DedA Protein Relates to Action-Mechanism of Halicyclamine A, a Marine Spongean Macrocyclic Alkaloid, as an Anti-dormant Mycobacterial Substance

Masayoshi Arai,Motomasa Kobayashi,Andi Setiawan,Takao Fujimoto,Liu Liu

doi:10.3390/md9060984

Abstract

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.

Highlights

Tuberculosis (TB) is one of the most common causes of morbidity and mortality in HIV-positive adults living in poverty [1]
To identify the gene that could confer a resistance to halicyclamine A, we prepared transformants of Mycobacterium smegmatis, which were transformed with the genomic DNA library of M. bovis BCG
Further study is necessary for the identification of the target molecule of halicyclamine A, these results strongly suggested that DedA protein correlates with the action mechanism of halicyclamine A as an anti-dormant mycobacterial substance

Summary

Introduction

Tuberculosis (TB) is one of the most common causes of morbidity and mortality in HIV-positive adults living in poverty [1]. We have established a screening system searching for anti-dormant mycobacterial substances [6,7,8]. In this assay system, M. tuberculosis H37Ra under hypoxic conditions, became highly resistant against isoniazid, which is one of the first-line drugs for TB. Halicyclamine A showed potent anti-microbial activity against M. smegmatis (MIC = 2.5 μg/mL), M. bovis BCG (MIC = 1.0 μg/mL), and M. tuberculosis H37Ra (MIC = 5.0 μg/mL) under standard aerobic growth condition as well as dormancy-inducing hypoxic condition [6]. The IMPDH over-expressing strains showed the same MIC value against halicyclamine A with that of the wild-type M. smegmatis [6]. Our continued study of the action-mechanism of halicyclamine A is presented

Results and Discussion

Identification of the Gene Causing Resistance to Halicyclamine A

Materials

Bacterial Culture

Conclusions

Methods