Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45 - PubMed (original) (raw)

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45

Yoshimasa Ishizaki et al. J Biol Chem. 2013.

Abstract

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate-GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Keywords: Antibiotics Action; Bacillus; CPZEN-45; Caprazamycin B; Cell Wall; Mycolylarabinogalactan; TagO; Teichoic Acid; Tuberculosis; WecA.

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Figures

FIGURE 1.

FIGURE 1.

Structures of caprazamycin A-G (1); caprazene (2), which is a product of the acid hydrolysate of caprazamycins; and CPZEN-45 (3), which is 4-butylanilide of caprazene.

FIGURE 2.

FIGURE 2.

Radar chart of antibacterial activity of caprazamycin B (gray line) and CPZEN-45 (black line).

FIGURE 3.

FIGURE 3.

Strategy for the construction of the M. smegmatis mc2155 derivative strain, containing the WecA of M. tuberculosis expressed from the multicopy plasmid instead of the original. The PCR fragment of wecA of M. smegmatis (wecAMsm) was amplified using the genomic DNA of the mc2155 strain as the template. The consequent DNA fragment was digested with restriction enzymes Age I and Ngo_M IV, and the 5′-end and 3′-end fragments were ligated to induce deletion from nucleotide positions 46–612 of wecAMsm. This fragment (Δ_wecAMsm) was inserted into a plasmid containing the hygromycin-resistant gene (hygR), the levansucrase gene (sacB), and pUC replicon, and the resulting plasmid, pUChphsacB-Δ_wecAMsm_, was introduced into M. smegmatis mc2155 by electroporation. Because pUChphsacB-Δ_wecAMsm_ does not have a replication origin for mycobacteria, only when the plasmid was integrated into the genome did the transformants show resistance to hygromycin. The resulting strain was transformed with another plasmid, p16Rkan-wecAMtb, by electroporation. This plasmid was constructed as follows: the PCR fragment of wecA of the BCG strain of M. bovis (wecAMbo) was amplified using the genomic DNA of BCG as the template. This fragment was cloned into a plasmid containing the kanamycin-resistant gene (kanR), the pAL5000 replicon for mycobacteria, and the pUC replicon. The resulting plasmid was used as template of another PCR to alter wecAMbo to wecA of M. tuberculosis (wecAMtb), which has 1 base difference from wecAMbo. Finally, the resulting strain, which is resistant to hygromycin and kanamycin, was challenged on agar plates containing 10% sucrose. Because the levansucrase encoded by the sacB gene leads to sucrose sensitivity, when integrated plasmid pUChphsacB-Δ_wecAMsm_ was excluded from the genome, the transformant became sucrose-resistant again. The resulting sucrose-resistant and hygromycin-sensitive transformants had wild-type or disrupted wecA; thus, the genotype of each cell was analyzed by Southern blotting and transcription analysis using RT-PCR and subsequent sequencing.

FIGURE 4.

FIGURE 4.

Effects of caprazamycin B, CPZEN-45, vancomycin, and tunicamycin on the incorporation of radiolabeled precursors into the macromolecules of B. subtilis 168. The percentage of the incorporation of [_methyl_-3H]thymidine (for DNA, squares), [5,6-3H]uridine (for RNA, diamonds),

l

-[4,5-3H]leucine (for protein, triangles), [1-14C]acetate (for fatty acids, crosses), _N_-acetyl-

d

-[1-14C]glucosamine (for peptidoglycan, closed circles), and [U-14C]glycerol (for teichoic acid, open circles) are shown. Each plot and error bars represent the average and standard deviation of three independent trials.

FIGURE 5.

FIGURE 5.

Effects of caprazamycin B, CPZEN-45, and tunicamycin on the activities of MraY and TagO in B. subtilis 168. The inhibitory curves of CPZEN-45 (diamonds), caprazamycin B (squares), and tunicamycin (triangles) on the activity of MraY and TagO are presented. Each plot and error bars represent the average and standard deviation of three independent trials.

FIGURE 6.

FIGURE 6.

Effects of caprazamycin B, CPZEN-45, and tunicamycin on the activities of WecA of M. tuberculosis expressed in M. smegmatis 8a10. The inhibitory curves of CPZEN-45 (diamonds), caprazamycin B (squares), and tunicamycin (triangles) on the activity are presented. Each plot and error bars represent the average and standard deviation of three independent trials.

FIGURE 7.

FIGURE 7.

Effect of CPZEN-45 on the WecA activity of cytoplasmic membranes of M. smegmatis mc2155. A, TLC, developed as described above and subjected to radioautography, shows the effects of CPZEN-45 on the synthesis of the immediate product of WecA, the polyprenyl-P-P-GlcNAc. Preparation of membranes, cell-free assay conditions, including use of UDP-[U-14C]GlcNAc, were as described (22). B, CPZEN-45 was calculated to inhibit WecA of M. smegmatis strongly with an IC50 of 4.4 ng/ml. Data shown here are typical of three independent trials.

FIGURE 8.

FIGURE 8.

Overview of the biosynthetic pathway of peptidoglycan and teichoic acid in B. subtilis 168 (modified from Ref. 13) (A) and the biosynthetic pathway of peptidoglycan and mycolylarabinogalactan in M. tuberculosis (B). MurX and WecA are the orthologs of MraY and TagO in B. subtilis, respectively.

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