Abstract
Tuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A three-gene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the iniBAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival.
Highlights
Our knowledge on cell wall biosynthesis in mycobacteria has greatly increased over the last decades, the bacterial stress responses that follow after treatment with antibiotics targeting the mycobacterial cell wall have yet to be elucidated
Carp leukocyte cells (CLC) and THP-1 macrophages were infected with M. marinum harboring the iniBAC reporter construct, and analysis of induction kinetics was tracked by flow cytometry
In this work we show that the induction of the iniBAC operon as a result of EMB or INH treatment takes place both in cultured M. marinum and in intracellular bacteria, confirming previous observations of iniBAC induction in Mycobacterium smegmatis and M. bovis BCG by Alland et al [12]
Summary
The acyl carrier protein kasA has been found to be up-regulated in response to isoniazid [10] Another set of genes that was found to be highly induced by sublethal concentrations of ethambutol and isoniazid in M. tuberculosis and Mycobacterium bovis BCG is the iniBAC operon [11, 12]. This operon was found to be induced by these two cell wall targeting antibiotics but not by general cell wall stress caused by disruption of the membrane integrity through the activity of granulysin or lysozyme [12]. Colangeli et al [16] have shown that lsr, among regulating a large set of other genes, negatively regulates iniBAC expression, whereas recent studies have implicated operon regulation by mtrA and the alleged INH binding regulation element inbR [17, 18]
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