Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

The Salmonella enterica SseK1 protein is a type three secretion system effector that glycosylates host proteins during infection on specific arginine residues with N-acetyl glucosamine (GlcNAc). SseK1 also Arg-glycosylates endogenous bacterial proteins and we thus hypothesized that SseK1 activities might be integrated with regulating the intrabacterial abundance of UPD-GlcNAc, the sugar-nucleotide donor used by this effector. After searching for new SseK1 substrates, we found that SseK1 glycosylates arginine residues in the dual repressor-activator protein NagC, leading to increased DNA-binding affinity and enhanced expression of the NagC-regulated genes glmU and glmS. SseK1 also glycosylates arginine residues in GlmR, a protein that enhances GlmS activity. This Arg-glycosylation improves the ability of GlmR to enhance GlmS activity. We also discovered that NagC is a direct activator of glmR expression. Salmonella lacking SseK1 produce significantly reduced amounts of UDP-GlcNAc as compared with Salmonella expressing SseK1. Overall, we conclude that SseK1 up-regulates UDP-GlcNAc synthesis both by enhancing the DNA-binding activity of NagC and by increasing GlmS activity through GlmR glycosylation. Such regulatory activities may have evolved to maintain sufficient levels of UDP-GlcNAc for both bacterial cell wall precursors and for SseK1 to modify other bacterial and host targets in response to environmental changes and during infection.

Section: Introductionmentioning

confidence: 99%

Arginine glycosylation regulates UDP-GlcNAc biosynthesis in Salmonella enterica

Qaidi

Scott

Hays

et al. 2022

“…Through the T3SS system, the enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC), Citrobacter rodentium NleB effectors, and Salmonella enterica SseK1/2/3 effectors are released in the mammalian cell cytoplasm and covalently attach N -acetylglucosamine (GlcNAc) to the conserved arginine residues in several host targets such as cell death domain-containing proteins to block host cell apoptosis or necroptosis. Unlike the canonical O-linked glycosyltransferase toxins at the serine/threonine residues, NleB/SseK orthologs modify the uncommon guanidine group of a target arginine residue, which is known to be a poor nucleophile with partially delocalized electrons at physiological pH 10 – 14 .…”

Section: Introductionmentioning

confidence: 99%

“…However, it is unclear why SseK2/NleB2 effectors show a weaker glycosyltransferase signal despite more than 60% sequence identity and less than 0.90 Å RMS structure similarity with the fatal SseK1/2 and NleB1 effectors 14 . As a major catalytic factor for deficient SseK2/NleB2 glycosylation, we focused on the catalytic Asp-x-Asp (DxD) motif, which is essential for binding Mn 2+ and UDP-GlcNAc, which is conserved throughout all mono-glycosyltransferase bacterial toxins regardless of the release mechanism and O-/N-glycosylation.…”

Section: Introductionmentioning

confidence: 99%

Catalytic DxD motif caged in Asx-turn and Met–aromatic interaction attenuates the pathogenic glycosylation of SseK2/NleB2 effectors

Koh

Kim

Cho

2022

Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto host crucial proteins, which allows adhesion to mammalian cells and modulates host cellular processes for pathogenesis. Escherichia coli NleB1, Citrobacter rodentium NleB, and Salmonella enterica SseK1/3 type III effectors fatally glycosyltransfer N-acetyl glucosamine (GlcNAc) from UDP-GlcNAc to arginine residues of death domain-containing proteins that regulate host inflammation, intra-bacterial proteins, and themselves, whose post-translational modification disrupts host immune functions and prolongs bacterial viability inside host cells. However, unlike the similar NleB1/SseK1/SseK3, E. coli NleB2 and S. enterica SseK2 show deficient GlcNAcylation and neither intra-bacterial glycosylation nor auto-glycosylation. Here, as the major factor in SseK2/NleB2 deficiency, we focused on the catalytic Asp-x-Asp (DxD) motif conserved throughout all O-/N-glycosyltransferases to coordinate Mn2+. All DxD motifs in apo-glycosyltransferases form Type-I-turns for binding Mn2+, similar to the ligand-bound DxD motif, whereas TcnA/SseK2/NleB2 DxD motifs form Asx-turns, which are unable to bind Mn2+. Interestingly, methionine of the NleB2 DMD motif forms triple Met–aromatic interactions, as found in age-associated diseases and tumor necrosis factor (TNF) ligand-receptor complexes. The NleB1 A222M mutation induces triple Met–aromatic interactions to steeply attenuate glycosylation activity to 3% of that in the wild type. Thus, the characteristic conformation of the DxD motif is essential for binding Mn2+, donors, and glycosylate targets. This explains why SseK2/NleB2 effectors with the DxD motif caged in the Asp-/Asn-turn (Asx-turn) and triple Met–aromatic interactions have lower glycosyltransferase activity than that of other fatal NleB1/SseK1/SseK3 toxins.

“…SseK and NleB are glycosyltransferases that glycosylates several host proteins and on specific arginine residues and interfere with their physiological function 6 – 8 . For example, SseK1 glycosylates the death domain containing protein TRADD (Tumor necrosis factor Receptor type 1-Associated DEATH Domain protein), and Tubulin Folding Cofactor TBCB 7 , 9 – 11 . SseK2 glycosylates FADD (FAS-Associated Death Domain protein) 7 .…”

Section: Introductionmentioning

confidence: 99%

Salmonella T3SS effector SseK1 arginine-glycosylates the two-component response regulator OmpR to alter bile salt resistance

Hasan

Scott

Hays

et al. 2023

Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.