Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages.

Jonna Idh,Thomas Schön,Johanna Raffetseder,Daniel Eklund,Blanka Andersson,Hanna Woksepp,Jim Werngren,Maria Lerm,Tommy Sundqvist,Olle Stendahl,Mikael Mansjö

doi:10.1371/journal.pone.0181221

Abstract

BackgroundDrugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis.MethodLuciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA.ResultsWhen M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO.ConclusionIn a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

Highlights

It has been estimated that only a few percent of individuals infected with Mycobacterium tuberculosis develop active tuberculosis (TB) during their lifetime
When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced nitric oxide (NO)-susceptibility in broth culture
Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth

Summary

Introduction

It has been estimated that only a few percent of individuals infected with Mycobacterium tuberculosis develop active tuberculosis (TB) during their lifetime This suggests that effective immune responses are protecting the host from developing disease [1]. The importance of NO is well established, and mice lacking iNOS develop a more disseminated disease with reduced survival following M. tuberculosis infection [6]. Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis.

Objectives

Methods

Results

Conclusion