Assembling of the Mycobacterium tuberculosis Cell Wall Core.

The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.

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.

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.

Cycloimidamicins, Novel natural lead compounds for translation inhibition in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most concerning pathogenic bacteria. We screened antibiotics using a highly drug-sensitive P. aeruginosa strain and an oligotrophic medium, and successfully isolated novel antibiotics, namely cycloimidamicins (CIMs), from a rare actinomycete strain, Lentzea sp. MM249-143F7. X-ray and nuclear magnetic resonance analyses revealed that CIMs possess a distinctive and unprecedented molecular structure, containing tetramic acid and an imidazole ring bound directly to indolone. The CIMs exhibited potent antibacterial activity against Gram-negative bacteria, as well as translation inhibition in Escherichia coli in both intact cells and in vitro. Additionally, E. coli strains resistant to known translation inhibitors did not exhibit cross-resistance to CIMs, suggesting that CIMs inhibit bacterial growth by blocking translation through a novel mechanism.

Amycolamicin: a novel broad-spectrum antibiotic inhibiting bacterial topoisomerase.

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Sealutomicins, new enediyne antibiotics from the deep-sea actinomycete Nonomuraea sp. MM565M-173N2.

A Nonomuraea sp. strain MM565M-173N2 was isolated from deep-sea sediment off the Sanriku coast, and new antibiotics were evaluated against carbapenem-resistant Enterobacteriaceae (CRE), which is a problematic group of bacteria because of their antimicrobial resistance. From 220 l of fermented broth from strain MM565M-173N2, we isolated four new antibiotics by gel filtration and HPLC, designated as sealutomicins A (1.8 mg), B (1.5 mg), C (0.8 mg), and D (0.8 mg). Their structures were determined from MS, NMR, and CD spectra. Sealutomicin A was found to be a new enediyne antibiotic, while sealutomicins B-D were aromatized products from sealutomicin A. Sealutomicin A showed strong antibacterial activity (MIC 0.05-0.2 µg ml-1) against CRE.

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug.

Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1-3, which converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate.

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.

Instability of the 16S rRNA methyltransferase-encoding npmA gene: why have bacterial cells possessing npmA not spread despite their high and broad resistance to aminoglycosides?

The NpmA bacterial 16S rRNA methyltransferase, which is identified from Escherichia coli strains, confers high resistance to many types of aminoglycoside upon its host cells. But despite its resistance-conferring ability, only two cases of its isolation from E. coli (14 years apart) have been reported to date. Here, we investigated the effect of the npmA gene on aminoglycoside resistance in Pseudomonas aeruginosa and Klebsiella pneumoniae and its stability in E. coli cells by comparing it with armA, another 16S rRNA methyltransferase gene currently spreading globally. As a result, we found that npmA conferred resistance to all types of aminoglycoside antibiotics we tested (except streptomycin) in both P. aeruginosa and K. pneumoniae, as well in E. coli. In addition, co-expression of armA and npmA resulted in an additive effect for the resistance. However, in return for the resistance, we also observed that the growth rates and the cell survivability of the strains transformed with the npmA-harboring plasmids were inferior than those of the control strains and that these plasmids were easily disrupted by IS10, IS1, and IS5 insertion sequences. We discuss these data in the context of the threat posed by pathogenic strains possessing npmA.

New antituberculous drugs derived from natural products: current perspectives and issues in antituberculous drug development.

Tuberculosis is one of the most common and challenging infectious diseases worldwide. Especially, the lack of effective chemotherapeutic drugs for tuberculosis/human immunodeficiency virus co-infection and prevalence of multidrug-resistant and extensively drug-resistant tuberculosis remain to be serious clinical problems. Development of new drugs is a potential solution to fight tuberculosis. In this decade, the development status of new antituberculous drugs has been greatly advanced by the leading role of international organizations such as the Global Alliance for Tuberculosis Drug Development, Stop Tuberculosis Partnership and Global Health Innovative Technology Fund. In this review, we introduce the development status of new drugs for tuberculosis, focusing on those derived from natural products.The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.126.

Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field.

When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed [Bioelectrochemistry 53 (2001) 149]. The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death [Bioelectrochemistry 54 (2001) 101]. These results suggest some possible changes in either the medium or the cells during the magnetic field exposure. When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E. coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field. When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed. This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression. It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression.

Novel antibacterial compounds specifically targeting the essential WalR response regulator.

The WalK/WalR (YycG/YycF) two-component system, which is essential for cell viability, is highly conserved and specific to low-GC percentage of Gram-positive bacteria, making it an attractive target for novel antimicrobial compounds. Recent work has shown that WalK/WalR exerts an effect as a master regulatory system in controlling and coordinating cell wall metabolism with cell division in Bacillus subtilis and Staphylococcus aureus. In this paper, we develop a high-throughput screening system for WalR inhibitors and identify two novel inhibitors targeting the WalR response regulator (RR): walrycin A (4-methoxy-1-naphthol) and walrycin B (1,6-dimethyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4-e][1,2,4]triazine-5,7-dione). Addition of these compounds simultaneously affects the expression of WalR regulon genes, leading to phenotypes consistent with those of cells starved for the WalK/WalR system and having a bactericidal effect. B. subtilis cells form extremely long aseptate filaments and S. aureus cells form large aggregates under these conditions. These results show that walrycins A and B are the first antibacterial agents targeting WalR in B. subtilis and S. aureus.