Lysocin E Targeting Menaquinone in the Membrane of Mycobacterium tuberculosis Is a Promising Lead Compound for Antituberculosis Drugs.

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.

The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity toward lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity. METHODS: MK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of colony-forming units. RESULTS: We found that Smith strain produced MKs with the length of the side chain ranging between 8 and 10, as opposed to other S. aureus strains that produce MKs 7-9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain harbored a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity toward lysocin E. CONCLUSION: We found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

[Development of Antibiotics Using Silkworm Bacteria and Fungi Infection Model].

　The emergence of antimicrobial resistant (AMR) bacteria has become a serious threat to public health. It is important that we find a mechanistically novel antibiotic to combat AMR. However, finding compounds which are both therapeutically effective and safe is difficult in the development of antibiotics. To solve these problems, we have focused on the silkworm model, which is economical and poses fewer ethical issues, as a means to evaluate the therapeutic effectiveness of test compounds in early stages of antibiotic development. Actually, the silkworm has pharmacokinetic parameters similar to mammals, and we revealed that antibiotics showed ED50s consistent with mammalian models. Thus, we screened therapeutically effective samples from natural products using the silkworm model, and found 23 candidates out of 15000 samples. We ultimately identified a novel antibiotic, lysocin E, and found that it demonstrates a potent therapeutic effect in the mouse systemic infection model. Furthermore, since the target of lysocin E is menaquinone on the bacterial membrane, it belongs to a novel class of antibiotics. In addition, we found a novel antibacterial agent named nosokomycin, GPI0363, and an antifungal agent, VL-2397 (ASP2397), using the silkworm model. In this report, we introduce the usefulness of the silkworm model in the development of antibiotics.

The Usefulness of Silkworms as a Model Animal for Evaluating the Effectiveness of Medicine and Food.

Development of novel medicines is an important responsibility of researchers in the field of pharmaceutical sciences. However, the discovery of new therapeutically effective without side effects is not an easy job. I think the limiting step of drug discovery is the process of evaluating the therapeutic effects of candidate drugs. To overcome this problem, I would like to propose a novel approach, "drug discovery with silkworms".

Advantages of the Silkworm As an Animal Model for Developing Novel Antimicrobial Agents.

The demand for novel antibiotics to combat the global spread of multi drug-resistant pathogens continues to grow. Pathogenic bacteria and fungi that cause fatal human infections can also kill silkworms and the infected silkworms can be cured by the same antibiotics used to treat infections in the clinic. As an invertebrate model, silkworm model is characterized by its convenience, low cost, no ethical issues. The presence of conserved immune response and similar pharmacokinetics compared to mammals make silkworm infection model suitable to examine the therapeutic effectiveness of antimicrobial agents. Based on this, we utilized silkworm bacterial infection model to screen the therapeutic effectiveness of various microbial culture broths and successfully identified a therapeutically effective novel antibiotic, lysocin E, which has a novel mode of action of binding to menaquinone, thus leading to membrane damage and bactericidal activity. The similar approach to screen potential antibiotics resulted in the identification of other therapeutically effective novel antibiotics, such as nosokomycin and ASP2397 (VL-2397). In this regard, we propose that the silkworm antibiotic screening model is very effective for identifying novel antibiotics. In this review, we summarize the advantages of the silkworm model and propose that the utilization of silkworm infection model will facilitate the discovery of novel therapeutically effective antimicrobial agents.