Limits...
A mycobacterium ESX-1-secreted virulence factor with unique requirements for export.

McLaughlin B, Chon JS, MacGurn JA, Carlsson F, Cheng TL, Cox JS, Brown EJ - PLoS Pathog. (2007)

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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Bacterial Two-Hybrid Analysis of Interaction of EspBT with Proteins of extRD1The target plasmid containing EspBT fused to the RNA polymerase alpha subunit was co-transformed with each of the bait plasmids containing the indicated extRD1 proteins fused to the lambda repressor into the reporter validation strain. Shown is the ratio of growth of the co-transformants on selective (+5 mM 3AT) versus non-selective plates. The experiment depicted is representative of three independent determinations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1937011&req=5

ppat-0030105-g004: Bacterial Two-Hybrid Analysis of Interaction of EspBT with Proteins of extRD1The target plasmid containing EspBT fused to the RNA polymerase alpha subunit was co-transformed with each of the bait plasmids containing the indicated extRD1 proteins fused to the lambda repressor into the reporter validation strain. Shown is the ratio of growth of the co-transformants on selective (+5 mM 3AT) versus non-selective plates. The experiment depicted is representative of three independent determinations.

Mentions: To learn more about the involvement of ESX-1 in EspB secretion, we tested whether EspB would interact with other ESX-1 genes by bacterial two-hybrid analysis (Figure 4). An advantage of the bacterial two-hybrid system is that it can allow detection of interactions of membrane-bound proteins [23]. In this assay, potential protein–protein interactions are assessed by determining the ratio of colonies that grow on selective medium to the number grown on non-selective medium. For each of the bait plasmids, co-transformation with an empty target resulted in a ratio of colonies on selective to non-selective medium of less than 0.1%, as did co-transformation of the EspBT target with an empty bait. In contrast, the Rv3879c bait and EspBT target resulted in a ratio of 7.6%, an increase of more than 75-fold. An Rv3876 bait also showed interaction above background with EspBT, but since the M. marinum Mh3876::tn mutant showed significant EspBM secretion (Figure 2C), any interaction between Rv3876 and EspBT is not likely to be required for EspB secretion and thus was not pursued.


A mycobacterium ESX-1-secreted virulence factor with unique requirements for export.

McLaughlin B, Chon JS, MacGurn JA, Carlsson F, Cheng TL, Cox JS, Brown EJ - PLoS Pathog. (2007)

Bacterial Two-Hybrid Analysis of Interaction of EspBT with Proteins of extRD1The target plasmid containing EspBT fused to the RNA polymerase alpha subunit was co-transformed with each of the bait plasmids containing the indicated extRD1 proteins fused to the lambda repressor into the reporter validation strain. Shown is the ratio of growth of the co-transformants on selective (+5 mM 3AT) versus non-selective plates. The experiment depicted is representative of three independent determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1937011&req=5

ppat-0030105-g004: Bacterial Two-Hybrid Analysis of Interaction of EspBT with Proteins of extRD1The target plasmid containing EspBT fused to the RNA polymerase alpha subunit was co-transformed with each of the bait plasmids containing the indicated extRD1 proteins fused to the lambda repressor into the reporter validation strain. Shown is the ratio of growth of the co-transformants on selective (+5 mM 3AT) versus non-selective plates. The experiment depicted is representative of three independent determinations.
Mentions: To learn more about the involvement of ESX-1 in EspB secretion, we tested whether EspB would interact with other ESX-1 genes by bacterial two-hybrid analysis (Figure 4). An advantage of the bacterial two-hybrid system is that it can allow detection of interactions of membrane-bound proteins [23]. In this assay, potential protein–protein interactions are assessed by determining the ratio of colonies that grow on selective medium to the number grown on non-selective medium. For each of the bait plasmids, co-transformation with an empty target resulted in a ratio of colonies on selective to non-selective medium of less than 0.1%, as did co-transformation of the EspBT target with an empty bait. In contrast, the Rv3879c bait and EspBT target resulted in a ratio of 7.6%, an increase of more than 75-fold. An Rv3876 bait also showed interaction above background with EspBT, but since the M. marinum Mh3876::tn mutant showed significant EspBM secretion (Figure 2C), any interaction between Rv3876 and EspBT is not likely to be required for EspB secretion and thus was not pursued.

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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus