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Single-cell analyses reveal an attenuated NF-?B response in the Salmonella-infected fibroblast.

ABSTRACT: The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-?B) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-?B activity in single cells during infection. Here, we investigated at the single cell level how NF-?B nuclear localization - a proxy for NF-?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-?B was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-?B to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-?B nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-? or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-?B target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-?B response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-?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-?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-?B signaling in fibroblasts, an effect only perceptible when ST+ 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.

SUBMITTER: Ramos-Marques E

PROVIDER: S-EPMC5626232 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast.

Ramos-Marquès Estel E Zambrano Samuel S Tiérrez Alberto A Bianchi Marco E ME Agresti Alessandra A García-Del Portillo Francisco F

Virulence 20160830 6

The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization - a proxy for NF-κB 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. Typhim ...[more]

PMID: 27575017

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Project description:Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.

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Single-cell analyses reveal an attenuated NF-?B response in the Salmonella-infected fibroblast.

Publications

Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast.

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