Genomic Insight into Primary Adaptation of Mycobacterium tuberculosis to Aroylhydrazones and Nitrofuroylamides In Vitro

Abstract

Background/Objectives: New anti-tuberculosis compounds are needed to treat patients infected with multi- or extensively drug-resistant Mycobacterium tuberculosis strains. Studies based on spontaneous in vitro mutagenesis can provide insights into the possible modes of action and resistance mechanisms of such new compounds. We evaluated the primary response of M. tuberculosis in vitro to the action of new aroylhydrazones and nitrofuroylamides. Methods: The reference strain H37Rv was cultured on solid media with compounds at increased concentrations relative to MIC. Resistant clones were investigated using whole-genome sequencing and bioinformatics tools to assess the role and potential impact of identified mutations. Results: Some of the mutations are significant (based on in silico analysis), located in essential genes, and therefore of particular interest. Frameshift mutations were observed in (i) Rv2702/ppgK, which is associated with starvation-induced drug tolerance and persistence in mice, and (ii) Rv3696c/glpK, which has been described as a switch on/off mutation associated with drug tolerance. Nonsynonymous substitutions were found in Rv0506/mmpS2, which belongs to the Mmp protein family involved in transport and drug efflux, and in infB, encoding the translation initiation factor IF-2. Conclusions: The primary adaptation of M. tuberculosis to the selective pressure of the tested compounds is complex and multifaceted. It involves multiple unrelated genes and pathways linked to non-specific drug tolerance, efflux systems, or mechanisms counteracting oxidative stress.

Keywords: Mycobacterium tuberculosis; adaptation; drug resistance; whole-genome sequencing.

Figure 1

Gene–gene network of genes mutated in aroylhydrazone-resistant clones in vitro. (A) Network of five genes detected in this study. (B) The same network with added inhA, coding for a target of aroylhydrazones InhA. The network was built using STRING (https://string-db.org/cgi/about.3 (accessed on 30 January 2025)). Note the lack of connecting links between the genes, which reflects that the genes are not interacting, to the best of current knowledge.

Figure 2

Gene–gene network of genes mutated in nitrofurane-resistant clones in vitro. (A) Network of eight genes, including two genes in this study and six previously reported genes [10]. (B) The same network with added ddn coding for the nitrofurane-activating enzyme Ddn. The network was built using STRING (https://string-db.org/cgi/about.3 (accessed on 30 January 2025)). Note the lack of connecting links between the genes for most of the gene pairs, which reflects that the genes are not interacting, to the best of current knowledge.