ESAT-6 (early secreted antigenic target, 6 kDa) proteins, including the previously mentioned CFP-10 (10 kDa short-term culture filtrate protein), form a large family that is defined on the following base: basis of protein size (about 100 amino acids); the occurrence of the cognate genes in pairs; their location downstream of a pe and ppe gene pair, which are coding mycobacterial protein with a characteristic proline-glutamic (PE) and proline-proline-glutamic (PPE) motif.
The interest in ESAT genes derives from the observation that esxA
genes are comprised in the RD1 (region of difference 1), whose deletion is thought to be responsible for the primary attenuation of M. bovis
in M. bovis
]. Complementation experiments have demonstrated that mutations that abolish production or secretion of RD1 ESAT-6 proteins confer an attenuated phenotype in various animal models, which in turn suggests that ESAT-6/CFP-10 play an important role in survival and multiplication of M. tuberculosis
within the host cell [20
Moreover, ESAT-6 proteins have been identified as strong targets for human B- and T-cell response, a finding which stimulates great interest in the potential of these antigens for vaccine use [22
]. Besides EsxA and EsxB, EsxH (Rv0288), included in cluster 3, has also been identified as a strong antigen in TB patient and BCG vaccinated donor [23
]. Two other ESAT proteins (Rv3017c, or EsxQ and Rv3019c, or EsxR), despite their high degree of identity with Rv0288, display a unique epitope pattern [24
]. These observations strengthen the hypothesis that these genes could encode proteins whose functions are similar, but whose recognition by the immune system differs; differential expression of individual genes could lead to antigenic variation, which would help mycobacteria to escape from the host defence. To better understand esx
genes function it is important to investigate their expression in varying conditions and in differing phases of the infective process.
esx genes were also identified in other mycobacteria; in particular the fast growing M. smegmatis contains three ESAT-6 gene clusters, which correspond to the previously identified regions 1 (encompassing region between msmeg0057 and msmeg0083 genes), 3 (msmeg0615-msmeg0625) and 4 (msmeg1534-msmeg1538) of M. tuberculosis. The finding that bacteria carrying ESAT-6 genes live in varying environmental niches suggests that, besides virulence, these proteins could have a more general role in mycobacterial physiology.
To better define the putative role of cluster 3 in mycobacterial pathogenicity and physiology, we decided to study ESAT cluster 3 gene regulation in M. smegmatis
and in M. tuberculosis
. As the rv0282
promoter region had been previously characterized [16
], we analysed msmeg0615
promoter region activity. Our results suggest that regulation differs in these organisms; while in M. tuberculosis
gene cluster 3 is controlled by IdeR and Zur regulators in an iron- and zinc-dependent manner, in M. smegmatis
only IdeR-dependent regulation is retained, while zinc has no effect on gene expression. Iron is a growth limiting factor both in the environment and during human infection. In mammalian hosts this metal is bound to high affinity iron-binding proteins, and abnormal high iron levels in serum are associated with exacerbation of the disease [25
]. It is worth noting that the differences in ESAT-6 cluster expression 3 in M. tuberculosis
and M. smegmatis
could be due to differences in the life styles of these organisms. As a pulmonary pathogen, M. tuberculosis
has to confront with a zinc-deficient environment, as this metal's concentration is low in lung alveoli [26
]. 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
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 msmeg0615
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
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
genes is essentially 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
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 complex in escaping from the phagosomal compartment of professional phagocytic cells [29
], 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
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
]. This apparent contradiction can be explained by direct or indirect inhibition exerted by environmental acid on IdeR function. Indeed, to date no data suggest the presence of an alternative pH-dependent promoter upstream of ESAT-6 cluster 3; msmeg0615
gene induction could be indirect, presumably as an effect of the environment on IdeR function or stability. Differential gene expression inside the ESAT-6 cluster could be related to the presence of the internal promoter pr2, whose activity diminishes under acid stress. As pr2 seems to be a weak promoter, its effect in M. tuberculosis
could be less evident, while in M. smegmatis
it could effectively reduce pr2-regulated genes expression. Unfortunately, it was not possible to identify pr2 promoter sequence in M. tuberculosis
, as 5' RACE experiments were unsuccessful; the probable reason is low expression levels. In M. smegmatis
, no SigA consensus
sequence could be found upstream of the 5' end of the transcript. We can hypothesize the involvement of an alternative sigma factor; indeed, this region showed sequence (boxed in Figure ) that resembled the sequence putatively recognized by M. tuberculosis
]. However, in this organism, SigH is induced by heat shock and oxidative stress [34
] and we are accordingly unclear as to the meaning of this observation. On the other hand, a bioinformatics search has predicted the existence of 26 sigma factors in M. smegmatis
, with a significant enrichment in the SigH subfamily [35
]. These paralogous members might have acquired specific functions, and might be induced in varying as yet unidentified conditions.