scholarly journals Distinct Roles of the Antiapoptotic Effectors NleB and NleF from Enteropathogenic Escherichia coli

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Georgina L. Pollock ◽  
Clare V. L. Oates ◽  
Cristina Giogha ◽  
Tania Wong Fok Lung ◽  
Sze Ying Ong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fas Ligand ◽  
Death Receptor ◽  
Effector Proteins ◽  
Host Cells ◽  
Caspase 8 ◽  
Death Domain ◽  
Content Type ◽  
Fas Signaling

ABSTRACT During infection, enteropathogenic Escherichia coli (EPEC) translocates effector proteins directly into the cytosol of infected enterocytes using a type III secretion system (T3SS). Once inside the host cell, these effector proteins subvert various immune signaling pathways, including death receptor-induced apoptosis. One such effector protein is the non-locus of enterocyte effacement (LEE)-encoded effector NleB1, which inhibits extrinsic apoptotic signaling via the FAS death receptor. NleB1 transfers a single N-acetylglucosamine (GlcNAc) residue to Arg117 in the death domain of Fas-associated protein with death domain (FADD) and inhibits FAS ligand (FasL)-stimulated caspase-8 cleavage. Another effector secreted by the T3SS is NleF. Previous studies have shown that NleF binds to and inhibits the activity of caspase-4, -8, and -9 in vitro. Here, we investigated a role for NleF in the inhibition of FAS signaling and apoptosis during EPEC infection. We show that NleF prevents the cleavage of caspase-8, caspase-3, and receptor-interacting serine/threonine protein kinase 1 (RIPK1) in response to FasL stimulation. When translocated into host cells by the T3SS or expressed ectopically, NleF also blocked FasL-induced cell death. Using the EPEC-like mouse pathogen Citrobacter rodentium, we found that NleB but not NleF contributed to colonization of mice in the intestine. Hence, despite their shared ability to block FasL/FAS signaling, NleB and NleF have distinct roles during infection.

scholarly journals Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

2016 ◽  
Vol 84 (5) ◽  
pp. 1346-1360 ◽  
Author(s):  
Tania Wong Fok Lung ◽  
Cristina Giogha ◽  
Kristina Creuzburg ◽  
Sze Ying Ong ◽  
Georgina L. Pollock ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fas Ligand ◽  
Effector Proteins ◽  
Single Amino Acid ◽  
Caspase 8 ◽  
Death Domain ◽  
Content Type ◽  
Functional Sites

EnteropathogenicEscherichia coli(EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a singleN-acetylglucosamine (GlcNAc) moiety in anN-glycosidic linkage to Arg117of the Fas-associated death domain protein (FADD). GlcNAcylation of FADD prevents the assembly of the canonical death-inducing signaling complex and inhibits Fas ligand (FasL)-induced cell death. Apart from the DXD catalytic motif of NleB1, little is known about other functional sites in the enzyme. In the present study, members of a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage showed that 17 mutant derivatives of NleB1, including the catalytic DXD mutant, did not inhibit caspase-8 activation. Regions of interest around the insertion sites with multiple or single amino acid substitutions were examined further. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63–66)AAAA(in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236–238)AAAmutant, did not bind to or GlcNAcylate FADD. Infection of mice with the EPEC-like mouse pathogenCitrobacter rodentiumexpressing NleBE253Aand NleBY219Ashowed that these strains were attenuated, indicating the importance of residues E253 and Y219 in NleB1 virulencein vivo. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that these are required for the virulence function of NleB1.

scholarly journals Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Maarten F. de Jong ◽  
Neal M. Alto
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
E Coli ◽  
Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

scholarly journals A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

2014 ◽  
Vol 82 (9) ◽  
pp. 3644-3656 ◽  
Author(s):  
Michael D. Engstrom ◽  
Christopher J. Alteri ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Upper Urinary Tract ◽  
Host Cells ◽  
Promoter Regions ◽  
Content Type ◽  
Tract Infections

ABSTRACTA heterogeneous subset of extraintestinal pathogenicEscherichia coli(ExPEC) strains, referred to as uropathogenicE. coli(UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associatedtosoperon is well expressedin vivobut poorly expressedin vitroand encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulatetosAand affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion oftosRalleviatestosArepression. Thetospromoter was localized upstream oftosRusing transcriptional fusions of putative promoter regions withlacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream oftosRinhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression offliC, encoding flagellin. Deletion oftosEFincreased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

scholarly journals Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1801-1812 ◽  
Author(s):  
Sylvia Kleta ◽  
Marcel Nordhoff ◽  
Karsten Tedin ◽  
Lothar H. Wieler ◽  
Rafal Kolenda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Single Infection ◽  
Content Type ◽  
E Coli ◽  
Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

scholarly journals Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Erez Mills ◽  
Kobi Baruch ◽  
Gili Aviv ◽  
Mor Nitzan ◽  
Ilan Rosenshine
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Public Health Problem ◽  
Effector Proteins ◽  
Host Cells ◽  
Global Public Health ◽  
Secretion Systems ◽  
Comprehensive Description ◽  
Content Type ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

scholarly journals Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

2016 ◽  
Vol 84 (11) ◽  
pp. 3220-3231 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Tomohiro Agata ◽  
Hirofumi Asano ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Antimicrobial Agents ◽  
Host Cells ◽  
Type I ◽  
Wild Type ◽  
Content Type ◽  
Type I Fimbriae ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

scholarly journals Salmonellaeffectors SseK1 and SseK3 target death domain proteins in the TNF and TRAIL signaling pathways

2018 ◽  
Author(s):  
Joshua P M Newson ◽  
Nichollas E Scott ◽  
Ivy Yeuk Wah Chung ◽  
Tania Wong Fok Lung ◽  
Cristina Giogha ◽  
...  
Keyword(s):  
Death Receptor ◽  
Distinct Type ◽  
Effector Proteins ◽  
Host Cells ◽  
Death Domain ◽  
Secretion Systems ◽  
Host Proteins ◽  
Tnf Superfamily ◽  
Arginine Residues ◽  
Death Receptor Signaling

AbstractStrains ofSalmonellautilise two distinct type three secretion systems to deliver effector proteins directly into host cells. TheSalmonellaeffectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 duringSalmonellainfection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, while ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376and Arg293respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggests that Salmonellae carryingsseK1andsseK3employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.

scholarly journals NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector

2021 ◽  
Vol 17 (6) ◽  
pp. e1009658
Author(s):  
Cristina Giogha ◽  
Nichollas E. Scott ◽  
Tania Wong Fok Lung ◽  
Georgina L. Pollock ◽  
Marina Harper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Host Cell ◽  
Peptide Sequencing ◽  
Effector Proteins ◽  
Host Protein ◽  
Death Domain ◽  
T3ss Effector ◽  
Arginine Residues

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.

Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells

1999 ◽  
Vol 276 (6) ◽  
pp. F837-F846 ◽  
Author(s):  
L. Richard Feldenberg ◽  
Sundararajah Thevananther ◽  
Marcela del Rio ◽  
Maryely de Leon ◽  
Prasad Devarajan
Keyword(s):  
Dna Cleavage ◽  
Serine Proteases ◽  
Fas Ligand ◽  
Mdck Cells ◽  
Morphological Changes ◽  
Critical Role ◽  
Atp Depletion ◽  
Caspase 8 ◽  
Death Domain

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (≈10–65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.

scholarly journals Vitamin C inhibited FasL-induced apoptotic death of mouse dendritic cells through c-FLIP expression

2020 ◽  
Vol 16 (4) ◽  
pp. 595-601
Author(s):  
Nguyen Thi Xuan ◽  
Le Thi Thu Hien
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Vitamin C ◽  
Fas Ligand ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Death Receptor ◽  
Caspase 8 ◽  
Mouse Bone Marrow ◽  
Fas Signaling

Vitamin C (VitC) is a potent antioxidant and contributes as an apoptosis inhibitor by preventing death receptor-triggered caspase 8 activity. Fas ligand (FasL) induces the apoptotic cell death via activation of Fas signaling, which is dependent on the expression level of anti-apoptotic molecule c-FLIP (FADD-like IL-1beta-converting enzyme-inhibitory proteins). The present study addressed the effects of VitC on survival of dendritic cells (DCs), a regulator of innate and adaptive immunity. To this end, mouse bone marrow cells were isolated and cultured to attain bone marrow-derived DCs (BMDCs). The cells were treated with FasL in the presence or absence of VitC. Real time RT-PCR, Western blotting and FACS analysis were performed to determine different hallmarks of DC apoptosis. As a result, FasL treatment resulted in activation of caspase 8 and stimulation of cell membrane scrambling, the effects were supressed when VitC was present in the cell culture or the cells were transfected with FLIP siRNA. In conclusion, VitC prevented FasL-triggered DC apoptosis mediated through the expression of c-FLIP.

Sign in / Sign up

Export Citation Format

Share Document