aureus USA300 (Osimertinib order Figure 2A). An additional immune reactive species was observed when EssB was overproduced from the plasmid (Figure 5A, white asterisk). Variants carrying the PTMD sequence, EssBNM and EssBMC, sedimented during ultracentrifugation, whereas EssBΔM, the variant that lacks the PTMD sequence, did not. Two proteins assumed aberrant find more behavior. The EssBN protein was either poorly produced or very unstable in S. aureus essB mutant (Figure 5A; white arrow). EssBC partitioned into both the soluble and the insoluble fractions. Perhaps, this domain interacts weakly with components of the secretion machine embedded in the membrane. Of note, only the plasmid encoding full-length
EssB restored EsxA secretion into the extracellular medium of essB mutant cultures (M); all other plasmids failed to complement Dactolisib datasheet essB for EsxA secretion (Figure 5B). As expected, the control ribosomal protein L6 was found in cell lysates (C) (Figure 5B). Figure 5 Complementation and dominant negative activity of truncated EssB variants. (A-B) Complementation studies. S. aureus USA300 lacking functional essB was transformed with vector carrying either no insert, or various truncated variants of EssB or full length EssB. (A) The subcellular localization of EssB immune
reactive species was assessed by subjecting cell lysates to ultracentrifugation to separate soluble (S) and (I) insoluble proteins and proteins in both extracts were resolved by SDS-PAGE followed by immunoblotting with specific antibodies Orotidine 5′-phosphate decarboxylase (α-SrtA is used for subcelluar fractionation control of an insoluble membrane protein). (B) Cultures were examined for production and secretion of EsxA. Cultures were spun to separate proteins in cells (C) from secreted
protein in the medium (M). α-L6 is used for fractionation control of a cytosolic protein. (C-D) Dominant negative studies. Truncated variants of EssB were examined for protein localization (C) and EsxA secretion (D) as described in panel A. All plasmids were transformed in wild-type strain USA300 (WT). All truncated variants with the exception of EssBΔM lacking PTMD prevented secretion of EsxA. The data for a duplicate of three independent experiments are shown. Arrows indicate proteins with correct mass found in reduced abundance (white arrow: EssBN; red arrow: EssBNM; blue and purple arrows: endogenous EssB). Protein products with aberrant mass are depicted with asterisks. When transformed into wild-type S. aureus USA300, plasmid produced EssB and variants fractioned as before following 100,000 × g ultracentrifugation (Figure 5C). Briefly, EssB, EssBNM and EssBMC were found in the sediment, EssBΔM remained soluble and EssBC fractionated equally in the soluble and insoluble compartments (Figure 5C). Expression of EssBNM led to some degradation of EssB (Figure 5C, black asterisk).
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