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. 2002 Aug;70(8):4165–4176. doi: 10.1128/IAI.70.8.4165-4176.2002

FIG. 4.

FIG. 4.

(A and B) Phagocytosis of the Y. enterocolitica ΔHOPEMT mutant overexpressing the effectors by J774 macrophages (A) or PMNs (B). J774 macrophages or PMNs were infected at an MOI of 50:1 for 30 min at 37°C with WT [MRS40(pYV40)] Y. enterocolitica, the yscN mutant [MRS4(pMSL41)], the ΔHOPEMT mutant [MRS40(pIML421)], or the ΔHOPEMT strain overexpressing YopE (pMSL68), YopH (pMSL69), YopT (pIML279), or YopO (pMSL34). Phagocytosis percentages were determined by double-immunofluorescence assay. Phagocytosis results are given as percentages of phagocytosed bacteria relative to the total number of cell-associated bacteria (∗, P < 0.05 compared to the ΔHOPEMT strain; n = 6). The values are the mean plus the standard error of the mean. For each experiment, at least 100 cells were counted. (C) Yop secretion by the Y. enterocolitica ΔHOPEMT mutant overexpressing the effectors. Western blot analysis (chemiluminescence detection) of proteins from culture supernatant corresponding to 2 ml of a culture of WT [MRS40(pYV40)] Y. enterocolitica, the yscN mutant [MRS40(pMSL41)], the ΔHOPEMT mutant [MRS40(pIML421)], or the ΔHOPEMT mutant overexpressing YopE (pMSL68), YopH (pMSL69), YopT (pIML279), or YopO (pMSL34) incubated for 4 h at 37°C in BHI-Ox. Immunoblot assay with rabbit anti-YopH, anti-YopT, and anti-YopO polyclonal antibodies and rat anti-YopE monoclonal antibodies (13A9). (D) Injection of the effectors by the various Y. enterocolitica ΔHOPEMT recombinant strains. J774 macrophages were infected at an MOI of 50:1 with WT Y. enterocolitica [MRS40(pYV40)], the ΔHOPEMT mutant [MRS40(pIML421)], the ΔHOPEMT mutant expressing YopE (pMSL68), the ΔHOPEMT mutant expressing YopH (pMSL69), the ΔHOPEMT mutant expressing YopT (pIML279), the ΔHOPEMT mutant expressing YopO (pMSL34), or the yopB mutant [MRS40(pPW401)] producing the same individual Yops. After 2 h, cytosolic fractions of J774 were prepared by Triton (0.1%) lysis and analyzed by SDS-PAGE and Western blotting with rabbit anti-YopH, anti-YopT, anti-YopO, and anti-SycE polyclonal antibodies and rat anti-YopE monoclonal antibodies (13A9). The absence of detection of SycE in the Triton-soluble fraction demonstrates that this fraction does not contain cytoplasmic proteins from Y. enterocolitica.