Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda

Abstract

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E . tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens.

Bacteriophage SPP1 Tail Tube Protein Self-assembles into β-Structure-rich Tubes

“…Interestingly, all Hcp-like proteins purified so far are hexamers in solution with the exception of EpvC from Edwardsiella tarda, which was found in both dimeric and hexameric states in solution (39). Furthermore, no in vitro self-association of these hexamers was reported to date.…”

Regulation of the Edwardsiella ictaluri Type III Secretion System by pH and Phosphate Concentration through EsrA, EsrB, and EsrC

“…1). In E. tarda, EvpC is a putative translocon protein that forms a tube-like structure (26) and is required for secretion of the E. tarda T6SS proteins EvpI and EvpP (56). Zheng et al (56) reported that individual mutations in 14 T6SS genes, including evpC, evpI, and evpP, attenuate virulence of E. tarda in fish, but the effects on intracellular replication were not reported.…”

TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System

“…The crystal structures of the Hcp (hemolysin coregulated protein) proteins present high similarity with the bacteriophage tail tube gpV protein (10,(13)(14)(15). Six Hcp assemble into rings of about 80 -90 Å wide with an internal diameter of about 30 -40 Å (13-15) that stack on each other to form tubes in vitro (9,(13)(14)(15)(16). The VgrG (valine glycine repeat protein) trimer is structurally homologous to the bacteriophage T4 gp27/gp5 spike complex which is used as a puncturing device to perforate the host cell envelope (9,17).…”