Bottom Line:
In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM.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.

Affiliation: Program in Microbial Pathogenesis and Host Defense, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACTSpecialized 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.

ppat-0030105-g005: GST Pulldown Analysis of EspB and Rv3879 Interactions(A) Agarose beads with immobilized GST, GST-SHP1, GST-syntaxin2, GST-Mh3879c, and GST-EspBM were incubated with lysates of E. coli that express V5-Mh3879c and V5-EspBM.(B) Agarose beads with immobilized GST, GST-SHP1, GST-syntaxin2, GST-Rv3879c, or GST-EspBT were incubated with lysates of E. coli that express V5-Rv3871, V5-Rv3879c, and V5-EspBT. Proteins from cell lysates retained on the beads after washing were separated by SDS-PAGE and detected by western blotting with an antibody against V5. To the right of each set of pulldowns, 0.1% of the input E. coli lysate was analyzed. EspBM physically interacts with Mh3879c, EspBT physically interacts with Rv3879c, and Rv3879c also interacts with Rv3871.

Mentions:
To test for an analogous interaction between EspBM and Mh3879c and to confirm the potential interaction between EspBT and Rv3879c suggested by the two-hybrid assay, we performed in vitro pull-down assays. All of the proteins used were expressed in Escherichia coli as GST- or V5-epitope-tagged fusions. Controls for nonspecific interactions included GST alone, as well as GST-syntaxin2, and GST-Shp1. As shown in Figure 5A, GST-tagged EspBM, but none of the GST controls, bound specifically to V5-tagged Mh3879c. In the reciprocal experiment, GST-tagged Mh3879c bound specifically to V5-EspBM. Similarly, as shown in Figure 5B, GST-tagged EspBT bound specifically to V5-tagged Rv3879c, and GST-tagged Rv3879c bound specifically to V5-tagged EspBT. These data demonstrate that recombinant EspBT and Rv3879c, as well as their M. marinum homologs, interact in vitro.

ppat-0030105-g005: GST Pulldown Analysis of EspB and Rv3879 Interactions(A) Agarose beads with immobilized GST, GST-SHP1, GST-syntaxin2, GST-Mh3879c, and GST-EspBM were incubated with lysates of E. coli that express V5-Mh3879c and V5-EspBM.(B) Agarose beads with immobilized GST, GST-SHP1, GST-syntaxin2, GST-Rv3879c, or GST-EspBT were incubated with lysates of E. coli that express V5-Rv3871, V5-Rv3879c, and V5-EspBT. Proteins from cell lysates retained on the beads after washing were separated by SDS-PAGE and detected by western blotting with an antibody against V5. To the right of each set of pulldowns, 0.1% of the input E. coli lysate was analyzed. EspBM physically interacts with Mh3879c, EspBT physically interacts with Rv3879c, and Rv3879c also interacts with Rv3871.

Mentions:
To test for an analogous interaction between EspBM and Mh3879c and to confirm the potential interaction between EspBT and Rv3879c suggested by the two-hybrid assay, we performed in vitro pull-down assays. All of the proteins used were expressed in Escherichia coli as GST- or V5-epitope-tagged fusions. Controls for nonspecific interactions included GST alone, as well as GST-syntaxin2, and GST-Shp1. As shown in Figure 5A, GST-tagged EspBM, but none of the GST controls, bound specifically to V5-tagged Mh3879c. In the reciprocal experiment, GST-tagged Mh3879c bound specifically to V5-EspBM. Similarly, as shown in Figure 5B, GST-tagged EspBT bound specifically to V5-tagged Rv3879c, and GST-tagged Rv3879c bound specifically to V5-tagged EspBT. These data demonstrate that recombinant EspBT and Rv3879c, as well as their M. marinum homologs, interact in vitro.

Bottom Line:
In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM.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.

Affiliation:
Program in Microbial Pathogenesis and Host Defense, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACTSpecialized 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.