Fig. 4: Tn-seq reveals a set of genes specifically essential for blood survival of S. aureus.

a The USA300 Tn library was cultured in human whole blood and plasma for 24 and 36 h, after which the bacteria were collected and prepared for Tn-seq. Image created in BioRender (Lee, W. (2025) https://BioRender.com/gwn8ee3). b The growth of USA300 was examined for 36 h. c Gene read counts from a 24 h post-inoculation sample were compared to those of the Tn library grown in TSB. Representative genes from the list of substantially depleted genes are presented on the plot. d Representative genes depleted in whole blood and plasma are expressed as a heat map (dots: genes that meet the log₂ fold change criterion but not the P value criterion; black lines: gene groups). e Representative genes required for intracellular (2 h) and plasma (36 h) survival are compared (red squares: genes for which the 4 or 8 h post-infection read counts were used since they were either defined as late essential or were not depleted at 2 h but rather at other time points). The mutants of selected genes were validated for their (f) essentiality and (g) non-essentiality for survival in whole blood. The mutants were cultured in whole blood for 24 h, and CFU were counted to measure the survival. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the USA300 strain. The exact P values are available in the Source Data file. h USA300 pre-exposed to 120 μM of oligomycin A (oligo A) was placed into whole blood in the presence of oligomycin A to investigate the essentiality of ATP production for blood survival. Data in b and f–h are presented as the mean ± SD (n = 3 biological replicates), and the P values are calculated by a two-sided, unpaired Student’s t-test. i The log₂ fold change of the mutants of genes essential for blood survival was expressed in a time course up to 36 h post-blood inoculation. Source data for b, c and f–i are available in the Source Data file.