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. 2012 Apr 13;287(24):20417–20429. doi: 10.1074/jbc.M111.336461

FIGURE 4.

FIGURE 4.

LOS biosynthesis mutants show reduced PE_PGRS and EspE release. A, whole cells pellets and supernatant from M. marinum E11 wild-type, ESX-5 mutant 7C1, and PE_PGRS mutants A2, A5, A8, and B1 grown in 7H9 broth without Tween 80 were analyzed by immunoblot for expression and secretion of PE_PGRS, ESAT-6, and EsxN. GroEL2 was used as a negative control for lysis. LOS-deficient mutants A2 and A5 show a clear effect on the secretion of PE_PGRS and EsxN, although LOS-IV-deficient mutants A8 and B1 are unaffected. Results are representative of three independent experiments. B, immunoblot of whole cells pellets and supernatant from M. marinum WT and mutants A5 and A5c shows that the secretion defect of mutant A5 can be complemented. C, genapol X-080-treated whole cell pellets (Gp) and cell surface extracts (Gs) of bacteria grown on solid agar. No reproducible differences between E11 wild type and the tested mutants were observed for both PE_PGRS and EspE extractability. D, selected LOS mutants A2, A4, A5, A6, A8, A9, B2, B7, and B10 were plated on a nitrocellulose filter together with E11 wild-type, ESX-5 mutant 7C1, and a pks12 mutant. All LOS mutants are reproducibly negative for EspE secretion on double filter assay, whereas all the controls are positive for secretion.