Walter, A.; Bischler, T.; Jungblut, M. J.; Breitsprecher, L.; Beck, J.; Schaefer, N.; Hofmann, L.; Ziesmann, T.; Haerteis, S.; Gadjalova, I.; Distler, U.; Beliu, G.; Psathaki, K.; Hensel, M.; Schneider-Brachert, W.; Graefenhan, T.; Stempfl, T.; Kieninger, B.; Muehlen, S.; Alt, V.; Mannala, G. K.; Fritsch, J.

Background: Staphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates 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: Four S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block face scanning electron microscopy (SBF SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. Results: All S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri- and putatively intranuclearly, revealing an unexpected intracellular niche. 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. Cell death outcomes were likewise isolate-dependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNF-R1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNF-R1 as a key regulator of U937 infection outcome. SaOS-2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. Conclusions: These results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, 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.