Intracellular Staphylococcus aureus triggers isolate-specific host transcriptional responses alongside TNF-R1 regulated cell fate decisions

Background Staphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. Many mechanisms underlying these differences remain poorly understood. This study investigated how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. Methods The intracellular localization of four S. aureus isolates was analyzed via transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. The transcriptional reprogramming of infected U937 monocytes was examined via mRNA sequencing. Infection outcomes and early TNF-R1 surface expression were characterized and compared with those of A549 and SaOS-2 cell lines employing flow cytometry and Western blot analysis, characterizing host cell death mechanisms in both wild-type and TNF-R1‒deficient backgrounds. Results All S. aureus isolates localized to the endo-lysosomal and cytosolic compartments but were also frequently detected in nucleo-proximal regions. In U937 monocytes, infection induced a conserved stress signature alongside isolate‑specific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronic‑infection isolate EDCC 5464. Similarly, cell death outcomes were isolate‑ and cell type-dependent, involved caspase activation consistent with intrinsic and extrinsic apoptosis signaling, and caspase-1 activation at distinct temporal dynamics. TNF‑R1 loss initially delayed but later increased later isolate-independent cytotoxicity. Higher TNF-R1 surface expression was triggered in U937 monocytes by all viable isolates, but only by EDCC 5464 in SaOS-2 and A549 cells. Viability of the latter was only marginally influenced by isolate or TNF-R1 loss. Conclusions These results highlight the multilayered determinants governing intracellular S. aureus survival, host cell susceptibility, and noncanonical intracellular localization that warrants further investigation. The TNF/TNF-R1 axis appears to critically influence regulated host defenses during early infection stages in a tissue-specific manner, though its mechanistic contribution remains to be fully elucidated. Together with distinct isolate-driven gene expression profiles of U937 monocytes, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies.