Laboratory of Microbiology

Research Outline

The Laboratory of Microbiology focuses on drug discovery. We have screened for novel bioactive molecules in microbial metabolites by developing a drug screening system based on the mechanism of pathogenesis of infectious diseases. Followed by detailed studies on the mode of action, the pharmacological and drug-like properties of potential molecules are commonly optimized in this institute. In addition, we have constructed a microorganism library containing various microbes from the soil, hydrosphere and so on.

Theme outline

1. Development of new antibiotics against XDR-TB

  • Tuberculosis (TB), caused by Mycobacterium tuberculosis, has been the most serious infectious disease for centuries. In 2013, 9.0 million people developed TB and 1.5 million died from the disease, 360,000 of whom were HIV-positive. In addition, the spread of multidrug-resistant tuberculosis (MDR-TB), including extensively drug-resistant tuberculosis (XDR-TB), which mainly results from inappropriate doses of chemotherapy, is now recognized as an additional problem. In response to this serious situation, we at BIKAKEN have explored antitubercular agents since the establishment of BIKAKEN in 1962 by Dr. Hamao Umezawa, who discovered kanamycin, the first antitubercular drug developed in Japan. Therefore, we have isolated promising candidate compounds named caprazamycins from the culture broth of a Streptomyces strain. We then modified them and finally established CPZEN-45 as the most promising derivative. CPZEN-45 is superior to caprazamycins in terms of some properties, including antitubercular activity against MDR- and XDR-TB. In addition, CPZEN-45 showed excellent therapeutic efficacy in the treatment of mice infected with XDR-TB.
  • We also evaluated the mechanism of action of CPZEN-45. We observed that CPZEN-45 strongly inhibited the activity of the integral membrane protein WecA transferase, involved in the biosynthesis of the cell envelope. This mechanism is different from any other currently studied mechanisms, implying that CPZEN-45 would be effective against most MDR-TB strains. In 2008, we joined “The Lilly TB Drug Discovery Initiative” and collaborated on further studies for developing this candidate drug in a short period of time.

Fig.1 Strucures of caprazamycins and CPZEN-45
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2. The antibacterial agent tripropeptin C

  • MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin resistant Enterococcus faecalis/faecium) are Gram-positive bacteria and two of the normal microbial flora; they are also known as opportunistic pathogens. They cause severe infections, such as sepsis, pneumonia, and endocarditis, in immunocompromised hosts and have the ability to acquire resistance to available drugs. Vancomycin has been the last resort for the treatment of MRSA, but its efficacy is limited because of the emergence of vancomycin intermediate and highly resistant bacteria. Thus, new drugs effective against these pathogens are urgently required.
  • Tripropeptin C, a cyclic lipopeptide antibiotic produced by Lysobacter sp., isolated from a soil sample collected in Okinawa prefecture, was discovered in 2001 (Fig. 1). The discovery of TPPC stemmed from research on compounds effective against MRSA without cross-resistance to existing drugs including vancomycin.
  • TPPC:
    - exhibited very potent antimicrobial activity against Gram-positive bacteria including resistant strains (MRSA, VRE, PRSP)
    - demonstrated excellent therapeutic efficacy in a mouse staphylococcal septicemia model when administered intravenously (45th ICAAC, Interscience Conference on Antimicrobial Agents and Chemotherapy) Distribution data supported a marked therapeutic efficacy of TPPC in mice.
    - inhibited of peptidoglycan biosynthesis with different mode of action from vancomycin and bacitracin.
    - revitalized and synergistically potentiated the activity of beta-lactams against MRSA. These results strongly indicate that TPPC is the promising candidate for novel anti-MRSA drug.
  • At present, the efficacies of TPPC and TPPC/beta-lactam combination treatments in a mouse-MRSA infection models are under evaluation.

Fig.2 Structure of tripropeptin C.
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3. A study on the spreading mechanism of NDM-1, a novel β-lactamase

Fig.3 Plasmid map of pNDM-1 Dok01 from Escherichia coli NDM-1Dok01.The gene bla NDM-1 was shown as red.
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  • The dissemination of drug-resistant pathogens has constituted a major threat to human life for a long period of time. Many of the genes responsible for the development of this resistance exist on movable plasmids and are transmissible between microorganisms. As a consequence, these genes have rapidly spread worldwide. Accordingly, it is considered that a study of the spreading mechanism may contribute to control of the proliferation of drug resistance in pathogens.
  • NDM-1 was first characterized as a novel class of β-lactamase in 2008. The gene blaNDM-1, which encodes NDM-1, exists on a mobile plasmid ; since its discovery this gene has disseminated throughout the world, particularly in South Asia and Europe. The first case in Japan was a NDM-1 producing strain of Escherichia coli, isolated at Dokkyo Medical University Hospital in 2009. One of our goals in collaboration with Dokkyo Medical University is to determine the very effective spreading mechanism of NDM-1. Until date, a large plasmid (ca. 200 kbp) encoding blaNDM-1 has been sequenced. We are now investigating the detail of the spreading mechanism using this sequence data.

4. Study of the aquatic and terrestrial microorganisms for drug discovery

  • We are expanding our stock culture library for the discovery of new bioactive metabolites and at present, we have > 40,000 pure cultures of soil bacteria. Fermentation broths of these bacteria are used for the screening of useful bioactive compounds. The screening results, therefore, directly depend on the quality and diversity of the library. Our library is composed mainly of actinomycetes, which are an excellent source for the discovery of novel secondary metabolites with diverse biological activities; hence, they are reg arded as the best screening sources for drug discov ery. At our inst itute, the clinically important drugs kanamycin, bleomycin, josamycin, and aclacinomycin were discovered from secondary metabolites of actinomycetes. High quality and as yet undiscovered isolates for screening sources are essential to the discovery of new metabolites. Our focus for isolating strains is based on poorly studied habitats within the extremobiosphere and underused samples as well as soi l samples. We are principally focusing attention on isolating bacteria from deep-sea sediment and aquatic organisms. To evaluate the diversity of these strains, we are performing a genotyping study based on 16S rRNA gene sequences of isolated bacteria. In addition, we have recently developed a new analysis method, MALDI-TOF MS typing, for act inomycetes.

Fig.4 Construction work flow of culture collection and broth library
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5. Screening for new antibiotics

  • Drug-resistant bacterial infections are a global concern for human health. The development of resistant bacteria has occurred in almost all major classes of antibiotics. The number of pathogenic bacteria, which have acquired tolerance to multiple classes of antibiotics, is steadily increasing worldwide in both developed and developing countries. In nosocomial infections, Enterococcus, Staphylococcus aureus , Klebsiella , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species are some of the most problematic. These pathogens are capable of not only producing the enzymes that inactivate antibiotics, such as extended spectrum β lactamases and aminoglycosidemodifying enzymes, but also altering antibiotics’ targets using their modification enzymes. This results in decreased affinity of the ant ibiotics for their targets.
  • Recently, 16S rRNA methylase was found to be one of the most problematic enzymes. This enzyme is produced by macrolide-resistant Staphylococcus and aminoglycoside-resistant Pseudomonas aeruginosa , Acinetobacter baumannii , and Enterobacter species. We have, therefore, been screening for new aminoglycoside antibiotics that are effective against the methylase-producing bacteria from microbial metabolites.

Fig.5 Screening for new aminoglycoside antibiotics
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