Fig. 1

Verification of B. thailandensis T6SS-5⁻ clpV-5-sfgfp and T6SS-5⁺ clpV-5-sfgfp mutants. a Confirmation of the deletion of the T6SS-5 gene cluster (size: ~ 28 kb) in B. thailandensis by PCR using genomic DNA of the indicated strains and primers specific for the T6SS-5 gene hcp-5 (yielding a 489 bp product for the wild type and no amplicon for the ΔT6SS-5 mutant) and genes flanking the T6SS-5 gene cluster (yielding a 28,201 bp and 1885 bp product for the wild type and ΔT6SS-5 mutant, respectively). b Representative images of RAW264.7 macrophages infected with B. thailandensis ΔT6SS-5 attTn7::P_S12_clpV-5-sfgfp (T6SS-5⁻ clpV-5-sfgfp) and a strain expressing a chromosomal clpV-5-sfgfp fusion (T6SS-5⁺ clpV-5-sfgfp) at MOI 2 for 13 h and stained with Giemsa. c Quantification of MNGC formation of RAW264.7 macrophages infected with the indicated B. thailandensis strains and stained as described in B. The ability of the T6SS-5⁺ clpV-5-sfgfp mutant to induce MNGC formation shows that ClpV-5 is functional when fused to sfGFP. The data shown are mean values +SD based on two experiments performed in triplicate and three randomly selected microscopic fields per sample (N > 7578 nuclei). ns, not significant/P = 0.498 (t-test); ****P < 0.0001 (Welch’s t-test). d Detection of ClpV-5-sfGFP fusion proteins in whole cell lysates of the indicated B. thailandensis strains by Western blot analysis using α-GFP antibody (MW: GFP, 27 kDa; ClpV-5, 101 kDa) and as loading control α-RpoB antibody (MW: RpoB, 153 kDa). The bacteria were grown to an OD_600nm of 1.0 in LB broth. The genes BTH_II0871 and BTH_II0872 encoding the two component system VirAG were overexpressed (p::virAG) in B. thailandensis T6SS-5⁺ clpV-5-sfgfp to induce production of the fusion protein outside the host cell environment