The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ϳ4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.Mycobacterium tuberculosis infects one-third of the world's population and kills 2 to 3 million people each year (13). The molecular and cellular mechanisms governing the pathogenesis of M. tuberculosis are beginning to be elucidated but are not fully understood. Mycobacterium marinum is a close relative of M. tuberculosis. M. marinum causes a tuberculosis-like disease in fish with symptoms similar to those of human tuberculosis and has been used as a surrogate model for studying the pathogenesis of M. tuberculosis (7,17,20,46,47).Previous studies have identified and partially characterized a specialized protein secretion system, ESX-1, in M. tuberculosis (14, 23, 24, 33, 44) and M. marinum (17, 50). This secretion system has recently been named the type VII secretion system (1). ESX-1 is encoded by genes of RD1 (region of difference 1) (24, 33, 44) and its surrounding region (23, 34), together termed extRD1 (4, 17). RD1 encompasses nine genes in M. tuberculosis (Rv3871 to Rv3879c) that are deleted from the attenuated vaccine Mycobacterium bovis BCG (2, 22). M. tuberculosis and M. marinum utilize ESX-1 to export virulence proteins that do not have the conventional SecA-dependent signal peptide sequences (17,24,33,44,50). The proteins that are secreted by ESX-1 and involved in virulence include ESAT-6, CFP-10, EspA, and Mh3881c (or EspB) (14,17,23,28,34,50). During secretion, Mh3881c is cleaved close to its C terminus to produce two fragments with apparent molecular masses of 50 and 11 kDa (28, 50). Inside the bacterial cytosol, the C-terminal sequence of Mh3881c interacts with ESA...

Mycobacterium tuberculosis (Mtb) extracellular DNA (eDNA) gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this eDNA of Mtb does not induce activation of the AIM2-inflammasome since AIM2 recognizes cytosolic DNA. Here we show that non-virulent mycobacteria such as M. smegmatis induce AIM2-inflammasome activation, which is dependent upon their strong induction of IFN-β production. In contrast, Mtb, but not an ESX-1 deficient mutant, inhibits the AIM2-inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that Mtb inhibits IFN-β production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of Mtb that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2-inflammasome activation during infection.

Background The HIV pandemic raised the potential for facultative-pathogenic mycobacterial species like, Mycobacterium kansasii , to cause disseminating disease in humans with immune deficiencies. In contrast, non-pathogenic mycobacterial species, like M. smegmatis , are not known to cause disseminating disease even in immunocompromised individuals. We hypothesized that this difference in phenotype could be explained by the strong induction of an innate immune response by the non-pathogenic mycobacterial species. Results A comparison of two rapid-growing, non-pathogenic species ( M. smegmatis and M. fortuitum ) with two facultative-pathogenic species ( M. kansasii and M. bovis BCG) demonstrated that only the non-pathogenic bacteria induced strong apoptosis in human THP-1 cells and murine bone marrow-derived macrophages (BMDM) and dendritic cells (BMDD). The phospho- myo -inositol modification of lipoarabinomannan (PI-LAM) isolated from non-pathogenic species may be one of the cell wall components responsible for the pro-inflammatory activity of the whole bacteria. Indeed, PI-LAM induces high levels of apoptosis and IL-12 expression compared to the mannosyl modification of LAM isolated from facultative-pathogenic mycobacteria. The apoptosis induced by non-pathogenic M. smegmatis was dependent upon caspase-3 activation and TNF secretion. Consistently, BALB/c BMDM responded by secreting large amounts of TNF upon infection with non-pathogenic but not facultative-pathogenic mycobacteria. Interestingly, C57Bl/6 BMDM do not undergo apoptosis upon infection with non-pathogenic mycobacteria despite the fact that they still induce an increase in TNF secretion. This suggests that the host cell signaling pathways are different between these two mouse genotypes and that TNF is necessary but not sufficient to induce host cell apoptosis. Conclusion These results demonstrate a much stronger induction of the innate immune response by non-pathogenic versus facultative-pathogenic mycobacteria as measured by host cell apoptosis, IL-12 and TNF cytokine induction. These observations lend support to the hypothesis that the strong induction of the innate immune response is a major reason for the lack of pathogenicity in fast-growing mycobacteria.

This study was performed to examine the adaptive immune response generated by three Mycobacterium bovis