Christian Rüter

Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines

Cell-permeable proteins, also called cell-penetrating peptides (CPPs), have the ability to cross cellular membranes, either alone or in association with bioactive cargo. We identified the Yersinia protein YopM as a novel bacterial cell-permeable protein. Here, we describe the ability of isolated recombinant YopM to enter host cells without a requirement for additional factors. This autonomous translocation of YopM was confirmed in several cell types, indicating that it is an intrinsic property of YopM. Using truncated versions of YopM, we show that either of the two N-terminal α-helices of YopM mediates translocation into the cells. Furthermore, the two α-helices are also able to deliver heterologous cargo, such as GFP or YopE. In addition, we found that, after entering the cells, YopM is functional and efficiently downregulates the transcription of pro-inflammatory cytokines (such as tumor necrosis factor-α and interleukins 12, 15 and 18). This finding suggests the potential use of YopM as a tool for protein delivery. Furthermore, it can lead to important advances in understanding and evaluating the intracellular and molecular function of YopM without the need for infection with Yersinia.

Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain

The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.

MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions

Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the ‘apical junctional complex—AJC’ with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the ‘Junctional Adhesion Molecules’ (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

Identification and characterization of Ibe, a novel type III effector protein of A/E pathogens targeting human IQGAP1

Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic Escherichia coli (ATEC) and enterohemorrhagic Escherichia coli (EHEC) belong to the family of attaching and effacing (A/E) pathogens. Pathogenicity is mediated by subversion of host cell functions involving type III secretion system (TTSS)‐dependent effector proteins. In this study, we have identified and characterized a novel TTSS‐dependent effector protein encoded at the 5′‐end of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) of ATEC strain 3431‐4/86 (O8:H‐). Using affinity purification we identified IQGAP1, a scaffolding protein involved in the regulation of the actin cytoskeleton, as a putative host cell target. Accordingly, we termed the novel effector protein ‘Ibe’ for IQGAP1‐binding effector. The interaction of Ibe and IQGAP1 was confirmed by co‐immunoprecipitation from ATEC‐infected cells and immunofluorescence analysis, which revealed colocalization of Ibe and IQGAP1 in ATEC‐induced pedestals and actin‐rich membrane ruffles. This suggests that the putative effector function of Ibe is mediated via IQGAP1. The Ibe‐independent recruitment of IQGAP1 to ATEC‐induced pedestals implies a general role for IQGAP1 in the subversion of host cell functions during infection. Homologues of the novel effector Ibe are widely distributed among EPEC, ATEC and EHEC strains but are not necessarily genetically linked to the LEE as they have occasionally also been found to be encoded within lambdoid prophages.

Esp-independent functional integration of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC) into host cell membranes.

The pathogenesis of enteropathogenic Escherichia coli (EPEC) is characterized by the type III secretion system‐dependent exploitation of target cells that results in attaching and effacing (A/E) lesions, actin rearrangements and pedestal formation. This pathology is mediated by effector proteins which are translocated by the type III secretion system into the host cell such as the translocated intimin receptor (Tir) and several E. coli secreted proteins (Esp). Secretion of virulence proteins of EPEC is tightly regulated. In response to Ca2+, Esp secretion is drastically reduced, whereas secretion of Tir is increased. Membrane insertion of Tir, secreted under low Ca2+ conditions, is therefore independent of Esp. Furthermore, espB and espD mutant strains of EPEC, unable to form the translocation pore, still translocate Tir into host cells membranes. This autointegrated Tir is functional, as it is able to complement a tir mutant strain in recruiting actin to bacterial contact sites. The uptake of Tir into the host cell appears to depend on the C‐terminal part of the protein, as deletion of this part of Tir prevents autointegration. Together, our results demonstrate that under conditions of limited Ca2+ an alternative mechanism for Tir integration can trigger the induction of A/E lesions.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.

Manipulation of pro-inflammatory cytokine production by the bacterial cell-penetrating effector protein YopM is independent of its interaction with host cell kinases RSK1 and PRK2.

The effector protein Yersinia outer protein M (YopM) of Yersinia enterocolitica has previously been identified and characterized as the first bacterial cell-penetrating protein (CPP). We found that recombinant YopM (rYopM) enters different eukaryotic cell types and downregulates the expression of several pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α]) after autonomous translocation. After infection with Y. enterocolitica or transfection of host cells, YopM interacts with isoforms of the two kinases ribosomal S6 protein kinase (RSK) and protein kinase C-related kinase (PRK). This interaction caused sustained RSK activation due to interference with dephosphorylation. Here we demonstrate by co-immunoprecipitation that rYopM interacts with RSK and PRK following cell-penetration. We show that autonomously translocated rYopM forms a trimeric complex with different RSK and PRK isoforms. Furthermore, we constructed a series of truncated versions of rYopM to map the domain required for the formation of the complex. The C-terminus of rYopM was identified to be essential for the interaction with RSK1, whereas any deletion in rYopM’s leucin-rich repeat domains abrogated PRK2 binding. Moreover, we found that the interaction of cell-penetrating rYopM with RSK led to enhanced autophosphorylation of this kinase at serine 380. Finally, we investigated whether downstream signaling of the trimeric rYopM-RSK/PRK complex modulates the expression of pro-inflammatory TNF-α. Here, we could exclude that interaction with RSK1 and PRK2 is essential for the anti-inflammatory effects of rYopM.

Current activities of the Yersinia effector protein YopM

Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogens virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed.