A Mycobacterium ESX-1–Secreted Virulence Factor with Unique Requirements for Export

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.

General secretion signal for the mycobacterial type VII secretion pathway

“…Another likely substrate is ESX-1-encoded EspJ, which was previously identified in M. tuberculosis cell envelope extracts (38). Finally, we also identified a T7S motif in EspB, one of the larger proteins secreted by ESX-1 (14). It is unknown whether EspB is recognized directly by ESX-1 or whether it is cosecreted with a small carrier protein as described for other T7S substrates (4).…”

“…To verify the role of the secretion motif in one of these putative substrates, an N-terminally HA-labeled version of M. marinum EspB and a corresponding Y81A mutant were generated. Strikingly, whereas secretion and concomitant C-terminal processing (14) was observed for WT EspB in M. marinum, the Y81A mutant was predominantly located in the bacterial cell pellet (Fig. 5C).…”

“…The archetypical T7S system, ESX-1, is responsible for the secretion of several proteins, including the ESX-1-encoded T-cell antigens ESAT-6 (early secreted antigenic target of 6 kDa) and CFP-10 (culture filtrate protein of 10 kDa) (7,(11)(12)(13)(14)(15). ESX-1 and its substrates play an important, albeit not yet fully understood role in M. tuberculosis virulence.…”

Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway

“…This could mean that processing of ESX-5 substrates is a common process, similar to the removal of a signal sequence. Interestingly, processing has also been observed for the ESX-1 substrate EspB (16), which was recently shown to be cleaved by the ESX-1-encoded protease MycP1 (47). To further investigate the nature of the LipY tub cleavage site, we altered the neighboring residues using site-directed mutagenesis.…”

“…Mycobacterial genomes contain up to five genetic loci coding for type VII secretion systems, named ESX-1 to ESX-5 (9, 10). The most well studied system, ESX-1, is responsible for the secretion of 10 substrates, including the important T-cell antigens ESAT-6 and CFP-10, and is required for full virulence of M. tuberculosis (7,(11)(12)(13)(14)(15)(16)(17). Phylogenetic analyses and comparative genomics suggest that the five ESX clusters have evolved by gene duplication and that ESX-5 is the result of the most recent duplication event (9).…”