Dimer recognition and secretion by the ESX secretion system inBacillus subtilis

Abstract

Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.WXG protein | type VII secretion system | protein translocation | YukE P rotein secretion is critical for protein targeting in any living cell and for its communication with the environment. Bacteria use a wide range of secretion mechanisms to export proteins out of the cytoplasm. Signals for secretion are most commonly primary amino acid sequences, but in some cases also may be formed through interacting surfaces of a substrate and its delivery effector. Some secretion systems unfold their substrates to translocate them across the membrane and cell wall. Other systems export folded proteins, sometimes in complex with bound cofactors. For example, the general secretory machinery (Sec) denatures the tertiary and secondary structure of its substrates to thread the polypeptide through the narrow opening of the integral membrane translocon complex, SecYEG (1). Type III secretion system (T3SS) machinery is thought to unfold the tertiary structure of its substrates, while preserving the secondary structure elements for the substrate recognition (2, 3). In contrast, the twin-arginine transport (Tat) system exports folded substrates (4) and is hypothesized to be able to translocate protein oligomers and complexes via a "hitchhiking" mechanism (5). Overall, these and other secretion types differ in the nature of substrate recognition signal and the mode of substrate translocation.Early secretory antigen 6 kDa (ESX, or type VII) secretion systems are widespread in actinomycetes and Gram-positive bacteria and affect a range of bacterial processes including sporulation, conjugation, and cell wall stability (6-10). In two notorious human pathogens, Mycobacterium tuberculosis and Staphylococcus aureus, ESX secretion was found to be crucial for establishing and maintaining the infection (11-15). Despite the importance of the ESX secretion for human health, the mechanism of this type of secretion is still largely unknown.Recent characterization of the ESX system in Bacillus subtilis confirmed that a functional system is encoded by the yuk/yue operon (16,17). Importantly, the B. subtilis system codes for a...

EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection

“…Homologs of the mycobacterial T7SS that transport WXG100 proteins have also been described in firmicutes, including Bacillus anthracis (32), Bacillus subtilis (33), and Staphylococcus aureus (14). Firmicutes carry some (EsxA, EsxB, and EccC), but not all, of the mycobacterial ESX-1 components required for secretion of ESAT-6 and CFP-10 (34), and their pathways are designated type VIIb (29).…”

The Enigmatic Esx Proteins: Looking Beyond Mycobacteria

“…Although a second potential 5 WXG100 substrate YfjA was identified in B. subtilis genome [10], there is no 6 evidence that the associated protein is exported in a YukBA-dependent 7 fashion [57,58]. Utilizing this simple Bacillus system Sysoeva et al [35] 8 demonstrated that YukE was secreted as a homodimer, and that the WXG100 9 motif and a C-terminal sequence are required for its secretion. Intriguingly, 10…”

“…secretion signal formed by both the WXG100 and C-terminal motifs, each 1 contributed by individual subunits of the substrate dimer [35]. Given the 2 conservation of these motifs across type VII substrates, they likely play a 3 crucial role in controlling type VIIb secretion [6].…”

Structure of a PE–PPE–EspG complex fromMycobacterium tuberculosisreveals molecular specificity of ESX protein secretion

“…"Type VII" secretion systems distantly related to mycobacterial ESX systems have been identified in numerous other Gram-positive bacteria (13)(14)(15)(16)(17). The protein components of type VII secretion systems differ considerably between bacterial species but generally include the following: (i) one or more small helical proteins of the WXG100 protein family (e.g., ESAT-6, YukE), (ii) an FtsK/SpoIII-type ATPase that is thought to drive protein secretion (e.g., EccC, YukB), and (iii) a multipass transmembrane protein that may form the pore of the translocon (e.g., EccD, YueB).…”