Evidence for Pore Formation in Host Cell Membranes by ESX-1-Secreted ESAT-6 and Its Role in Mycobacterium marinum Escape from the Vacuole

Abstract

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 ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

“…This strongly suggests that, regardless of the general similarity, the capacity to interact with the membranes in response to acidification is a hallmark of the ESAT-6 molecules associated with virulence. These findings are consistent with the membrane interaction studies of MmESAT-6, the reportedly membrane-interacting molecule from the pathogenic M. marinum (29). It would be interesting to look into the membrane interaction of other orthologous ESAT-6 proteins from both pathogenic and non-pathogenic species in relation to virulence.…”

ESX-1 dependent impairment of autophagic flux byMycobacterium tuberculosisin human dendritic cells

“…In this context, it is conceivable that the membrane-lysing or pore-forming activity exerted by ESAT-6 and/or other effector proteins of the ESX-1 system might be responsible for autophagy blockage during the early phases of infection while at later stages it allows the phagosome-to-cytosol translocation of Mtb. [40][41][42][43][44] Although in our experimental model it is not yet clear whether ESAT-6 modulates directly or indirectly the autophagic process, the results obtained so far strongly suggest that the functional interaction between ESAT-6 and the DC autophagosome/autolysosome machinery is crucial to influence the immune response against TB. 30 We also showed that rapamycin treatment restores a functional autophagic flux in Mtb-infected cells, as evaluated by the analysis of SQSTM1 levels and the colocalization of MAP1LC3 with the lysosomal markers LAMP1 and cathepsin B.…”

“…[40][41][42][43][44] However, since Mtb is still detected in vesicle structures 16 h after infection (ref. 44 and data not shown) when autophagy block is already fully visible, it is likely that the effect on autophagy was not exerted by cytosolic Mtb.…”

The crystal structure of the mycobacterium tuberculosis Rv3019c‐Rv3020c ESX complex reveals a domain‐swapped heterotetramer

“…24 The ESAT-6 homolog from the ESX-1 system of M. marinum has also been implicated in disrupting host cell membranes and a genetic deletion of the ESAT-6 gene has been shown to eliminate the pore-forming ability of M. marinum. 8 To date…”

“…The canonical ESX complex, EsxAB, is encoded within the 9.5 kb region of difference 1 (RD1), which is the only common genetic deletion found in two attenuated mycobacterial vaccine strains [M. bovis bacillus Calmette-Guérin (BCG) and M. microti]. In vivo studies have determined that the RD1 locus is partially responsible for the virulence of M. tuberculosis, M. bovis, M. marinum, and M. microti [3][4][5][6][7][8] and complementation of BCG or the avirulent M. microti vaccine strain with RD1 increased the virulence of these strains. 6,9 Biochemical and bioinformatic analyses have determined that the RD1 locus encodes, in addition to EsxAB, the components of a protein secretion system, ESX-1, which is the prototypical member of the Type VII secretion system family (T7S system; reviewed in 10 ).…”