Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages
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.