Iron-Regulated Protein HupB of Mycobacterium tuberculosis Positively Regulates Siderophore Biosynthesis and Is Essential for Growth in Macrophages

Abstract

dMycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe 3؉ -specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 g Fe ml ؊1 ), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression

“…The growth of mycobacterial strains in high (8.0 mg Fe ml 21 )-and low (0.02 mg Fe ml 21 )-iron media and the assay of the siderophores were as described in our previous study (Pandey et al, 2014).…”

“…This was done essentially as described in our previous study (Pandey et al, 2014). In brief, the cDNA obtained by converting 1 mg total RNA isolated from high-and low-iron organisms using the SuperScript III First-Strand Synthesis System was used for qRT-PCR (Invitrogen).…”

“…Major advances have been made in the understanding of iron acquisition in this class of bacteria (Quadri & Ratledge, 2005;Ratledge & Dover, 2000;, and important milestones include deciphering the biosynthetic pathway of mycobactin (Quadri et al, 1998) and demonstrating its importance for the survival of the pathogen in vivo (De Voss et al, 2000). Our recent findings on the potentiating effect of HupB, a 28 kDa iron-regulated protein of M. tuberculosis, on mycobactin biosynthesis has contributed further to the understanding of regulation of expression of these iron-chelating molecules (Pandey et al, 2014).…”

“…The observed negative correlation of antiHupB antibodies with serum iron status in patients with tuberculosis was indicative of the iron-limited environment of the pathogen inside the human host. The objective of the present study was to investigate how iron influenced the transcription of hupB, and we demonstrated IdeR-dependent repression of hupB and, more interestingly, identified the 10 bp HupB box, first identified in the mbtB promoter (Pandey et al, 2014), in the hupB promoter DNA.…”

“…HupB is implicated in other roles, including adhesion of Mycobacterium leprae (Soares de Lima et al, 2005) and mycobacterial cell wall synthesis (Katsube et al, 2007). We first reported HupB as an ironregulated protein expressed by iron-limited Mtb (Yeruva et al, 2006), and recently we generated a hupB knock-out strain of Mtb and unambiguously proved the role of HupB as a positive regulator of mycobactin biosynthesis (Pandey et al, 2014); the study also reflected the essential role of HupB in vivo as the mutant failed to survive inside macrophages. HupB can bind iron (Takatsuka et al, 2011) and its ability to hold about 81 iron [Fe(III)] atoms led the authors to propose an iron storage role for the protein.…”