While ESAT-6 cluster 1 is known to be essential to virulence, the role of cluster 3 is still to be defined; nevertheless, iron- and zinc-dependent expression strongly suggest a high level expression

in the lung during the infective process, and hence a contribution to the antigenic profile throughout the course of infection [22]. To better understand the expression of ESAT-6 cluster 3 genes, it was important to verify whether internal promoters appear within this region; in both organisms, the presence of promoter upstream of msmeg0620 and rv0287 coding regions suggests that gene expression within ESAT-6 gene cluster could be differential. To better define the effect of each promoter on overall esx gene regulation, we compared NU7441 msmeg0615 and msmeg0620 expression in varying conditions by means of relative quantitative PCR. As an internal control to normalize loaded RNA we used sigA, which encodes the mycobacterial major sigma factor [27, check details 19]. sigA is widely used as a standard in qPCR because its expression is constitutive in various growth phases and under differing stress conditions. An approximate 3-fold decrease in sigA transcript was reported in M. tuberculosis during the stationary growth phase [28]; these data do not seem to affect our results significantly, as we observed increased repression of this promoter in the stationary phase. The expression of msmeg0615 and msmeg0620 genes is essentially

Fludarabine similar; they appear to be repressed in most of the tested conditions, with the exception of acid stress (pH 4.2). These data suggest the presence of two transcriptional units: the first, regulated by pr1 (msmeg0615

promoter), encompasses the whole cluster, while the second, regulated by pr2, includes the msmeg0620 downstream genes. Although previous studies [16] noted the coordination of all genes expression within cluster 3 under Zur regulation, divergence between rv0282 and rv0287 induction levels under acid stress and the appearance of an internal promoter also suggest that two overlapping transcriptional units exist. As regards the hypothetical role of the CFP-10/ESAT-6 Liothyronine Sodium complex in escaping from the phagosomal compartment of professional phagocytic cells [29, 30], the finding of cluster 3 gene induction in acidic pH condition is surely noteworthy. Acidification may indeed be a signal for the induction of genes needed in phagosome survival. A previous transcriptional analysis by means of microarray failed in the identification of rv0282 and rv0287 among M. tuberculosis genes induced under acid stress [31]. This discordance could be explained with different sensitivity of the methodologies used in these investigations. Both IdeR and iron-regulated genes were previously reported to be upregulated during macrophage infection [32, 33]. This apparent contradiction can be explained by direct or indirect inhibition exerted by environmental acid on IdeR function.