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

Abstract

Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE. These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ⌬eseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.T he type III secretion system (T3SS) is a contact-dependent translocation system. It forms a syringe-like structure spanning the inner and outer membranes of bacteria and induces pore formation on host cells (1). Through these pores, an array of bacterial proteins (effectors) are delivered into host cells, where they manipulate host cell signaling pathways to promote bacterial survival (2).Edwardsiella tarda is a Gram-negative intracellular pathogen that can cause hemorrhagic septicemia in fish (3), as well as gastroand extraintestinal infections in humans (4, 5). The T3SS is well known to be one of the most important virulence factors in E. tarda (6, 7). It facilitates the survival and replication of E. tarda in host cells (6-9). E. tarda T3SS contains 34 open reading frames (ORFs), which encode the apparatus, chaperones, effectors, and regulators (6, 10). The expression of E. tarda T3SS is controlled by many factors, such as the two-component system esrA-esrB (6) and esrC (11) inside the T3SS gene cluster, as well as phoP-phoQ (12) and phoR-phoB (13) located outside the T3SS gene cluster. Intriguingly, our group recently showed that a type III secretion system-secreted protein (EscE) also functions as a regulator controlling the injection of effectors and secretion of translocators (14).EseB, EseC, and EseD, secreted by the E. tarda T3SS, are homologous ...

Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

“…Many previous reports have shown that bacterial T3SS effectors play critical roles in bacterial pathogenesis in vivo (D'Cruze et al, 2011;Kang, Vellasamy, Chua, & Vadivelu, 2015). E. piscicida T3SS has been well defined to be an important virulence factor, which significantly affects bacterial colonisation in multiple fish infection models, including turbot (Yang et al, 2015), naive blue gourami (Yi et al, 2016), and zebrafish (Wang et al, 2010). Here, we examined whether E. piscicida T3SS effector EseK and its chaperones EscH and EscS contribute to bacterial colonisation in vivo.…”

“…Data are representative of at least three independent experiments including turbot (Yang et al, 2015), naive blue gourami (Yi et al, 2016), and zebrafish (Wang et al, 2010). (a) MBP pull-down assays using purified proteins MBP-EscH and His sumo-EseK-His incubated with MBP beads.…”

“…Here, we examined whether E. piscicida T3SS effector EseK and its chaperones EscH and EscS contribute to bacterial colonisation in vivo. We compared the abilities of the wild-type strain and ΔT3SS, ΔeseK, ΔescH, ΔescS, or ΔescHΔescS to colonise zebrafish via a competitive index assay as described previously (Wang et al, 2010;Yi et al, 2016). Compared to the wild-type strain, ΔT3SS showed a greatly impaired colonisation ability in zebrafish (Fig.…”

The Edwardsiella piscicida Type III Translocon Protein EseC Inhibits Biofilm Formation by Sequestering EseE

“…Interestingly, eseB, eseD, and eseE are all located in the escC-eseE operon, whereas eseG and eseJ are located in other operons ( Fig. 4C) (23,24). This suggests that EseC may selectively control the expression of proteins encoded by the escC-eseE operon.…”

“…We next determined if the loss of eseC would have an overall effect on the production and secretion of T3SS-encoded proteins, including translocon proteins (EseB/C/D), effector proteins (EseG/J) (11,12), and EseE (a chaperone protein for EseC) (23). EvpC, a T6SS-encoded protein, was used as a loading control.…”