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

Abstract 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. Graphical Abstract Isolate- and cell type- specific host responses to internalized S. aureus across subcellular localization, transcriptional programs, host fates, and the role of TNF-R1 signaling in infection outcomes of U937 monocytes. In U937 wt , SaOS-2 wt , and A549 wt cells, intracellular S. aureus resides in (frequently LAMP-1–decorated) membrane-enclosed compartments or directly in the cytosol at 2 hpi, but also close to host nuclear structures across cell lines. TNF-R1 surface expression is increased by all viable isolates in U937 wt cells, but only by EDCC 5464 in A549 wt and SaOS-2 wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937 wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates. This response is weakest for the chronic infection isolate EDCC 5464. Isolate-dependent cell death (marked by Annexin V/7-AAD staining; not shown) of U937 wt cells involves heterogenous activation of caspase-8, caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in earlier stages of infection (6 hpi). The extent of activation then barely further increases until 24 hpi and includes changes in the mitochondrial trans-membrane potential (ΔΨm) and additional caspase-1 activation, consistent with engagement of apoptotic signaling alongside other pathways at distinct stages of infection. S. aureus -induced cell death and caspase activation is abolished in (B) U937 ΔTNF-R1 at 6 hpi, but is significantly reinforced across the tested effectors differences at 24 hpi, whereas isolate-specific differences observed in U937 wt are lost. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549 wt cells that correlates with Annexin V/-7-AAD staining (not shown) but is accompanied by minimal caspase activation. The number of coloured arrows indicate the mean proportion of effector-positive cells (↑ ~20-40%, ↑ ↑ 40-60%, ↑ ↑ ↑ >60%), the number of coloured plus-signs (+) indicate a trend for the extent of the effect; colours represent each S. aureus isolate: SH1000 (green), EDCC 5055 (blue), EDCC 5458 (orange), EDCC 5464 (yellow). Created with BioRender.com.