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Van-M-02, a novel glycopeptide, was revealed to exert potent activities against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA). A crude assay system was then used to study the mode of action of Van-M-02 as a peptidoglycan synthesis model of both vancomycin-susceptible and -resistant strains. The results suggested that Van-M-02 inhibits the synthesis of lipid intermediates irrespective of their termini. This inhibitory activity may contribute to the anti-VRE and anti-VRSA activities observed.
The increasing incidence of vancomycin resistance in clinical settings has prompted research into new antibiotics against vancomycin-resistant strains (7, 12). We previously reported the synthesis of a novel glycopeptide, Van-M-02 (Fig. (Fig.1),1), during the course of our study of a vancomycin dimer (8). In this report, we describe the potent activities of Van-M-02 against the Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA), and the investigation of its mode of action using a crude assay system.
MICs were determined by the broth dilution method in accordance with CLSI document M7-A7 (2). Van-M-02 showed potent activities against VRSA, VanA-type VRE, and constitutive VanB-type VRE, with MICs of 4 μg/ml or less despite its structural similarity to vancomycin (Table (Table1).1). In order to assess the contribution of the d-Ala-d-Ala binding pocket to the activity of Van-M-02, N-terminally degraded Van-M-02 (ΔN-Van-M-02, Fig. Fig.1)1) was prepared (see Materials and Methods in the supplemental material). In a previous report, the corresponding compound ΔN-vancomycin (6) lost its antibacterial activities even against vancomycin-sensitive strains due to the lack of tight binding to d-Ala-d-Ala. In the present study, however, ΔN-Van-M-02 was effective against both vancomycin-sensitive (MIC = 2 μg/ml) and -resistant strains (MICs of 4 to 16 μg/ml).
To further investigate the mode of action of Van-M-02, a macromolecule precursor incorporation assay was performed. Van-M-02 specifically inhibited the incorporation of N-acetyl-d-[1-3H]glucosamine into cells of S. aureus and VRE (see Materials and Methods and Fig. S1A and D in the supplemental material). ΔN-Van-M-02 retained the specific inhibition of incorporation (see Fig. S1B and E in the supplemental material). These results suggested that cell wall synthesis is the primary target by which Van-M-02 exerts its antibacterial activity.
We next evaluated the inhibition of in vitro peptidoglycan synthesis by Van-M-02 with a crude assay system using wall membrane particulate-containing membrane enzymes of S. aureus and UDP-MurNAc-pentapeptide (for the susceptibility model) or UDP-MurNAc-depsipeptide (for the resistance model). UDP-MurNAc-pentapeptide (d-Ala-d-Ala terminus) was prepared from S. aureus, and UDP-MurNAc-depsipeptide (d-Ala-d-lactate terminus) was prepared from VRE (see Materials and Methods in the supplemental material). The formation of lipid intermediates (d-Ala-d-Ala terminus or d-Ala-d-lactate terminus) and nascent peptidoglycan (d-Ala-d-Ala terminus or d-Ala-d-lactate terminus) via successive reactions catalyzed by several enzymes, i.e., MraY, MurG, FemX, FemA, FemB, and transglycosylase, etc., were detected by thin-layer chromatography (TLC) utilizing the incorporation of [14C]glycine into them (see Fig. S2 in the supplemental material). In this report, “lipid intermediates” refers to lipid I and lipid II. Among the lipid intermediates, only lipid II can be glycylated, as reported previously (11). Lipid II was the lipid intermediate detected by TLC in the present study. The amounts of radioactivity incorporated into lipid intermediates and peptidoglycan in the resistance model were slightly reduced, by 14% and 22%, respectively, compared to those in the susceptibility model, which could result in a reduction of the growth rate of VRSA (4). To the best of our knowledge, this is the first report that the UDP-MurNAc-depsipeptide served as a substrate in an enzymatic assay for the peptidoglycan synthetic pathway in S. aureus.
In the susceptibility model of the crude assay system, the inhibition of peptidoglycan formation by vancomycin was far more potent (50% inhibitory concentration [IC50] = 4.5 ± 0.4 μg/ml) than that of the lipid intermediates (IC50 = 180 ± 46 μg/ml) (9). This result is reasonable, because the primary target of vancomycin is transglycosylase, an enzyme catalyzing the formation of peptidoglycan from lipid intermediates. The inhibition of peptidoglycan formation by Van-M-02 was similarly potent (IC50 = 4.2 ± 0.1 μg/ml). The inhibition of lipid intermediate formation was moderate in this case (IC50 = 32 ± 7.0 μg/ml) (Table (Table2).2). Thus, peptidoglycan polymerization by transglycosylase may also be a primary target of Van-M-02 in the susceptibility model. In the resistance model, on the other hand, the levels of the inhibitory activities of Van-M-02 were not very different between lipid intermediate formation (IC50 = 76 ± 0.6 μg/ml) and peptidoglycan formation (IC50 = 36 ± 4.2 μg/ml). Therefore, the processes of lipid intermediate formation would also be important targets of Van-M-02, in addition to transglycosylase, in the resistance model. It is also noteworthy in lipid intermediate formation that the IC50 of Van-M-02 (IC50 = 76 ± 0.6 μg/ml) was significantly lower than that of vancomycin (IC50 = 2,300 ± 1,400 μg/ml). ΔN-Van-M-02, which lacks a d-Ala-d-Ala binding motif, also showed inhibition of peptidoglycan and lipid intermediate formation in both the susceptibility and resistance models (Table (Table22).
We next examined whether the antibacterial activity of Van-M-02 was antagonized by external Nα,N-diacetyl-l-Lys-d-Ala-d-Ala (dKAA) or Nα,N-diacetyl-l-Lys-d-Ala-d-lactate (dKAL) in order to investigate the contribution of substrate-binding properties to the mechanism of Van-M-02. In the presence of 64 μg/ml dKAA, the antibacterial activity of vancomycin was decreased considerably (MIC = 16 μg/ml) but the activity of Van-M-02 was not substantially affected (MIC = 0.5 μg/ml) (Table (Table3).3). Thus, Van-M-02 should exert its antibacterial activities via a less substrate-dependent mechanism than vancomycin. Since dKAA had a smaller effect on the antibacterial activity of Van-M-02 than on that of vancomycin (Table (Table3),3), the potent activity of Van-M-02 would not be attributable to its enhanced binding affinity for the d-Ala-d-Ala terminus of the lipid intermediates of vancomycin-susceptible strains. In other words, another target of Van-M-02 may exist.
Since dKAL did not decrease the antibacterial activities of Van-M-02 (Table (Table3),3), the activities of Van-M-02 would not be attributable to high binding affinity for the d-Ala-d-lactate terminus of the lipid intermediates of VRE (depsilipids I and II).
These in vitro crude assays and antagonism studies suggested that Van-M-02 inhibits the lipid intermediate synthesis irrespective of the termini of lipid intermediates in bacteria. The inhibitory activity may contribute to anti-VRE and -VRSA activities.
Although the modified semiquantitative crude assay system enabled us to obtain insights into the actions of Van-M-02, their molecular mechanisms have not been fully characterized. Elucidation of the molecular target(s) of Van-M-02 would be a worthy topic of future research. Possible targets would be (i) enzymes catalyzing the synthesis of lipid intermediates (MurG, etc.) and transglycosylase, (ii) lipid intermediates, or (iii) the bacterial membrane.
In conclusion, inhibition of lipid intermediate synthesis should be considered a possible antibacterial target of Van-M-02 for vancomycin-susceptible and -resistant strains. We are currently working to establish quantitative assays with purified enzymes involved in cell wall synthesis (MurG, transglycosylase, etc.) and their substrates (depsilipid I, depsilipid II, etc.) that should shed light on the mechanism of action of modified vancomycins.
This study was supported in part by Grants-in-Aid for Scientific Research (16310150, 17035039, 18032010, and 21310136) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Yamada Science Foundation; the Mochida Memorial Foundation; and the Uehara Memorial Foundation (to H.A.).
We are grateful to Kazuo Kuruma for his technical assistance. Strain HIP11714 was provided by the network on antimicrobial resistance in Staphylococcus aureus (http://www.narsa.net/content/default.jsp).
Published ahead of print on 23 November 2009.
†Supplemental material for this article may be found at http://aac.asm.org/.