Journal
VIRULENCE
Volume 8, Issue 6, Pages 719-740Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/21505594.2016.1229727
Keywords
attenuation; cell sorting; fibroblast; intracellular; live cell imaging; NF-kappa B response; Salmonella; single-cell analysis
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Funding
- Spanish Ministry of Economy and Competitiveness [BIO2013-46281-P, IPT-2012-0213-060000]
- Marie Curie Grant [2011298447]
- 'Formacion de Personal Investigador' (FPI) Program of the Spanish Ministry of Economy and Competitiveness
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The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-kappa B) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-kappa B activity in single cells during infection. Here, we investigated at the single cell level how NF-kappa B nuclear localization - a proxy for NF-kappa B activity - oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-kappa B was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (STC) cells translocate NF-kappa B to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST-fibroblasts exhibited higher NF-kappa B nuclear translocation than the STC population, with these latter cells turning refractory to external stimuli such as TNF-alpha or a second infection. Sorting of STC and ST-cell populations confirmed enhanced expression of NF-kappa B target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-kappa B response in the infected fibroblast. Higher expression of SOCS3, encoding a suppressor of cytokine signaling, was also observed in the STC population. Intracellular S. Typhimurium subverts NF-kappa B activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-kappa B target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-kappa B signaling in fibroblasts, an effect only perceptible when STC and ST-populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.
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