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 4224 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.
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