Substrate Interaction with the EssC Coupling Protein of the Type VIIb Secretion System

Abstract

Staphylococcus aureus employs the type VIIb secretion system (T7SSb) to secrete effector proteins that either have antibacterial activities or promote bacterial persistence in mouse infection models. Here, we present the crystal structure of the ATPase domain D3 of the EssC coupling protein from S. aureus USA300_FPR3757, an integral component of the T7SSb complex, resolved at a 1.7-Å resolution. EssC-D3 shares structural homology with FtsK/SpoIII-like ATPase domains of T7SSa and T7SSb and exhibits a conserved pocket on the surface with differential amino acid composition. In T7SSa, substrate EsxB interacts with the D3 domain through this pocket. Here, we identify amino acids in this pocket that are essential for effector protein secretion in the T7SSb. Our results reveal that the adjacent ATPase domain D2 is a substrate binding site on EssC and that substrates bound to D2 require domain D3 for further transport. Point mutations in the Walker B motif of domain D3 have diametric effects on secretion activity, either abolishing or boosting it, pointing to a critical role of domain D3 in the substrate transport. Finally, we identify ATPase domain D3 as a virulence determinant of S. aureus USA300_FPR3757 using an invertebrate in vivo infection model. IMPORTANCE The emergence of antibiotic-resistant bacteria poses a rising problem in antibiotic treatment (S. Boyle-Vavra and R. S. Daum, Lab Invest 87:3–9, 2007, https://doi.org/10.1038/labinvest.3700501). We have used the multidrug-resistant S. aureus USA300_FPR3757 as a model organism to study the T7SSb. Effector proteins of this system have been associated with abscess formation and bacterial persistence in mouse models (M. L. Burts, A. C. DeDent, and D. M. Missiakas, Mol Microbiol 69:736–746, 2008, https://doi.org/10.1111/j.1365-2958.2008.06324.x; M. L. Burts, W. A. Williams, K. DeBord, and D. M. Missiakas, Proc Natl Acad Sci U S A 102:1169–1174, 2005, https://doi.org/10.1073/pnas.0405620102). We determined the structure of the essential ATPase domain D3 of the T7SSb at atomic resolution and validated a surface-exposed pocket as a potential drug target to block secretion. Furthermore, our study provides new mechanistic insights into the T7SSb substrate transport.

Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism

“…It lies at the centre of a multisubunit membrane complex that mediates protein transport across the cytoplasmic membrane [15][16][17]. The protein has four ATP-binding domains in its cytoplasmic C-terminus, each of which is essential for secretion activity [13,14,[16][17][18][19], and the most C-terminal of these domains also plays a role in substrate recognition [13,20,21]. Substrates are recognized through a short C-terminal signal sequence, and secretion appears to be post-translational because at least some substrates are exported in a folded form [22][23][24].…”

The conserved core component EsaA mediates bacterial killing by the type VIIb secretion system

“…is also unable to secrete effector proteins, but it expresses a full-length EsaA 19,32 . In this case, the competition assay showed a very efficient capacity of S. aureus USA300 ΔessC to kill the prey bacterium, comparable to that of the wild type S. aureus USA300, suggesting that the inability of USA300 ΔesaAex to kill the prey bacterium is likely due to the absence of the extracellular EsaAex.…”

A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

“…In Firmicutes, the mechanism and components of the T7SSb have been most extensively studied in Staphylococcus aureus 12–16 , in which the core machinery consists of four membrane proteins: EsaA, EssA, EssB, and EssC, as well as a cytoplasmic protein, EsaB 17,18 . While deletion of any one of these core components can abrogate T7SSb activity 13,19,20 , the hexameric, membrane-bound ATPase EssC is considered the driver of T7SSb, of which the ATP-binding domains in the C-terminal region are required for substrate secretion and the C-terminal region is also necessary for substrate recognition and specificity 15,21–23 . It has been shown in several Gram-positive bacterial species that the C-terminal sequence of EssC is highly variable across strains, resulting in EssC subtypes that are typically accompanied by a unique set of putative secreted effector-encoding genes 17,24,25 .…”