This Article Full Text (PDF) Other Versions of this Article: AEM.02663-07v1 74/11/3512    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pawaria, S. Articles by Dikshit, K. L. Search for Related Content PubMed PubMed Citation Articles by Pawaria, S. Articles by Dikshit, K. L. Agricola Articles by Pawaria, S. Articles by Dikshit, K. L.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol. doi:10.1128/AEM.02663-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Response of Mycobacterium tuberculosis hemoglobin promoters to in vitro and in vivo growth conditions

Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India

^* To whom correspondence should be addressed. Email: kanak{at}imtech.res.in.

	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 considered to play pivotal roles in its cellular metabolism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusion of the promoters of glbN and glbO genes with green fluorescent protein were constructed and their response was monitored in M. smegmatis and M. tuberculosis H37Ra, exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophage. Promoter activity of glbN increased substantially during stationary phase and was nearly 3-3.5-fold higher than glbO promoter, which remained more or less constant during different growth phases of M. smegmatis as well as M. tuberculosis H37Ra. In both the mycobacterial hosts, glbN promoter activity was induced 1.5-2-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 as compared to SNP, although the overall increase in its activity was at a much lower level in comparison to glbN promoter. Additionally, the glbN promoter remained insensitive to oxidative stress generated by H₂O₂ but the glbO promoter activity increased to nearly 1.5-fold under similar conditions suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress. In contrast, the transition metal-induced hypoxia enhanced activity of both glbN and glbO promoters at all growth phases, with glbO promoter being induced to 2.3-fold, which was found highest for this promoter under all the conditions evaluated. The addition of iron along with nickel reversed the induction in both the cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron were depleted in the growth media by addition of an iron chelator. These results suggested the involvement of an iron/heme-containing oxygen sensor in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. Mode of promoter regulation under different physiological conditions was found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra indicating that these promoters might be regulated by components that are common to both the systems. Confocal microscopy of the THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times points up to 48 hr through FACS analysis of the intracellular M. tuberculosis cells indicated the glbN promoter to be active at all time points assessed, whereas, the glbO promoter remained at a steady state level upto 24 hr post infection and increased by 2-fold after 48 hr of infection. Overall regulation pattern of M. tuberculosis trHb gene promoters, thus, correlates not only with the stresses that tubercle bacillus is likely to encounter once inside the macrophage environment but also with the present day knowledge of their functionalities. The in vivo studies, demonstrating for the first time the expression of trHbs during macrophage infection of M. tuberculosis, strongly point towards the requirement and thus, the importance of these hemoglobins during intracellular regime of the bacterium. The present study conducted herein on transcriptional regulation of M. tuberculosis hemoglobins, in vitro under various stress conditions and in vivo after macrophage infection, substantiate that biosynthesis of both trHbs, trHbN and trHbO, in its native host is regulated via the environmental signals that tubercle bacillus faces during macrophage infection and growth in its human host.