Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in ASM journals Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kurokawa, K. Articles by Sekimizu, K. Search for Related Content PubMed PubMed Citation Articles by Kurokawa, K. Articles by Sekimizu, K.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, September 2009, p. 4025-4027, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00200-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Target Specificity of Antibacterial Agents Using Staphylococcus aureus ddlA Mutants and D-Cycloserine in a Silkworm Infection Model

Kenji Kurokawa,^1,2* Hiroshi Hamamoto,¹ Miki Matsuo,^1, Satoshi Nishida,^1, Noriko Yamane,³ Bok Luel Lee,² Kazuhisa Murakami,^3, Hideki Maki,³ and Kazuhisa Sekimizu¹

Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,¹ National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, South Korea,² Discovery Research Laboratories, Shionogi and Co., Ltd., 3-1-1 Futaba-Cho, Toyonaka, Osaka 561-0825, Japan³

Received 13 February 2009/ Returned for modification 12 March 2009/ Accepted 10 June 2009

Top ABSTRACT INTRODUCTION REFERENCES

 
The availability of a silkworm larva infection model to evaluate the therapeutic effectiveness of antibiotics was examined. The 50% effective doses (ED₅₀) of D-cycloserine against the Staphylococcus aureus ddlA mutant-mediated killing of larvae were remarkably lower than those against the parental strain-mediated killing of larvae. Changes in MICs and ED₅₀ of other antibiotics were negligible, suggesting that these alterations are D-cycloserine selective. Therefore, this model is useful for selecting desired compounds based on their therapeutic effectiveness during antibiotic development.

Top ABSTRACT INTRODUCTION REFERENCES

 
The spread of multidrug-resistant strains continues to cause serious clinical problems (2, 4). Due to the expansion of the compound library in recent years, high-throughput screening of these compounds for their effectiveness against defined target enzymes has been performed as the first step in antibacterial agent development (1, 9). While many inhibitory compounds are discovered by this approach, further analysis of the compounds for structure, antibacterial effect, and therapeutic effectiveness in animal models is important for identifying the most promising candidates. Among them, evaluation of a compound's therapeutic effectiveness in mammal models is usually carried out for only a limited number of candidates because of cost and ethical concerns. However, it is extremely significant, because pharmacological actions of drugs are affected by various factors, such as absorption, distribution, metabolism, excretion, and drug interactions in the body.

We previously reported a silkworm larva infection model in which inoculation of pathogenic bacteria or true fungi into larval hemolymph leads to larval death (6, 7) but in which death can be prevented by coinjection of antibiotics or antifungal agents (3). Fifty percent effective doses (ED₅₀) for antibiotics tested on silkworm larvae were similar to those reported for antibiotics tested on mice. The large size of silkworm larvae and their slow movement allow quantitative evaluation of pathogens and drugs by injection (such as of 50 µl) into the hemolymph. In addition, the midgut or the fat body (equivalent to the liver) can be removed and used for pharmacological experiments. Owing to these characteristics, the therapeutic effectiveness of candidates can be evaluated using the silkworm larva model (3).

We have identified Staphylococcus aureus genes essential for cell growth or viability by isolating temperature-sensitive S. aureus mutants (12). S. aureus is a gram-positive pathogenic bacterium that causes abscesses, pneumonia, endocarditis, and food poisoning and has received increasing attention because it has rapidly gained resistance to various currently available antibiotics. Because temperature-sensitive mutants often have altered sensitivity to antibiotics, these alterations can be exploited for evaluation of the target specificity of antibacterial agents. In this study, we examined the antibacterial effect and therapeutic effectiveness of D-cycloserine against ddlA mutants of S. aureus. The D-Ala-D-Ala ligase encoded by the ddlA gene synthesizes a D-Ala-D-Ala dimer, which is then added to UDP-N-acetylmuramyl-tripeptide (10) and is thereby an essential intermediate during peptidoglycan biosynthesis. D-Cycloserine inhibits both D-Ala-D-Ala ligase and alanine racemase and thus attenuates peptidoglycan biosynthesis (8, 11).

First, we examined whether ddlA mutations altered the MIC of D-cycloserine selectively or not. The three temperature-sensitive S. aureus ddlA mutants that we used here were newly acquired by a previously described method (5); each of the mutants had a single transition mutation (G304A, C532T, and G115A), resulting in single amino acid substitutions (Asp102Asn, Pro178Ser, and Asp39Asn in the TS2921, TS5337, and TS10007 strains, respectively). Although the MIC of D-cycloserine against the parent RN4220 strain was 100 µg/ml, the MIC for TS2921 decreased to one-eighth of that value, 12.5 µg/ml (Table 1). Decrease in the MIC of D-cycloserine for TS5337 was also evident, while that of the MIC for TS10007 was not significant. The TS2921 phenotype of high susceptibility to D-cycloserine was reversed by introduction of the pSddlA plasmid or by addition of D-Ala-D-Ala to the medium (Table 1), suggesting that the susceptible phenotype is caused by a reduced ability to produce the D-Ala-D-Ala dimer of the mutant enzyme. MICs of vancomycin, tetracycline, and norfloxacin against these ddlA mutants were the same as those of these drugs against the parent strain. Also, the differences between the MICs of flomoxef or chloramphenicol against the parent strain and the MICs against the ddlA mutants were not significant (Table 1). Therefore, these results suggest that the S. aureus ddlA TS2921 and TS5337 mutants were susceptible to D-cycloserine and that the high sensitivity of the TS2921 mutant to D-cycloserine is antibiotic specific.

View this table: [in this window] [in a new window]

TABLE 1. MICs of antibiotics against S. aureus ddlA mutantsa

Next, we determined the therapeutic effectiveness of several antibiotics against S. aureus parent- or ddlA mutant-mediated larval death according to our previously established method (3, 7), except that bacterial cells were suspended in 0.9% NaCl, and we compared the values between the parent strain and the mutants. When silkworm larvae were inoculated with 3 x 10⁷ CFU of S. aureus parental and mutant cells, their 50% lethal times, the times at which half of larvae were dead, were much the same between the parent strain and the three ddlA mutants (Table 2), suggesting that they have comparable lethal effects on silkworm larvae. The ED₅₀ of D-cycloserine against ddlA mutant-mediated larval death, which was determined from survival curves, was 12.5% or <5.6% of that against larval death mediated by the parent strain (Table 2). In all ddlA mutants tested, the decrease in ED₅₀ of D-cycloserine compared with that of the parent starin was more noticeable than the decrease in the MIC. In contrast, the difference between the parent and the ddlA mutants with respect to the ED₅₀ of vancomycin, flomoxef, tetracycline, or chloramphenicol was within twofold, suggesting a selective effectiveness of D-cycloserine against all the ddlA mutants tested. These results suggest that the silkworm model is useful to examine the target-mediated antibacterial and therapeutic actions of the compounds. Differences between the S. aureus ddlA mutants were observed with regard to MIC and ED₅₀. Therefore, using multiple mutants that have distinct mutations makes it possible to identify a wider variety of candidate compounds during screening.

View this table: [in this window] [in a new window]

TABLE 2. Therapeutic effectiveness of antibiotics against S. aureus ddlA mutants in a silkworm larva infection modela

We questioned why the decreases in ED₅₀ were larger than the decreases in MIC against the ddlA mutants. Two explanations can be proposed. One is that the innate immunity of the silkworm larvae suppressed bacterial cell growth. The other is that it is not bacterial cell growth itself but rather host-killing ability that is being evaluated in the silkworm model.

In conclusion, we report here that comparison studies of therapeutic effectiveness of the compounds against parental and mutant bacterial strains can be helpful for evaluating their target-specific actions. These merits make this model worthy of attention as a secondary screening method for candidate antibacterial agents identified through target-based high-throughput screening.

 
We thank Makiko Miyatani, Hiromi Komaki, Kozue Saito, Kaori Hayasaka, Aya Yoshino, Yumiko Matsuzawa, and Kiyomi Kyogoku for their technical assistance.

This work was supported in part by Grants-in-Aid for Scientific Research from the JSPS, by the Industrial Technology Research Grant Program in 2004 from NEDO of Japan, by the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, and by grants from Genome Pharmaceuticals Institute Co. Ltd.

 
* Corresponding author. Mailing address: Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-5841-4821. Fax: 81-3-5684-2973. E-mail: kurokawa{at}mol.f.u-tokyo.ac.jp

Published ahead of print on 22 June 2009.

Present address: Department of Bacteriology, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

Present address: Department of Chemistry, The University of Chicago, Chicago, IL 60637.

Present address: Genome Pharmaceuticals Institute Co. Ltd., The University of Tokyo Entrepreneur Plaza, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Ehmann, D. E., J. E. Demeritt, K. G. Hull, and S. L. Fisher. 2004. Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-L-alanine ligase. Biochim. Biophys. Acta 1698:167-174.[Medline]
2
2. Gold, H. S., and R. C. Moellering, Jr. 1996. Antimicrobial-drug resistance. N. Engl. J. Med. 335:1445-1453.[Free Full Text]
3
3. Hamamoto, H., K. Kurokawa, C. Kaito, K. Kamura, I. M. Razanajatovo, H. Kusuhara, T. Santa, and K. Sekimizu. 2004. Quantitative evaluation of the therapeutic effects of antibiotics using silkworms infected with human pathogenic microorganisms. Antimicrob. Agents Chemother. 48:774-779.[Abstract/Free Full Text]
4
4. Hiramatsu, K., L. Cui, M. Kuroda, and T. Ito. 2001. The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol. 9:486-493.[CrossRef][Medline]
5
5. Inoue, R., C. Kaito, M. Tanabe, K. Kamura, N. Akimitsu, and K. Sekimizu. 2001. Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus. Mol. Genet. Genomics 266:564-571.[CrossRef][Medline]
6
6. Kaito, C., K. Kurokawa, Y. Matsumoto, Y. Terao, S. Kawabata, S. Hamada, and K. Sekimizu. 2005. Silkworm pathogenic bacteria infection model for identification of novel virulence genes. Mol. Microbiol. 56:934-944.[CrossRef][Medline]
7
7. Kurokawa, K., C. Kaito, and K. Sekimizu. 2007. Two-component signaling in the virulence of Staphylococcus aureus: a silkworm larvae-pathogenic agent infection model of virulence. Methods Enzymol. 422:233-244.[CrossRef][Medline]
8
8. Lambert, M. P., and F. C. Neuhaus. 1972. Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W. J. Bacteriol. 110:978-987.[Abstract/Free Full Text]
9
9. Liu, S., J. S. Chang, J. T. Herberg, M. M. Horng, P. K. Tomich, A. H. Lin, and K. R. Marotti. 2006. Allosteric inhibition of Staphylococcus aureus D-alanine:D-alanine ligase revealed by crystallographic studies. Proc. Natl. Acad. Sci. USA 103:15178-15183.[Abstract/Free Full Text]
10
10. Neuhaus, F. C. 1962. The enzymatic synthesis of D-alanyl-D-alanine. I. Purification and properties of D-alanyl-D-alanine synthetase. J. Biol. Chem. 237:778-786.[Free Full Text]
11
11. Neuhaus, F. C., and J. L. Lynch. 1964. The enzymatic synthesis of D-alanyl-D-alanine. 3. On the inhibition of D-alanyl-D-alanine synthetase by the antibiotic D-cycloserine. Biochemistry 3:471-480.[CrossRef][Medline]
12
12. Oku, Y., K. Kurokawa, M. Matsuo, S. Yamada, B. L. Lee, and K. Sekimizu. 2009. Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells. J. Bacteriol. 191:141-151.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, September 2009, p. 4025-4027, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00200-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in ASM journals Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kurokawa, K. Articles by Sekimizu, K. Search for Related Content PubMed PubMed Citation Articles by Kurokawa, K. Articles by Sekimizu, K.