Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

Identification of Mycobacterium avium genes up-regulated in cultured macrophages and in mice.

To investigate Mycobacterium avium gene expression upon infection of macrophages, we created a M. avium-promoter library upstream of a promoter-less gene encoding the green fluorescent protein (GFP) in Mycobacterium smegmatis. Clones were evaluated for increased expression of GFP after infection of U937 macrophages. A number of M. avium genes were up-regulated more than 3-fold after 24 and 48 h following macrophage infection. M. avium genes expressed by M. smegmatis during growth in macrophages include genes encoding transport/binding proteins, synthesis, modification and degradation of macromolecules, and a great majority of genes for which no function is currently known. For some of the unknown genes, homologues were identified in bacteria such as Mycobacterium leprae, Salmonella typhimurium and Agrobacterium tumefaciens. In order to investigate if these genes were also expressed in M. avium during macrophage infection in vitro and in vivo, transcripts of selected genes were quantified using real time RT-PCR. Evaluation of most expressed genes in M. smegmatis confirmed their up-regulation in M. avium after 24 h infection of macrophages in vitro and mice.