Molecular Characterization of the eis Promoter of Mycobacterium tuberculosis

Abstract

To further understand. Experiments were done to characterize the eis promoter in M. smegmatis and M. tuberculosis H37Ra. The putative ؊10 and ؊35 regions matched the Escherichia coli 70 consensus 67 and 83%, respectively, making it a group A/SigA-like mycobacterial promoter. Expression of site-directed variants of the core promoter region, determined by flow cytometry using gfp as a reporter, showed that the putative ؊10 region is essential for eis expression. In addition, site-directed alteration of the eis promoter to the consensus E. coli 70 promoter elements increased gfp transcription to levels similar to that driven by the heat shock promoter, phsp60, of Mycobacterium bovis BCG. Upstream promoter deletion analysis showed that a 200-and 412-bp region of the promoter was necessary for maximum expression of gfp in M. smegmatis and M. tuberculosis H37Ra, respectively. Random mutagenesis of the 412-bp eis promoter, using a catechol 2,3-dioxygenase screen and activity assay, defined nucleotides upstream of the core promoter region that are essential to eis expression in both M. smegmatis and M. tuberculosis H37Ra, including a region homologous to a DinR cis element.Tuberculosis (TB) continues to be the world's most destructive human bacterial infectious disease. Current estimates show that more than two million people die from TB each year, and TB remains a major cause of premature death (18). Mortality due to TB is a major global health crisis due to AIDS and the increasing prevalence of multidrug-resistant strains of Mycobacterium tuberculosis, although effective treatments are available. Despite the elucidation of the genome sequence of several M. tuberculosis strains (5, 14) and available genetic tools to identify genes involved in TB pathogenesis (13,16,23,26), the molecular basis of its ability to survive within host cells and evade host immune responses is unknown. Understanding the molecular mechanisms of pathogenesis is essential for the development of better methods of diagnosis, treatment, and prevention. One way to heighten our understanding of M. tuberculosis pathogenesis is to examine the regulation of potential virulence genes, in particular the promoters and other elements that govern their expression.Approximately 130 mycobacterial promoters have been characterized to date, but only 76 have been categorized into the four groups (A to D) of mycobacterial promoters (15). Identified promoters comprise less than 3% of the potential promoters in the genome of M. tuberculosis. The majority of the categorized promoters are from Mycobacterium smegmatis and Mycobacterium paratuberculosis, with less than 25% derived from M. tuberculosis itself. This indicates that we know little about promoter function in mycobacteria and even less about promoters from M. tuberculosis. Therefore, there is a need for detailed studies on M. tuberculosis promoter characterization and function, especially for genes that may play a role in the survival of M. tuberculosis within macrophages.The eis gene of M. tuberculosis H37Rv...

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

“…The biological function of Eis in Mtb has been the subject of recent interest (6,7,(22)(23)(24)(25)(26)(27)(28)(29). The biochemical and structural studies described in this report provide clear evidence for an unprecedented multiacetylation capability of Eis that inactivates AG antibiotics.…”

Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

“…For example, evidence indicates that SigA regulates expression of some virulence genes through interaction with the transcription factor WhiB3 (Steyn et al, 2002). Mutational analyses of the eis promoter show that sequences upstream of the 235 region are required for optimal expression of a reporter gene in mycobacteria (Roberts et al, 2004), suggesting that as yet unidentified transcriptional activators contribute to eis expression.…”

“…An earlier report indicated that the 235 and 210 regions of the eis promoter resemble the consensus sequence for SigA-like promoters (Roberts et al, 2004). To determine whether SigA directly regulates eis, we performed ChIP on brothgrown TB294, using a monoclonal anti-SigA antibody to recover SigA-DNA complexes (Fig.…”

“…Although SigA is predicted to control housekeeping functions, our previous study (Wu et al, 2004) and work by others (Collins et al, 1995;Steyn et al, 2002) suggest that SigA also regulates genes involved in host-pathogen interactions. We wanted to identify genes regulated by SigA that enhance the ability of M. tuberculosis strain 210 to grow in host cells.An earlier report suggested that the eis gene (Rv2416c) of M. tuberculosis is a member of the SigA regulon, given the similarity of the putative 210 and 235 regions of the eis promoter to SigA-like promoters (Roberts et al, 2004). The eis gene was originally identified by the Friedman laboratory based on the ability of the M. tuberculosis gene to enhance survival of the non-pathogenic species Mycobacterium smegmatis in macrophages, hence the name 'eis' for 'enhanced intracellular survival ' (Wei et al, 2000).…”

“…An earlier report suggested that the eis gene (Rv2416c) of M. tuberculosis is a member of the SigA regulon, given the similarity of the putative 210 and 235 regions of the eis promoter to SigA-like promoters (Roberts et al, 2004). The eis gene was originally identified by the Friedman laboratory based on the ability of the M. tuberculosis gene to enhance survival of the non-pathogenic species Mycobacterium smegmatis in macrophages, hence the name 'eis' for 'enhanced intracellular survival ' (Wei et al, 2000).…”