EseG, an Effector of the Type III Secretion System of Edwardsiella tarda , Triggers Microtubule Destabilization

Abstract

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD 50 ) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 ␤-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. (EseG 142-192 ), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

“…pACYC-eseG::cyaA and pACYC-eseJ::cyaA, respectively expressing the chimeric proteins EseG-CyaA and EseJ-CyaA (10), were thus introduced into the E. tarda wild-type, ⌬eseE, and ⌬esaN strains. The ⌬esaN strain was unable to secrete or translocate T3SS effectors (10,17). The plasmid-carrying strains were used to infect J774A.1 cells.…”

“…Aside from these well-characterized translocon proteins, two E. tarda T3SS effectors have been identified. The first is EseG, which interacts with ␣-tubulin and destabilizes microtubules (17). The other one, EseJ, inhibits bacterial adherence to epithelioma papillosum of carp (EPC) cells but facilitates E. tarda replication in EPC cells and J774A.1 murine macrophages (10).…”

“…Both EscA and EscC prevent the degradation of their target substrates (16,19). EscB, the chaperone for EseG, is required for intracellular EseG stability (17).…”

Transcriptomic dissection of the horizontally acquired response regulator EsrB reveals its global regulatory roles in the physiological adaptation and activation of T3SS and the cognate effector repertoire inEdwardsiella piscicidaduring infection toward turbot

“…Thus, TEM-1 based effector translocation assays were performed to determine whether any of the co-upregulated genes are potential translocated effectors. In this assay, effector-b-lactamase fusions are translocated into host cells, where their b-lactamase activity results in cleavage of the CCF2 substrate and emission of blue light at 450 nm; in the absence of b-lactamase activity and the preservation of intact CCF2, excitation at 409 nm results in intramolecular FRET and the emission of green light at 520 nm 29,48 (Fig. S5A).…”

“…EseG (ETAE_0866) is a T3SS effector that localizes the host membrane fraction after translocation; it destabilizes microtubules when overexpressed in mammalian cells through a conserved domain that includes amino acid residues 142-192. 28,29 EseJ (ETAE_0888) is also a translocated T3SS effector; it reduces bacterial adhesion to EPC cells and facilitates intracellular bacterial replication. 30 ETAE_1757 was recently identified as a novel T3SS effector that is translocated into the host nucleus and inhibits the phosphorylation of MAPK in host cells.…”