Responses ofMycobacterium tuberculosisHemoglobin Promoters to In Vitro and In Vivo Growth Conditions

Abstract

The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3-to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5-to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H 2 O 2 , but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ϳ2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of th...

Comparative Analysis of Mycobacterial Truncated Hemoglobin Promoters and thegroEL2Promoter in Free-Living and Intracellular Mycobacteria

“…The genetic organization of glbO is largely conserved across various mycobacterial species in comparison to that of glbN. Among mycobacteria, adaptation to obligate parasitism has resulted in the loss of trHb genes, as in the case of M. leprae, which retained only glbO (4,6,27). Since M. leprae has retained only a minimalistic repertoire of functional genes, trHbO can be regarded as an essential product for intracellular parasitism, while trHbN appears to be dispensable.…”

“…The genome of M. tuberculosis contains glbO and glbN genes, encoding truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), respectively, which belong to a new family of hemoproteins that are extensively distributed in eubacteria, cyanobacteria, protozoans, and plants (27,29,38). The amino acid sequences of trHbs are shorter and share very low similarity to the sequences of classical vertebrate hemoglobins and myoglobins.…”

An Unconventional Hexacoordinated Flavohemoglobin from Mycobacterium tuberculosis

“…The transcriptional fusion was made by PCR amplification of a sequence encompassing Ϫ240/ϩ26 relative to the translational start codon of the Rv0385 gene using complementary oligonucleotide primers (supplemental Table S2) essentially as described earlier (29). The recombinant plasmid pSCFHb P thus created was transformed into M. tuberculosis H37Ra using standard protocol.…”

“…Transcriptional Fusion and Determination of Promoter Activity-The transcriptional fusion of MtbFHb was made in an E. coli-M. tuberculosis shuttle vector, pSC301 (29), by replacing the superoxide dismutase gene promoter, upstream of GFP, with upstream sequences of MtbFHb-encoding gene. The transcriptional fusion was made by PCR amplification of a sequence encompassing Ϫ240/ϩ26 relative to the translational start codon of the Rv0385 gene using complementary oligonucleotide primers (supplemental Table S2) essentially as described earlier (29).…”

“…All measurements were carried out in triplicate. For in vivo analysis of promoter activity, cells of M. tuberculosis H37Ra, carrying pSC301 and pSCHmp P , were infected into THP-1 macrophages and analyzed through a flow cytometer following the earlier described procedure (29).…”