Comprehensive analysis of iron utilization by Mycobacterium tuberculosis

Author summary Tuberculosis is caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious disease, resulting in approximately 1.5 million deaths per year worldwide. M. tuberculosis resides in granulomas which are formed in the lung in an attempt to wall off the infection. Recently, it was shown that iron-sequestering proteins accumulate in these granulomas to high concentrations, establishing an environment devoid of free iron. In this study, we examined the contribution of each gene towards the utilization of different iron sources by M. tuberculosis. We identified 165 genes involved in iron utilization by Mtb, 66 of which were classified as essential or as required for optimal growth of M. tuberculosis in the presence of different iron sources. Our study provides an unprecedented insight into the genetic determinants of iron utilization by M. tuberculosis. Iron is essential for nearly all bacterial pathogens, including Mycobacterium tuberculosis (Mtb), but is severely limited in the human host. To meet its iron needs, Mtb secretes siderophores, small molecules with high affinity for iron, and takes up iron-loaded mycobactins (MBT) and carboxymycobactins (cMBT), from the environment. Mtb is also capable of utilizing heme and hemoglobin which contain more than 70% of the iron in the human body. However, many components of these iron acquisition pathways are still unknown. In this study, a high-density transposon mutagenesis coupled with deep sequencing (TnSeq) showed that Mtb exhibits nearly opposite requirements for 165 genes in the presence of heme and hemoglobin versus MBT and cMBT as iron sources. The ESX-3 secretion system was assessed as essential for siderophore-mediated iron uptake and, surprisingly, also for heme utilization by Mtb. Predictions derived from the TnSeq analysis were validated by growth experiments with isogenic Mtb mutants. These results showed that (i) the efflux pump MmpL5 plays a dominant role in siderophore secretion, (ii) the Rv2047c protein is essential for growth of Mtb in the presence of mycobactin, and (iii) the transcriptional repressor Zur is required for heme utilization by Mtb. The novel genetic determinants of iron utilization revealed in this study will stimulate further experiments in this important area of Mtb physiology.