Mapping ofMycobacterium tuberculosis katGPromoters and Their Differential Expression in Infected Macrophages

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Abstract
Intracellular pathogenic bacteria, includingMycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant inM. tuberculosisis thekatGgene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence renderingM. tuberculosisexquisitely sensitive to this compound. In this context,katGexpression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH. Thus, it is important to delineate the regulation and expression ofkatG, as this not only can aid understanding of howM. tuberculosissurvives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of thekatGpromoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped thekatGmRNA 5′ ends and demonstrated that two promoters, P1furAand P1katG, control transcription ofkatG. ThefurAandkatGgenes are cotranscribed from P1furA. Both P1furAand P1katGpromoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcriptional fusions P1furA-gfp, P1katG-gfp, and P1furA-P1katG-gfpconfirmed the existence of twokatGpromoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulentM. tuberculosisH37Rv. P1furAis induced early upon infection, and P1katGbecomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of thefurA-katGregion and indicate differential regulation in vivo of the twokatGpromoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.