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

Abstract

Enteropathogenic Escherichia 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 single Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrheagenic pathogen of children that utilizes a type III secretion system (T3SS) to inject virulence effector proteins directly into host intestinal cells. These effectors subvert host cell function and are encoded by genes either within the locus of enterocyte effacement (LEE) pathogenicity island (1) or outside the LEE (non-Lee-encoded [Nle] effectors) (2). A number of non-LEEencoded effectors that disrupt host cell signaling have been characterized in recent years (3, 4). For example, both NleC and NleE inhibit NF-B signaling and, consequently, block the production of inflammatory cytokines, such as interleukin-8 (5-7). NleB1 was recently characterized to be a glycosyltransferase effector that modifies a conserved arginine residue in the Fas-associated death domain protein (FADD) as well as the death domain-containing proteins TRADD and RIPK1, thereby blocking host death receptor signaling and apoptosis (8, 9). Modification of FADD prevents its recruitment to the Fas receptor and the subsequent recruitment and activation of caspase-8 (8, 9).Glycosyltransferases are enzymes that catalyze the transfer of a sugar moiety from an activated sugar donor substrate containing a phosphate-leaving group, such as nucleoside diphosphate sugars, to a specific acceptor substrate (10, 11). Cytosolic glycosylation is a vital molecular mechanism by which various bacterial toxins and effector proteins subvert eukaryotic host cell function (12). For example, Clostridium difficile toxins A and B modify small Rho GTPases by mono-O-glucosylation at specific threonine residues (13). Glycosylated Rho GTPases are locked in an inactive state, inhibiting downstream signaling pathways, resulting in the disruption of the cell cytoskeleton, the induction of apoptotic cell death, and fluid accumulation in the large intestine (14).The transfer of carbohydrate components to proteins usually occurs at the oxygen nucleophile of the hydroxyl group of a serine or threonine side chain, resulting in the formation of an O-linked glycosidic linkage, or at the nitrogen nucleophile of the amide group of an asparagine side chain, forming an N-linked glycosidic linkage (15)(16)(17)(18)(19). O-GlcNAc modification, unlike classic O-or Nlinked protein glycosylation, involves the transfer of a single GlcNAc moiety to the hydroxyl group of a serine or threonine residue, and this modification is not elongated (17,20). A nonclassical N-linked modification involves the addition of single glycosyl moieties from activated nucleotide sugars to asparagine residues of the target protein (21-23). NleB1 catalyzes an unusual glycosylation reaction that results in the addition of GlcNAc in an N-glycosidic linkage to arginine 117 (R117) of FADD (8,9). Th...

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

“…Mutation of a conserved DxD catalytic motif within SseK1 (Asp 223 -Ala 224- Asp 225 ) and SseK3 (Asp 226 -Ala 227 -Asp 228 ) abrogated their glycosyltransferase activity [9], consistent with findings for the homologous T3SS effector, NleB1, from EPEC [7, 8]. Mutation of a single glutamic acid (Glu 253 ) also impairs the ability of NleB1 to inhibit NF-κB activation following TNF stimulation of mammalian cells [8], and renders NleB1 catalytically inactive [10]. Here, we confirmed the importance of this conserved glutamic acid to the biochemical activity of SseK1 and SseK3 by infecting RAW264.7 cells with a Salmonella Typhimurium SL1344 triple ΔsseK123 deletion mutant [6] complemented with either native SseK1, −2 or −3, catalytic triad mutants, or mutants lacking the conserved glutamic acid (SseK1 E255A , SseK2 E271A , and SseK3 E258A ).…”

“…Mutation of a conserved DxD catalytic motif within SseK1 and SseK3 abrogates their glycosyltransferase activity [9], consistent with findings for NleB1 [7, 8]. These studies describing catalytically important regions of the glycosyltransferases provide opportunities to better understand the function of these novel enzymes [10].…”

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

“…In 2016 Wong Fok Lung and co-workers mutated nleB and identified certain residues in NleB either interfering with FADD binding or preventing GlcNacylation. 52 Among the non-functional NleB protein variants they confirmed the importance of two invariant aspartate residues D221 and D223. 30 A catalytic Asp-X-Asp motif is featured by various GT-A glycosyltransferases.…”

Citrobacter rodentium NleB Protein Inhibits Tumor Necrosis Factor (TNF) Receptor-associated Factor 3 (TRAF3) Ubiquitination to Reduce Host Type I Interferon Production

“…This N-GlcNAcylation disrupts TNF signaling in EPEC-infected cells, thus impacting NF-B activation, apoptosis, and necroptosis (28,29). A recent paper defined numerous amino acids that are important to the N-GlcNAcylation of FADD by EPEC NleB1 and also used the C. rodentium infection model to show the role of these amino acids in virulence (30 (28). Labeling of an arginine residue by NleB1 was also detected for TNFR1 and RIPK1 (28).…”