Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1-secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1-dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1-mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1-mediated lysis.Mycobacterium tuberculosis | Mycobacterium marinum | ESAT-6 | ESX-1 secretion system | cell membrane lysis

The ciliate Tetrahymena thermophila, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how Tetrahymena can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro-and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) ␣-and 11 imp ␤-like proteins revealed that imp ␣-like proteins are nucleus specific-nine localized to the germ line micronucleus-but that most imp ␤-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp ␣ adapters. The different imp ␣ proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the IMA10 gene encoding an imp ␣-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in Tetrahymena are regulated by diverse, specialized karyopherins.The regulated transport of macromolecules into and out of the nucleus controls many cellular processes. The import of proteins containing nuclear localization signals (NLSs) is facilitated by karyopherin proteins, also known as importins. Karyopherins shuttle their NLS-containing cargo through nuclear pore complexes (NPCs), protein channels that permeate the nuclear membrane. Two classes of karyopherins, imp ␣ and ␤ proteins, collaborate to orchestrate specific nucleocytoplasmic trafficking (reviewed in reference 13). To transport proteins containing a classical NLS (cNLS), imp ␣ binds to the cargo's cNLS and associates with imp ␤ in a ternary complex that docks at an NPC (reviewed in reference 23). The karyopherin-cargo complex is directed through the NPC, primarily through interactions of imp ␤ with NPC proteins (6, 7, 26). Nuclear proteins may, alternatively, contain nonclassical NLSs that mediate direct association with imp ␤ proteins without the imp ␣ adapter.Eukaryotic cells typically encode multiple karyopherins. In yeast and humans, which contain at least 14 and 20 karyopherins, respectively, most are members of the imp ␤ family. Yeast encodes just one imp ␣, whereas humans possess three distinct classes (reviewed in reference 23). The imp ␣ proteins have a characteristic structure consisting of an amino (N)-terminal imp ␤-binding (IBB) domain of ϳ100 amino acids (aa) that is followed by 8 to 10 alpha-helical armadillo (ARM) repeats that span most of the remainder of the protein. The ARM repeats are responsible for recognition ...

Significance Tuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis , and its close pathogenic relative Mycobacterium marinum , initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane–bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.