SummaryInitiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis , which encompasses RD1 ( Rv3871-Rv3879c ), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants . M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6 / CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c , not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo , suggesting that this gene plays additional roles in M. marinum survival in the host.

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...

No abstract

Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.