DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae

Hyun Kim,Youn Uck Kim,Yasuhiko Suzuki,Chie Nakajima,Noboru Nakata,Keigo Shibayama,Shigetarou Mori,Yasuo Fukutomi

doi:10.1038/s41598-019-47364-5

Hyun Kim, Youn Uck Kim + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-47364-5

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Abstract

Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25–30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

Highlights

Leprosy is a chronic human infectious disease caused by Mycobacterium leprae (Mle), with approximately 200,000 new cases reported worldwide every year[1]
The construction of expression vectors encoding Mle- and Mycobacterium tuberculosis (Mtb)-DNA gyrase is detailed in our previous reports[25,26,27]
The identity and integrity of these genes after cloning were confirmed by sequencing and alignment to reference sequences; the results showed that mutations were not introduced during polymerase chain reaction (PCR)

Summary

Introduction

Leprosy is a chronic human infectious disease caused by Mycobacterium leprae (Mle), with approximately 200,000 new cases reported worldwide every year[1]. Mle and Mycobacterium tuberculosis (Mtb) express only DNA gyrase[19,20,21] from a gyrB-gyrA contig in the complete genome; this enzyme is the sole target of fluoroquinolones[19,22]. GyrA2GyrB2 heterotetrameric structure[23,24], is an ATP-dependent enzyme that transiently cleaves and unwinds double-stranded DNA24 to catalyse DNA negative supercoiling. This enzyme is essential for efficient DNA replication, transcription, and recombination. We constructed and expressed the Mle-DNA gyrase as a recombinant enzyme, determined its activities in vitro and/or in vivo by radiorespirometry analysis for Mle viability and allelic exchange into other mycobacteria, and further evaluated its contribution to the growth and survival of Mle bacilli. We propose a novel therapeutic strategy—thermal therapy—against leprosy

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