The regulation and function of the ESAT-6 gene cluster operons of Mycobacterium tuberculosis
Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2006.
The ESAT-6 gene cluster regions are duplicated 5 times in the genome of Mycobacterium tuberculosis. ESAT-6 gene cluster region 1 is the most frequently studied region as it contains RD1 (region of difference 1). RD1 is a 9.5 Kb deletion region confirmed to be involved in mycobacterial virulence and pathogenesis, and is present in virulent M. bovis strains, yet absent in all attenuated M. bovis BCG vaccine strains. The antigens CFP-10 and ESAT-6, which both evoke strong T-cell responses in experimental animals and humans, are situated in the RD1 region, and are thought to be key antigens in mycobacterial virulence. The absence of this region from the genomes of all BCG vaccine strains, led to the conclusion that the mechanism of attenuation of M. bovis BCG was due to the loss of RD1. Studies have shown that this attenuation is attributed to the loss of cytolytic activity mediated by secreted ESAT-6 (and some of the genes responsible for its secretion), which in turn results in reduced tissue invasiveness. The potent T-cell antigens ESAT-6 and CFP-10 are secreted without ordinary sec-dependent secretion signals. A study of the potential functions of the proteins encoded by the ESAT-6 gene clusters shows that most of these proteins have a potential to function in a protein-dependent ATP-binding cassette active transport system. It has been shown that ESAT-6 gene cluster region 1 is responsible for the secretion of the ESAT-6 and CFP-10 genes contained in this region, explaining the absence of any ordinary sec-dependent secretion signals in the amino acid sequences of members of this family. In order to elucidate the regulation of expression of the ESAT-6 gene cluster region 1, shown to encode for a secretion system for ESAT-6 and CFP-10 and to be involved in virulence, an operon analysis and promoter identification experiments were carried out in this study. The analysis of the ESAT-6 gene cluster region 1 showed the existence of more than one operon in this region and three constitutively-expressed promoters driving the expression of the genes in the operons. These results provide insight into the functional relationship (regulatory and secretory mechanisms) between the genes contained within ESAT-6 gene cluster region 1.None of the other four ESAT-6 gene cluster regions have been proven to also encode secretion systems. Preliminary studies indicated that the ESAT-6 gene cluster region 3 is expressed in its entirety as one single operon and a strong promoter involved in the expression of this region was identified. Mtb9.9A (the ESAT-6 antigen of the ESAT-6 gene cluster region 5) have also been shown to evoke strong T cell responses and to be secreted without any ordinary secretion signal. During the present study, we thus aimed to investigate the secretion of Mtb9.9A in order to determine whether it is also secreted by a dedicated secretion system encoded by ESAT-6 gene cluster region 5. The fact that region 5 was shown to be the last of the four duplications is important, as a positive result with this region would indicate whether the other four gene clusters share a similar secretion function. ESAT-6 gene cluster regions 2, 4 and 5 were isolated in the present study to form part of subsequent ESAT-6 gene cluster region secretion studies. Mtb9.9A was cloned, expressed and purified for antibody-generation, Resulting antibodies were used in an antigen secretion analysis. The secretion analysis entailed the integration of the isolated ESAT-6 gene cluster region 5 into the genome of M. smegmatis and investigation of the influence of the genes (contained in region 5) on the secretion of a heterologously expressed Mtb9.9A-HA-tagged fusion protein. We therefore attempted to show whether the proteins encoded by the ESAT-6 gene cluster region 5 also function together as a mycobacterial membrane-bound complex involved in protein-dependent transport and if so, whether this transport system is responsible for the active secretion of the native ESAT-6 antigen (designated Mtb9.9A) of region 5. This study opens the way for the understanding of the regulation, transport- and secretion mechanisms of important T-cell antigens of the mycobacteria, thereby giving insight into and building onto our understanding of the pathogenicity of Mycobacterium tuberculosis. A better understanding of these mechanisms could lead to the development of efficient strategies to either terminate or enhance secretion of antigens, which in turn will have an impact on drug and vaccine design and development.