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.

Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone-like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1-mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell-cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell-cell adhesion ability.

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR–Fe2+ complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml−1; 0.36 µM Fe) was still detectable at 8 µg Fe ml−1 (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml−1 (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein–DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.

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