Analysis of T3SS-independent Autonomous Internalization of the Bacterial Effector Protein SspH1 from Salmonella typhimurium

Abstract

Previous studies identified the effector protein YopM of Yersinia enterocolitica as a novel bacterial cell-penetrating protein. YopM's ability to translocate across the host cell plasma membrane independently of Yersinia's type III secretion system (T3SS) is mediated by its two N-terminal α-helices. The SspH1 effector protein of Salmonella typhimurium shares significant homology in sequence and structure with YopM, which prompted us to investigate potential cell-penetrating abilities of this effector protein. For this, we recombinantly expressed SspH1 in Escherichia coli and analyzed a potential T3SS-independent translocation of the protein by cell fractionation of HeLa cells, immunofluorescence microscopy and FACS analyses. The functionality of the recombinant protein as an E3 ubiquitin-ligase was determined using in vitro-ubiquitination assays. Additionally, an effect of the recombinant protein on the expression of pro-inflammatory cytokines was analyzed by quantitative real time PCR. In this study, we could show that the SspH1 effector protein of Salmonella typhimurium is able to translocate autonomously into eukaryotic cells without requiring additional factors. Furthermore, we could show that recombinant SspH1 is a functional E3 ubiquitin ligase that is able to undergo auto-ubiquitination following T3SS-independent translocation and to reduce the expression of Interleukin-8 in IL-1β stimulated cells.

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

“…Plasmids for the overexpression of proteins were constructed using the pET24b(+) 3xFLAG vector (Lubos et al, 2014) in order to generate rNleC or rTat NleC with C-terminal 3xFLAG tags for detection of the protein and 6xHis for purification. To insert the nlec gene into the vector, a restriction-free cloning approach was chosen (Chen et al, 2000; van den Ent and Löwe, 2006) using the primer pairs F-NleC 3xFLAG/R-NleC 3xFLAG or F-Tat NleC 3xFLAG/R-NleC 3xFLAG (Table 1) and genomic EPEC E2348/69 DNA.…”

“…To insert the nlec gene into the vector, a restriction-free cloning approach was chosen (Chen et al, 2000; van den Ent and Löwe, 2006) using the primer pairs F-NleC 3xFLAG/R-NleC 3xFLAG or F-Tat NleC 3xFLAG/R-NleC 3xFLAG (Table 1) and genomic EPEC E2348/69 DNA. Restriction-free cloning was performed as described in Lubos et al (2014). Deletion mutants were generated using an inverse PCR approach with the insertion of restriction sites for Eco RI using deletion primer pairs (F-NleC Δ183–187/R-NleC Δ183–187 or F-NleC Δ208–257/R-NleC Δ208–257) employing pET24b(+): NleC 3xFLAG as a template.…”

“…Recently, ours and other laboratories have identified several virulence proteins of different bacterial pathogens including the T3SS-dependent effector proteins YopM from Yersinia enterocolitica (Rüter et al, 2010), SspH1 from Salmonella enterica serovar Typhimurium (Lubos et al, 2014), or the Tir protein of enteropathogenic E. coli (Michgehl et al, 2006) and TcpC from uropathogenic E. coli (UPEC) (Cirl et al, 2008; Yadav et al, 2010) that are able to translocate autonomously across eukaryotic membranes without a need for a T3SS or any other bacterial factor. These T3SS-associated effector proteins exhibit properties of cell-penetrating peptides (CPPs) and have been proposed to represent a novel class of “cell-penetrating effectors” or CPEs (Rüter et al, 2010; Yadav et al, 2010; Snyder et al, 2013; Lubos et al, 2014).…”

“…These T3SS-associated effector proteins exhibit properties of cell-penetrating peptides (CPPs) and have been proposed to represent a novel class of “cell-penetrating effectors” or CPEs (Rüter et al, 2010; Yadav et al, 2010; Snyder et al, 2013; Lubos et al, 2014). Although the relevance of type-3-secretion-independent translocation during bacterial infections has not been unraveled yet, these findings prompted the development of innovative strategies of applying CPEs as self-delivering therapeutics against autoimmune diseases.…”

Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike

“…13,14 The latter three effectors share high sequence homology in their N-terminal regions, which are dominated by an a-helical structure, followed by leucine rich repeats. 11,13,15 Besides, a growing number of biologic or synthetic cell-penetrating peptides is available, which can be fused either genetically or chemically to heterologous cargo-proteins for delivery into target cells and also specific target cell compartments of interest (for a review see refs. 16,17).…”