Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

Abstract

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.

Cell Penetrating Peptides, Novel Vectors for Gene Therapy

“…Interestingly, even certain pathogenic bacteria use CPPs for delivery of bacterial effector proteins into different types of mammalian cells. For example, the pathogenic bacteria Yersinia enterocolitica encodes for the anti-inflammatory protein YopM with two alpha helices, α1H and α2H, in its amino terminus that function as CPPs similar to Antp [20][21][22]. Similarly, Shigella and Salmonella encode for immune effector proteins that can also enter cells efficiently to modulate the immune response [22].…”

Nuclear trafficking of bacterial effector proteins

“…CPEs are thought to enter eukaryotic cells via endocytosis, involving different uptake mechanisms depending upon the target cell type (Norkowski et al, 2018). The concentration of the CPEs and their associated cargo was also suggested to influence uptake (Norkowski et al, 2018).…”

Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization

“…Notably, by comparing the ratio of fluorescence from internalized rhodamine-labeled molecules to that of internalized naphthofluorescein-labeled molecules, the relative efficiencies of endosomal escape for different molecules-of-interest could be estimated. 35,163,[165][166][167] While these assays were designed to report exclusively on cytosolic localization, they required additional independent information, and they lacked absolute quantitation. Recently, a new fluorescence-based method was reported that provided absolute quantitation of fluorophore concentration in the cytosol (Figure 3c).…”