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, 9 (1), 3607

Nucleolar Fibrillarin Is an Evolutionarily Conserved Regulator of Bacterial Pathogen Resistance

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Nucleolar Fibrillarin Is an Evolutionarily Conserved Regulator of Bacterial Pathogen Resistance

Varnesh Tiku et al. Nat Commun.

Abstract

Innate immunity is the first line of defense against infections. Pathways regulating innate responses can also modulate other processes, including stress resistance and longevity. Increasing evidence suggests a role for the nucleolus in regulating cellular processes implicated in health and disease. Here we show the highly conserved nucleolar protein, fibrillarin, is a vital factor regulating pathogen resistance. Fibrillarin knockdown enhances resistance in C. elegans against bacterial pathogens, higher levels of fibrillarin induce susceptibility to infection. Pathogenic infection reduces nucleolar size, ribsosomal RNA, and fibrillarin levels. Genetic epistasis reveals fibrillarin functions independently of the major innate immunity mediators, suggesting novel mechanisms of pathogen resistance. Bacterial infection also reduces nucleolar size and fibrillarin levels in mammalian cells. Fibrillarin knockdown prior to infection increases intracellular bacterial clearance, reduces inflammation, and enhances cell survival. Collectively, these findings reveal an evolutionarily conserved role of fibrillarin in infection resistance and suggest the nucleolus as a focal point in innate immune responses.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
fib-1/fibrillarin regulates bacterial infection resistance in C. elegans. a, b fib-1 knockdown improves survival of wild-type N2 worms upon S. aureus and E. faecalis infection (P < 0.0001). c, d ncl-1 mutants (e1865 and e1942) are short-lived compared to wild-type N2 upon infection with S. aureus and E. faecalis (P < 0.0001). hlh-30(tm1978) served as a control for infection. Survival experiments were performed three times independently. P-values were calculated by log-rank test
Fig. 2
Fig. 2
FIB-1/fibrillarin and nucleolar size are reduced upon bacterial infection. a, b FIB-1 levels are significantly reduced in wild-type N2 after S. aureus and E. faecalis infection. FIB-1 is modestly reduced in ncl-1 mutants after infection but the levels are still higher relative to wild-type N2 after infection. Error bars represent mean ± s.e.m. of three independent biological replicates. c FIB-1::GFP shows reduced fluorescent signal after infection with S. aureus. d, e Nucleolar size in hypodermal cells of wild-type N2 worms is reduced after 12-h infection with S. aureus and E. faecalis. Nucleolar size in hypodermal cells of ncl-1 mutants after infection remains unaffected. Error bars represent mean ± s.d. f S. aureus infection for 12 h reduces 26S and 18S rRNA levels in wild-type C. elegans. RNA extracted from equal number of uninfected and infected worms was analyzed using a bioanalyzer. Scale bars represent 20 (c) and 5 μm (d). *P < 0.05, **P < 0.01, ns—non-significant, unpaired t-test
Fig. 3
Fig. 3
fib-1/fibrillarin reduction improves resistance of infection-sensitive mutants. ad fib-1 RNAi significantly improves the survival of infection-sensitive pmk-1(km25) (P = 0.0001), hlh-30(tm1978) (P = 0.0021), daf-16(mu86) (P < 0.0001) and ncl-1(e1865 and e1942) mutants (P < 0.0001) upon S. aureus infection. Survival experiments were performed three times independently. P-values were calculated by log-rank test
Fig. 4
Fig. 4
fib-1/fibrillarin reduction induces translation suppression. a, b Twelve-hour S. aureus infection induces Pirg-1GFP reporter. Error bars represent mean ± s.e.m. ce fib-1 knockdown induces Pirg-1GFP reporter and mRNA expression of irg-1. Error bars represent mean ± s.e.m. f fib-1 RNAi reduces the levels of 26S and 18S rRNA in worms. RNA extracted from equal number of worms was analyzed using a bioanalyzer. g, h fib-1 RNAi treatment reduced puromycin incorporation suggestive of reduced translation. No puromycin and cycloheximide treatments served as controls. Error bars represent mean ± s.e.m. i ifg-1(cxTi9279) exhibits significantly extended survival compared to wild-type N2 upon S. aureus infection (P < 0.0001, log-rank test). j, k fib-1 knockdown significantly increases the survival of wild-type N2 (P < 0.0001, log-rank test) but not of ifg-1(cxTi9279) (P = 0.74, log-rank test) upon S. aureus infection. Survival experiments were performed three times independently. Scale bars represent 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns—non-significant, unpaired t-test
Fig. 5
Fig. 5
Fibrillarin reduction protects mammalian cells against bacterial pathogens. a S. aureus infection leads to a reduction of fibrillarin levels in HeLa cells. b, c Mouse bone marrow-derived macrophages show a reduction in fibrillarin 24 h post infection with S. aureus, E. faecalis, S. typhimurium and L. monocytogenes. d, e Twenty-four hours of S. aureus infection leads to a reduction in nucleolar size of THP1 macrophages. Error bars represent mean ± s.d. f Fibrillarin siRNA reduces cytotoxicity relative to control siRNA after 24 h of S. aureus infection (MOI 10) in murine bone marrow-derived macrophages. Error bars represent mean ± s.e.m., unpaired t-test. g Fibrillarin siRNA leads to a better clearance of intracellular pathogens thereby lowering the number of CFU per mL after 6 and 24 h of S. aureus infection (MOI 10) in murine bone marrow-derived macrophages. Error bars represent mean ± s.e.m., unpaired t-test. h Six hours after S. aureus infection, ELISA results show a decrease in pro-inflammatory cytokines interleukin 6 and 8 after fibrillarin siRNA treatment in HeLa cells. Error bars represent mean ± s.e.m., unpaired t-test. i ELISA results show an increase in anti-inflammatory cytokine interleukin 10, 24 h post infection with S. aureus upon fibrillarin knockdown in mouse bone marrow-derived macrophages. Error bars represent mean ± s.e.m, unpaired t-test. j, k HeLa cells infected with GFP-labeled S. aureus and stained with lysotracker show increased co-localization of bacteria with lysosomes in cells treated with fibrillarin siRNA. **P < 0.01, ***P < 0.001, unpaired t-test. Scale bars represent 4 (d) and 10 μm (j). UI uninfected, HPI hours post infection, MOI multiplicity of infection

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