Figure 3. Identification of host RIDD targets during Brucella infection.

(A) Venn diagram showing numbers of candidate RIDD genes identified in the indicated datasets. (B) Common candidate RIDD genes identified in Bm16M-infected cells and other conditions in the indicated datasets. (C) Interaction network analysis of candidate RIDD genes (Bloc1s1 associated genes) identified in Brucella-infected cells and the corresponding enriched KEGG pathways. Different pathways are distinguished by different colors. Interacting genes are shown with the smallest sized of dot with gene names. The upper-left-corner panel: enriched KEGG pathways and interacting candidate RIDD genes (%). qRT-PCR validation of RIDD candidate genes Bloc1s1 (D), Diras2 (E), Cd300lf (F), and Txnip (G) identified from RNA-seq analysis. Relative mRNA expression levels in potential RIDD targets from control and m-IRE1α BMDMs infected with Bm16M at 16 and 48 h.p.i. were measured by qRT-PCR. (H) qRT-PCR analysis of expression levels of Bloc1s1 in ΔXbp1 BMDMs that were either uninfected (control), infected with Bm16M, or treated with tunicamycin (Tm, an UPR inducer, 5 μg/ml) at 4-hr post infection/treatment. Expression levels of the indicated genes were normalized to Gapdh expression. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and ***: significance at p < 0.05, 0.01, and 0.001, respectively.

Figure 3—figure supplement 1. IRE1α activation is Brucella Type 4 secretion system (T4SS)-dependent and gene profiling of host cells infected by Brucella.

(A) Determination of IRE1α RNase activity in host cells via analysis of Xbp1 splicing. Murine J774.A1 macrophages were infected with Bm16M, Bm16MΔvirB2, or treated with Tm (2.5 μg/ml); at the indicated h.p.i. or treatment, the infected or drug-treated cells were harvested for Xbp1 splicing analysis. Representative images from one of three independent experiments are shown. (B) Strategy for gene profiling via RNA-seq analysis. (C) Heatmap of differentially expressed genes (DEGs) in the indicated host cells infected with Bm16M at 4 and 24 h.p.i. DEGs from triplicate samples are shown. (D) Heatmap of endoplasmic reticulum (ER)-associated DEGs in the wt-IRE1α (WT) host cells infected (In) and uninfected (Un) with Bm16M at 24 h.p.i. (upper panel) or at 4 and 24 h.p.i. (lower panel). (E) Fold changes of representative DEGs.

Figure 3—figure supplement 2. KEGG pathway network analysis of the candidate RIDD genes identified via RNA-seq analysis from host cells infected or uninfected with Bm16M and/or treated or untreated with 4μ8C at 4 and/or 24 h.p.i.

The functionally grouped network was visualized using Cytoscape (https://cytoscape.org/) based on the degree of connectivity (node size) between the KEGG pathways and RIDD genes (p < 0.05, nodes). Different KEGG pathways are distinguished by different colors. (A) The identified 847 candidate RIDD genes are mainly enriched in the KEGG pathways of ribosome (cellular component organization and biogenesis), spliceosome (RNA metabolism), oxidative phosphorylation, thermogenesis, and retrograde endocannabinoid signaling, etc., in which, a total of 142 RIDD candidate gene were enriched. Interaction networks of candidate RIDD genes enriched in the KEGG pathways of ribosome and oxidative phosphorylation (B), and spliceosome (C). KEGG pathway network analysis of the identified candidate RIDD genes (p < 0.05) involved in ribosome biogenesis and ribosomal RNA processing (D) or spliceosome associated factors (E).

Figure 3—figure supplement 3. Validation of RIDD target genes.

Real-time reverse transcription-PCR (qRT-PCR) assay for the expression levels of RIDD target genes Bloc1s1 (A), CD3300lf (B), Diras2 (C), and Txnip (D) in host cells infected with B. abortus strains S19 (BaS19) (A–C) or BaS2308 (D). Relative mRNA expression levels of the potential RIDD targets in RAW264.7 cells pretreated with 4μ8C (50 μM) or treated with tunicamycin (5 μg/ml) 1 hr before and during infection with the indicated Brucella strains. Gene expression was normalized to Gapdh. Un: uninfected cells. For information of WT Bloc1s1 and mBloc1s1 cells, please see the following Figure 4—figure supplement 1. (E) Relative microRNA miR-17-5p expression in the indicated cells untreated or treated with 4μ8C and infected with BaS2308. (F) The expression levels of Bloc1s1 in host cells treated with heat-killed B. melitensis (strains Bm16M or Bm16M∆Vjbr) determined by qRT-PCR. Data represent the means ± standard error of mean (SEM) from at least three independent experiments. *, **, and ***: significance at p < 0.05, 0.01, and 0.001, respectively.