Microbiology and Infectious Disease

RNase III in Salmonella Enteritidis enhances bacterial virulence by reducing host immune responses

Bill Kwan-wai Chan
Yingxue Li
Hongyuhang Ni
Edward Wai-chi Chan
Xin Deng
Linfeng Huang author has email address
Sheng Chen author has email address

State Key Laboratory of Chemical Biology and Drug Discovery and the Department of Food Science and Nutrition, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Wang-Cai Biochemistry Lab, Division of Natural and Applied Sciences, Duke Kunshan University, No.8 Duke Ave, Kunshan, Jiangsu, China. Zip code: 215316
Department of Infectious Diseases and Public Health, City University of Hong Kong, Kowloon, Hong Kong
Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong
Global Health Research Center, Duke Kunshan University, No.8 Duke Ave, Kunshan, Jiangsu, China. Zip code: 215316
Shenzhen Key Laboratory of Food Biological Safety Control, The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen, China;

https://doi.org/10.7554/eLife.102755.1

Open access
Copyright information

Figures and data

The macrophage internalization and replication rate of six S. Enteritidis strains.

RNase III was over-expressed in high virulence strains and was responsible for degradation of dsRNA.
(a) Expression level of the rnc gene in six S. Enteritidis isolates. (b) Two clinical isolates (SEBL, SEST), and their corresponding rnc knockout mutants (SEBLΔrnc, SESTΔrnc) were cultured for 3h and 12h. Their total RNAs were extracted and the dsRNA was detected using J2 antibody. Same amount of total RNA from each sample were loaded. The specificity of J2 antibody on dsRNA was checked by using commercial dsRNA and ssRNA ladder.

Virulence assays of Salmonella strains carrying different constructs.
(a) RAW264.7 invasion rate of strains carrying different constructs. (b) RAW264.7 intracellular survival and replication rate.

Expression of immune signals in macrophage RAW264.7 and colon epithelial cells Caco-2 upon being transfected by total RNA extracted from Salmonella strains carrying different constructs.
RNA was transfected into the cell using Lipofectamine 2000 transfection reagent. (a) Expression of (a)TNF-α, (b)IL-1β, (c)MDA-5, (d)IFN-β in RAW264.7 cell line; Expression of (e)IFN-β, (f)MDA-5, (g)RIG-1 in Caco-2 cell line. The cells of no-bacteria control were treated with Lipofectamine 2000 transfection reagent without RNA.

Expression of immune signals in macrophage RAW264.7 and colon epithelial cells Caco-2 upon being transfected by total RNA extracted from clinical Salmonella isolates.
(a to d) RNAs extracted from strain SEBL and SEST, and their corresponding knockout mutants SEBLΔrnc and SESTΔrnc were transfected into two cell lines. The expression levels of immune signal were detected. (e)Total RNAs of the four Salmonella strains were extracted and treated with RNase III (dsRNA endonuclease) and exonuclease T (single-stranded RNA nuclease) respectively, followed by transfection into Caco-2 cell line to elicit different expression level of IFN-β. The cells treated with Lipofectamine 2000 only were used as control.

Virulence assays of the high virulence food isolate 654 and the corresponding rnc gene knockout mutant 654Δrnc.
(a)Macrophage internalization and replication assay. (b)Murine sepsis infection assay. The vector pET-rnc which carried the rnc gene was transformed into strain 654Δrnc to produce 654Δrnc::pET-rnc, which was then used in a complementation test. (c)ROS level in Salmonella isolates at different time intervals upon treatment with H₂O₂. (d)Western blot of the SodA protein in strains carrying different constructs. GAPDH was used as endogenous control. (e)The mRNA transcript level of the sodA gene in strains carrying different constructs. (f) The level of ROS in strains carrying different constructs.

Mechanism underlying the contributions of RNase III to Salmonella virulence.
(a) Demonstration of gene expression inhibition due to the accumulation of dsRNA. (b) Illustration of how host immune response triggered by dsRNA from low virulent and high virulent Salmonella strains.

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