Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.

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The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1β, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.

The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system.

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.