induced a reduction in the levels of surface-associated GAPDH, a putative plasminogen-binding protein, likely contributing to the observed invasion defect. Overall, these findings highlight the power of automated high-throughput microscopy screening to dissect host–pathogen interactions, identify novel L. monocytogenes genes required for cell infection, and uncover an unexpected role for PurA in maintaining GAPDH surface localization and promoting bacterial entry into host cells. • Development of an automated high-throughput microscopy pipeline combining GFP-tagged L. monocytogenes and machine learning–based image analysis. • Screening of 4,224 mariner transposon mutants identifies 14 mutants with reduced numbers of intracellular bacteria. • Novel infection-related genes include clpX, fur, lisK, prfB, proAB, purAB, and lmo2217 , several linked to metabolic and regulatory pathways. • Deletion of purA impairs bacterial growth, intracellular proliferation, virulence in mice, and unexpectedly compromises entry into host cell. • purA controls surface GAPDH levels, revealing a mechanistic link between purine biosynthesis, GAPDH localization, and bacterial invasion.

Source: Microbiological Research

DOI: 10.1016/j.micres.2026.128442