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. 2012 Apr 9;2:71–75. doi: 10.1016/j.fob.2012.03.003

Fig. 2.

Fig. 2

Overproduction of VirB5 and presence of multiple copies of the virB promoter region attenuate wild type B. suis. (A) Replication kinetics of wild type 1330, virB5, 1330 (pBBRpvirB) or (pBBRpvirB–virB5) in J774A.1 macrophages. A representative experiment is shown. The virB5 mutant and wild type 1330 (pBBRpvirB–virB5) were significantly attenuated (P = 0.001, 2.7 × 10−5 at 24 hpi and P = 2.5 × 10−5, 5.2 × 10−4 at 48 hpi, respectively). 1330 is significantly more virulent than 1330 (pBBRpvirB) (P = 0.011 at 24 hpi and P = 0.001 at 48 hpi). (B) Immuno blot analysis of wild type 1330, 1330 (pBBRpvirB–virB5) and 1330 (pBBRpvirB). All strains were grown under acid growth conditions. Expression was detected with polyclonal antisera raised against VirB5, VirB9, VirB10 and Bcsp31 (loading control). (C) Immuno blot analysis of wild type 1330, the virB5 mutant, virB5 (pGLpvirB–virB5), and virB5 (pBBRpvirB–virB5). Expression was detected with polyclonal antisera raised against VirB5, VirB9, VirB10 and Bcsp31. D. Replication kinetics in J774 macrophages of wild type 1330, the virB5 mutant, virB5 (pBBRpvirB–virB5) and virB5 (pBBRplac–virB5), the latter induced at different time points (0, 2, 5, and 24 hpi) with IPTG. At 48 hpi, wild type 1330 and virB5 (pBBRplac–virB5) induced at 0 hpi, 2 hpi and 5 hpi are significantly different from virB5, virB5 (pBBRplac–virB5) and virB5 (pBBRplac–virB5) non-induced or induced at 24 hpi (P = 0.027 between virB5 (pBBRplac–virB5) induced at 0 hpi and virB5 (pBBRplac–virB5) induced at 24 hpi).