Edwardsiella ictaluri type III secretion system (T3SS) effector EseN is a phosphothreonine lyase that inactivates ERK1/2

Abstract

EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vivo in head kidney derived macrophages. Interaction of EseN with phosphorylated ERK1/2 (pERK1/2) was also confirmed using PLA, suggesting that MVP serves as a signaling scaffold for ERK1/2 and EseN. Channel catfish Ictalurus punctatus infected with E. ictaluri strains lacking the eseN gene had reduced numbers of E. ictaluri in the tissues following infection and reduced mortality compared to fish infected with the wild-type. Our results indicate that eseN encodes a PTL domain that interacts with MVP as a possible scaffold protein and inactivates pERK1/2 to ERK1/2, resulting in increased proliferation of E. ictaluri and, ultimately, death of the host.

Delivering the pain: an overview of the type III secretion system with special consideration for aquatic pathogens

“…In contrast, transcription and assembly of the related Salmonella SPI-2 T3SS only requires acidification of the SCV [ 104 ]. The EseN effector plays an important role in E. ictaluri invasion, where it activates extracellular replication in catfish head kidney by dephosphorylation in vivo in the head kidney of infected fish, and ex vivo in macrophages derived from the head kidney [ 105 ]. EseN can also shut down signal-regulated kinases 1 and 2 (ERK1/2) early in the infectious process [ 80 , 105 ].…”

“…The EseN effector plays an important role in E. ictaluri invasion, where it activates extracellular replication in catfish head kidney by dephosphorylation in vivo in the head kidney of infected fish, and ex vivo in macrophages derived from the head kidney [ 105 ]. EseN can also shut down signal-regulated kinases 1 and 2 (ERK1/2) early in the infectious process [ 80 , 105 ]. Furthermore, there is evidence of reduced numbers of bacteria in infected tissue and in mortality in channel catfish ( Ictalurus punctatus ) infected with bacteria with a deleted eseN gene bacteria compared to wild-type infected tissue [ 105 ].…”

“…EseN can also shut down signal-regulated kinases 1 and 2 (ERK1/2) early in the infectious process [ 80 , 105 ]. Furthermore, there is evidence of reduced numbers of bacteria in infected tissue and in mortality in channel catfish ( Ictalurus punctatus ) infected with bacteria with a deleted eseN gene bacteria compared to wild-type infected tissue [ 105 ]. Finally, low pH and low phosphate triggers the expression of the T3SS effectors EseB, EseC, and EseD expression as well as the PEI1and PEI2 encoded, ESeH and ESeI [ 106 ].…”

Edwardsiella ictaluri T3SS Effector EseN Modulates Expression of Host Genes Involved in the Immune Response

“…It was reported that in many pathogens secreted effectors can affect intracellular uptake, surface colonization of the cell without uptake, adherence to macrophages and inhibition of phagocytosis, cytotoxicity, vesicular trafficking, control of programmed cell death, manipulation of gene expression, and up- and down-regulation of inflammatory cytokines [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. The E. ictaluri effector EseN is a phosphothreonine lyase (PTL) that inactivates extracellular mitogen-activated protein kinases (ERK1/2) [ 12 ]. Based on similarity to other effectors, EseN can also inactivate other mitogen-activated-protein-kinases (MAPKs) [ 13 , 14 ].…”

“…Because EseN is important for the virulence of E. ictaluri [ 2 , 12 ] and the virulence mechanisms of this factor are unknown, RNAseq and transcriptome analysis were used to identify potential genes and pathways that are modulated by EseN. We used RT-qPCR to confirm RNAseq data and to study extended time points of infection.…”