Figure 1.
Preparation of biotinylcyslabdan.
The structures of cyslabdan and biotinylcyslabdan are shown. The carboxyl moiety of the N-acetylcysteine residue of cyslabdan was modified with amine-(PEO)2-biotin to synthesize biotinylcyslabdan.
Table 1.
MIC values of β–lactams and other antibiotics against MRSA in the absence and presence of cyslabdan.
Figure 2.
Identification of cyslabdan-binding proteins.
Biotinylcyslabdan was attached to avidin beads and incubated with an MRSA lysate. After washing nonspecific proteins with PBS, the bound proteins were recovered from the beads via denaturation with Laemmli sample buffer and were then analyzed via SDS–PAGE. Typical data from five separate experiments are shown. The arrow indicates a band around 50 kDa that was detected reproducibly.
Figure 3.
Binding of cyslabdan to hexahistidine-tagged SAR1388 and its related proteins.
Biotinylcyslabdan-immobilized avidin beads were used to analyze the interaction between cyslabdan and the proteins of FemA, FemX and FemB. The bound proteins were recovered by denaturing with Laemmli sample buffer and were analyzed using SDS–PAGE.
Figure 4.
Effect of cyslabdan on FemX, FemA and FemB activities.
The amount of monoglycyl lipid II, triglycyl lipid II and pentaglycyl lipid II produced by FemX-, FemA- and FemB-catalyzed reaction was compared in the presence and absence of cyslabdan (0.8 mM). The amount of each product formed in the absence of cyslabdan is set up at 100%, and each product formed in the presence of cyslabdan is expressed as a percentage of control.
Figure 5.
HPLC analysis of the peptidoglycan composition of cyslabdan-treated MRSA.
(A) HPLC analysis of the muropeptides extracted from MRSA peptidoglycans. Peptidoglycans were prepared from MRSA in the presence or absence of cyslabdan (4 µg/mL). After treating the peptidoglycans with mutanolysin and sodium tetrahydroborate, the resultant monomer-, dimer-, trimer-, and oligomer-type muropeptide products were analyzed by HPLC under the established conditions using an ODS column. The upper chromatogram indicates the results obtained for control MRSA, whereas the lower chromatogram indicates those obtained for cyslabdan-treated MRSA. (B) Structures of monomer-type muropeptides identified by ESI-MS. The HPLC results obtained for the monomeric fraction have been enlarged on the left-hand side. Peaks 1–3 were recovered and analyzed by ESI-MS. The structures of peaks 1–3 are shown on the right-hand side. Peaks 1 and 2 (asterisks) accumulated in the cyslabdan-treated MRSA.
Figure 6.
Proposed mechanism underlying the effect of cyslabdan on the activity of imipenem against MRSA.
In MRSA, the first Gly is extended from the l-Lys of the pentapeptide region of the murein monomer (MurNAc-GlcNAc-pentapeptide) by FemX. The introduction of the second and third Gly is catalyzed by FemA, and the fourth and fifth Gly are added by FemB, to form (Gly)5-murein monomers. PBP and/or PBP2′ finally catalyze the cross-linking of the fifth Gly and fourth d-Ala of the pentapeptide region in the next murein monomer together with the concomitant release of the terminal d-Ala of the pentapeptide. Imipenem alone: β-lactam inhibited PBP, but insensitive PBP2′ was able to cross-link (Gly)5-murein monomers. Therefore, MRSA can grow even in the presence of imipenem. Cyslabdan alone: the nonantibiotic compound cyslabdan inhibited FemA, causing the accumulation of Gly-murein monomers, but PBP and/or PBP2′ were able to cross-link them; hence, MRSA can grow even in the presence of cyslabdan. Combination of imipenem and cyslabdan: MRSA was not able to grow, indicating that PBP2′ cannot cross-link Gly-murein monomers. In this illustration, the red cross indicates the observation that PBP2′ was not able to cross-link the resulting monoglycyl murein monomer to the next murein monomer.