Figure 1.
Brucella infection of macrophages induces ER structural reorganization.
RAW 264.7 macrophages were uninfected or infected with YFP expressing B. melitensis (green) for 24 h. Arrow indicates condensation and fragmentation of the ER, visualized with anti-calreticulin antibody (red). Results are representative of 5 independent experiments.
Figure 2.
Brucella infection activates the UPR in macrophages in vitro.
RAW 264.7 macrophages were uninfected (NI) or infected with 100 MOI B. melitensis (B. mel). A) After 24 h, cells were resuspended in TRIzol for RNA processing. Relative expression of reverse transcribed cDNA was determined by quantitative PCR (qPCR) with normalization to 18S rRNA or hprt. Bars are combined mean fold inductions for 4–5 independent experiments (NI = 1) ± sem. *P<0.05, **p<0.003. B) Cells were infected with 100 MOI for 24 h and processed for RNA. XBP1 spliced and unspliced mRNA species were resolved by high-density agarose gel or detected by qPCR. %Splicing = spliced/total×100. Bars represent combined mean ± sem from 2–4 independent experiments. Representative gel is shown below. C) 16 h post infection, RAW cells were lysed and lysates resolved by SDS PAGE. CHOP or β-actin proteins were detected by immunoblot. Results are representative of 3 experiments.
Figure 3.
Brucella induces the UPR in vivo.
BALB/c mice were injected ip with PBS (NI) or 107 B. melitensis (B. mel). After 24 h, CD11b+ cells were isolated from pooled spleens and cells were resuspended in Trizol for RNA purification. TNF-α, BiP, CHOP, and ERdj4 gene expression was detected by qPCR with normalization to 18S rRNA. Error bars denote standard deviations between 2 pools (7 mice each). ERdj4 expression is from 1 pool each (NI or B. mel) of 4 mice. Results represent 2 independent experiments. *p = 0.01.
Figure 4.
MyD88 deficiency impairs Brucella induced XBP1 splicing but not induction of other UPR genes.
Bone marrow derived macrophages from MyD88−/− or wild type mice (MyD88+/+) were uninfected (NI) or infected with 100 MOI of B. melitensis (B. mel). After 24 h, cells were processed for RNA isolation. Relative gene expression was determined by qPCR with normalization to 18S rRNA or hprt. Percent XBP1 splicing was quantified using Agilent and high-percent agarose gel. A sample agarose gel image shows corresponding unspliced and spliced (XBP1(u) and XBP1(s)) mRNA species. Data is combined from 3 (IL-6, BiP, XBP1 splicing) or 4 (CHOP, ERdj4) infected sets (one each) of wild type and MyD88−/− mice by normalizing fold induction to the non-infected control in each set (NI = 1). Error bars represent standard error of the mean. *P≤0.04 vs. infected MyD88+/+. **P≤0.02. ***P = 0.001.
Figure 5.
TcpB mutation reduces Brucella-induced UPR target gene expression.
A) RAW 264.7 macrophages were uninfected (NI) or infected with 100 MOI B. melitensis (WT), heat killed Brucella (HK), a VirB deletion mutant (ΔVirB), or TcpB deletion mutant (ΔTcpB) for 24 h (legend at bottom of panel A). Cells were processed for RNA and relative UPR target gene expression (BiP, CHOP, and ERdj4) was determined by quantitative PCR (qPCR). XBP1 splicing was detected by qPCR. To combine independent experiments, WT induced UPR gene expression was set = 100%. Bars represent combined means of 3–4 (ΔTcpB) or 4 (HK, ΔVirB) experiments ± sem. *P<0.04, **p<0.02, ***p<0.009 vs. WT. B) RAW 264.7 or J774 cells were either uninfected (NI) or infected for 24 h with 100 MOI of 16M B. melitensis (WT), the TcpB deletion mutant (ΔTcp) or the complemented TcpB deletion mutant (C). RNA expression was normalized to 18S rRNA and WT 16M (set = 100%). Bars represent combined means of 14 (NI, WT) and 10 (ΔTcpB) experiments, and 5 complemented TcpB (C) mutant experiments. *P≤0.03, ***p = 0.008 comparing ΔTcpB mutant and complemented mutant gene expression.
Figure 6.
TcpB mutation results in less ER structural disruption following infection.
RAW 264.7 cells were infected with an YFP-expressing TcpB deletion mutant (ΔTcpB) or wild type (WT) B. melitensis (green) for 24 h. The ER is visualized with anti-calreticulin (red). Arrow indicates ER condensation and fragmentation. Confocal microscopy results are representative of 5 independent experiments. Scale bar is 20 µM.
Figure 7.
TcpB protein induces UPR and ER restructuring.
A) RAW 264.7 cells were untreated (NT) or stimulated with 50 µg/mL MBP or MBP-TcpB (TcpB). After 24 h, cells were processed for RNA and relative expression of BiP, CHOP, ERdj4, or spliced XBP1 (XBP1s) determined by qPCR, with normalization to 18S rRNA. Bars depict combined means from 2 independent experiments, and are representative of 3 experiments, *p<0.004 vs. MBP. For ERdj4, p value is not significant vs. MBP, but P = 0.03 vs. non-treated cells. B) RAW 264.7 cells were untreated (Control), or stimulated with 10–50 µg/mL purified MPB-TcpB for 24 h. Cells were then fixed, stained for calreticulin, (red) and nuclei counterstained with DAPI (blue). Cells were imaged at 60× in close up (Zoom, right) or without additional digital zoom (Broad Field, left).
Figure 8.
TcpB induced ER restructuring is not dependent on the UPR.
A) RAW264.7 macrophages were pre-treated with 500 µg/mL TUDCA 30 min., followed by 6 h 10 µg/mL tunicamycin (Tm) as indicated, and then harvested for RNA. Relative UPR gene expression was assessed by qPCR. Results were combined from 2 independent experiments, *p = 0.01 and **p = 0.001 vs. Tm treatment only and NS = not significant vs. untreated cells. B) RAW 264.7 cells were pre-treated with 500 µg/mL TUDCA (TUDCA +) or not pre-treated (TUDCA −) for 60 min prior to stimulation with 50 µg/mL TcpB, 10 µg/mL tunicamycin (Tm), or media (Control). Cells were then fixed, stained for calreticulin (red), and counterstained with DAPI. Images are 100×.
Figure 9.
The TcpB mutant displays altered growth in macrophages.
RAW264.7 macrophages were infected with wild type (B. mel, black) or TcpB mutant Brucella (ΔTcpB, gray). Select TcpB mutant cultures were also pre-treated with tunicamycin at 0.01 µg/mL (dashed lines). At different times following infection, cells were lysed and CFU determined by transfer to dilution plates. Error bars depict standard deviations of quadruplicate determinations. *P≤0.001. Results are representative of 4 independent experiments showing altered late growth of ΔTcpB.
Figure 10.
The chemical chaperone TUDCA inhibits BiP and CHOP induction and Brucella replication.
A) RAW cells were pre-treated with 500 µg/mL TUDCA, and then infected with 100 MOI Brucella (B. mel) for 30 min., washed, and incubated 24 h prior to harvesting for RNA analysis by qPCR. Results were combined from 3–4 independent experiments by normalization to B. melitensis induced UPR gene expression ( = 100%), *p≤0.04 and p = 0.00003 vs. B. melitensis infection and NS vs. uninfected. For XBP1s and ERdj4, N = 2 independent experiments. B) RAW cells were not treated (black circles) or pre-treated with TUDCA (gray squares), infected as in (A), and lysed at different times following infection. CFU (colony forming units) were determined by transfer to dilution plates. *P≤0.04. Similar results were obtained with 10 MOI Brucella (not shown). Results are representative of 6 independent experiments. C) D17 osteosarcoma cells were untreated (left) or pre-treated with 500 µg/mL TUDCA (right), infected with Brucella-YFP (green) for 24 h, fixed, and stained with anti-calreticulin (red).