Fig. 1: Methicillin-resistant S. aureus USA300 establishes infection in mammalian cells.

a To determine genes essential for the early entry into and late survival in host cells, A549 cells were infected with the USA300 Tn library for 1.5 h, after which extracellular bacteria were removed by gentamicin. Samples collected right after gentamicin were defined as 0 h throughout the manuscript. Image created in BioRender (Lee, W. (2025) https://BioRender.com/bytpvde). Extent of infection (b) of USA300 into host cells (A549, Raw264.7, and HaCaT) and (c) of MRSA and MSSA strains into A549 cells were determined by cell infection assay (\(\%\;{\rm{entry}}=\,\frac{{\rm{CFU}}\; {\rm{recovered}}\; {\rm{at}}\;0\;{\rm{h}}}{{\rm{input}}\; {\rm{CFU}}}\times 100\)). d The CFU were counted at 0–8 h post-infection as a measure of USA300 survival inside the A549 cells. Data in b–d are presented as the mean ± SD (n = 3 biological replicates); P values are calculated by a two-sided, unpaired Student’s t-test. Source data for b–d are available in the Source Data file. e The intracellular bacteria were examined by transmission electron microscopy at 4 h post-infection (scale bar = 1 μm). f Entry and survival of FtsZ-GFP-expressing USA300 in A549 cells were inspected by fluorescence microscopy. The cell membrane and bacteria are shown in red and green, respectively (scale bar = 5 μm). The images in e–f are representative of three independent experiments. g The entire sequenced genome at 0 h was plotted using Circos with the green peaks representing read counts for each TA site and blue and orange outer squares representing genes with counts lower (depleted) and higher (enriched) than those of the control library, respectively. The % TA sites (% TA sites detected out of all TA sites in the genome) show the coverage of the genome by Tn-seq and are presented in a table (\( * \;{\rm{relative}}\%=\,\frac{\%{\rm{TA}}\; {\rm{sites}}\; {\rm{of}}\; {\rm{an}}\; {\rm{experimental}}\; {\rm{sample}}}{\%{\rm{TA}}\; {\rm{sites}}\; {\rm{of}}\; {\rm{a}}\; {\rm{control}}\; {\rm{Tn}}\; {\rm{library}}\;(54.3\%)} \times 100\)). h The read counts generated by sequencing in both forward (red) and reverse (blue) directions for each TA site within the saeQRS locus of the control and 4 h samples were plotted using R.