Effects of Salmonella enterica serovar typhimurium sseK1 on macrophage inflammation-related cytokines and glycolysis.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important virulent intracellular pathogen, causes inflammatory gastroenteritis or typhoid. Macrophages play a key role in innate immunity against Salmonella. Salmonella secreted effector K1 (SseK1) encoded by SPI2 has been identified a novel translocated protein. To investigate the role of Salmonella enterica serovar Typhimurium sseK1 about the inflammation and glycolysis in macrophages, the levels of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and Nitric Oxide in macrophages infected by S. Typhimurium SL1344 wild-type (WT) group, ΔsseK1 mutant group and sseK1-complemented group were measured. And the glycolysis level was determined in RAW 264.7 cells infected with these different Salmonella strains. The results showed that groups infected by wild-type strain, sseK1 mutant and sseK1-complemented strain upregulated the production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO at 3 h, 6 h and 12 h, respectively. The production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO in wild-type strain group were significantly decreased compared with the ΔsseK1 mutant group, which suggested that sseK1 down-regulated the production of related inflammatory factors. Moreover, hexokinase, lactic acid and pyruvic acid levels significantly decreased by infection with sseK1 mutant compared to the wild-type strain. The ATP level of ΔsseK1 mutant group was remarkably increased than WT group and sseK1-complemented group. These indicated that the sseK1 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. In summary, the results demonstrated that sseK1 can down-regulate the inflammation-related cytokines and enhance the glycolysis level in macrophages infected by S. Typhimurium, which may be beneficial for S. typhimurium survival in macrophages.

Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways.

Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors 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 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas 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 Arg376 and Arg293 respectively. 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 suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.

Salmonella enterica serovar Typhimurium sseK3 induces apoptosis and enhances glycolysis in macrophages.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen that can survive and replicate in macrophages. Glycolysis is essential for immune responses against S. Typhimurium infection in macrophages, and is also associated with apoptosis. S. Typhimurium secreted effector K3 (SseK3) was recently identified as a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis in cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in S. Typhimurium-infected macrophages. RESULTS: Macrophages were infected with S. Typhimurium SL1344 wild-type (WT), ΔsseK3 mutant or sseK3-complemented strain, and the effects of sseK3 on apoptosis and glycolysis were determined. The adherence and invasion in the ΔsseK3 mutant group were similar to that in the WT and sseK3-complemented groups, indicating that SseK3 was not essential for the adherence and invasion of S. Typhimurium in macrophages. However, the percentage of apoptosis in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. Caspase-3, caspase-8, and caspase-9 enzyme activity in the ΔsseK3 mutant group were significantly lower than in the WT group and sseK3-complemented groups, indicating that sseK3 could improve the caspase-3, caspase-8, and caspase-9 enzyme activity. We also found that there were no significant differences in pyruvic acid levels between the three groups, but the lactic acid level in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. The ATP levels in the ΔsseK3 mutant group were remarkably higher than those in the WT and sseK3-complemented groups. These indicated that the sseK3 enhanced the level of glycolysis in macrophages infected by S. Typhimurium. CONCLUSIONS: S. Typhimurium sseK3 is likely involved in promoting macrophage apoptosis and modulating glycolysis in macrophages. Our results could improve our understanding of the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3.

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

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.

The impact of sseK2 deletion on Salmonella enterica serovar typhimurium virulence in vivo and in vitro.

BACKGROUND: Salmonella enterica is regarded as a major public health threat worldwide. Salmonella secretes the novel translocated effector protein K2 (SseK2), but it is unclear whether this protein plays a significant role in Salmonella enterica Typhimurium virulence. RESULTS: A ΔsseK2 mutant of S. Typhimurium exhibited similar growth curves, adhesion and invasive ability compared with wild-type (WT) bacteria. However, deletion of sseK2 rendered Salmonella deficient in biofilm formation and the early proliferative capacity of the ΔsseK2 mutant was significantly lower than that of the WT strain. In vivo, the LD50 (median lethal dose) of the ΔsseK2 mutant strain was increased 1.62 × 103-fold compared with the WT strain. In addition, vaccinating mice with the ΔsseK2 mutant protected them against challenge with a lethal dose of the WT strain. The ability of the ΔsseK2 mutant strain to induce systemic infection was highly attenuated compared with the WT strain, and the bacterial load in the animals' internal organs was lower when they were infected with the ΔsseK2 mutant strain than when they were infected with the WT strain. CONCLUSIONS: We conclude that sseK2 is a virulence-associated gene that plays a vital role in Salmonella virulence.

Role of the sseK1 gene in the pathogenicity of Salmonella enterica serovar enteritidis in vitro and in vivo.

Salmonella enteritidis is a common food-borne pathogen associated with consumption of contaminated poultry meat and eggs, which frequently causes gastroenteritis in humans. Salmonella secreted effector K1 (SseK1), as a translocated and secreted protein has been identified to be essential for the virulence of Salmonella typhimurium in host cells. However, the role of the sseK1 gene in the pathogenicity of S. enteritidis remain unclear. In this study, a sseK1 deletion mutant of S. enteritidis was constructed and its biological characteristics were examined. It was found that the sseK1 deletion mutant did not affect the growth, adherence and invasion of Salmonella enteritidis when compared to the wild-type S. enteritidis. However, the mutant showed decreased formation of biofilm and significantly reduced intracellular survival of bacteria in activated mouse peritoneal macrophages, as well as showed reduced pathogenicity to a murine model by increasing the lethal dose 50% (LD50) value and decreasing the proliferation ratio of bacteria in vivo. Taken together, this study determined an important role for SseK1 in the pathogenicity of S. enteritidis in vitro and in vivo.

Modulation of extrinsic apoptotic pathway by intracellular glycosylation.

The importance of post-translational modifications (PTMs), particularly O-GlcNAcylation, of cytoplasmic proteins in apoptosis has been neglected for quite a while. Modification of cytoplasmic proteins by a single N-acetylglucosamine sugar is a dynamic and reversible PTM exhibiting properties more like phosphorylation than classical O- and N-linked glycosylation. Due to the sparse information existing, we have only limited understanding of how GlcNAcylation affects cell death. Deciphering the role of GlcNAcylation in cell fate may provide further understanding of cell fate decisions. This review focus on the modulation of extrinsic apoptotic pathway via GlcNAcylation carried out by O-GlcNAc transferase (OGT) or by other bacterial effector proteins.

Arginine GlcNAcylation and Activity Regulation of PhoP by a Type III Secretion System Effector in Salmonella.

Salmonella type III secretion system (T3SS) effector SseK3 is a glycosyltransferase delivered directly into the host cells to modify host protein substrates, thus manipulating host cellular signal transduction. Here, we identify and characterize the Arg-GlcNAcylation activity of SseK3 inside bacterial cells. Combining Arg-GlcNAc protein immunoprecipitation and mass spectrometry, we found that 60 bacterial proteins were GlcNAcylated during Salmonella infection, especially the two-component signal transduction system regulatory protein PhoP. Moreover, the Arg-GlcNAcylation of PhoP by SseK3 was detected in vivo and in vitro, and four arginine residues, Arg65, Arg66, Arg118, and Arg215 were identified as the GlcNAcylation sites. Site-directed mutagenesis showed that the PhoP R215A change significantly reduced the DNA-binding ability and arginine to alanine change at all four sites (PhoP 4RA) completely eliminated the DNA-binding ability, suggesting that Arg215 is essential for the DNA-binding activity of PhoP and GlcNAcylation of PhoP affects this activity. Additionally, GlcNAcylation of PhoP negatively regulated the activity of PhoP and decreased the expression of its downstream genes. Overall, our work provides an example of the intra-bacterial activities of the T3SS effectors and increases our understanding of endogenous Arg-GlcNAcylation.

Arg-GlcNAcylation on TRADD by NleB and SseK1 Is Crucial for Bacterial Pathogenesis.

Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domain (DD) proteins through arginine-GlcNAcylation. Here, we performed a substrate screen on 12 host DD proteins with conserved arginine during EPEC and Salmonella infection. NleB from EPEC hijacked death receptor signaling through tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNF signaling through the modification of TRADD Arg235/Arg245 and TNFR1 Arg376, respectively. Furthermore, mouse infection studies showed that SseK1 but not SseK3 rescued the bacterial colonization deficiency contributed by the deletion of NleBc (Citrobacter NleB), indicating that TRADD was the in vivo substrate. The result provides an insight into the mechanism by which attaching and effacing (A/E) pathogen manipulate TRADD-mediated signaling and evade host immune defense through T3SS effectors.

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity.

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets.

Auto Arginine-GlcNAcylation Is Crucial for Bacterial Pathogens in Regulating Host Cell Death.

Many Gram-negative bacterial pathogens utilize the type III secretion system (T3SS) to inject virulence factors, named effectors, into host cells. These T3SS effectors manipulate host cellular signaling pathways to facilitate bacterial pathogenesis. Death receptor signaling plays an important role in eukaryotic cell death pathways. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) are T3SS effectors. They are defined as a family of arginine GlcNAc transferase to modify a conserved arginine residue in the death domain (DD) of the death receptor TNFR and their corresponding adaptors to hijack death receptor signaling. Here we identified that these enzymes, NleB, SseK1, and SseK3 could catalyze auto-GlcNAcylation. Residues, including Arg13/53/159/293 in NleB, Arg30/158/339 in SseK1, and Arg153/184/305/335 in SseK3 were identified as the auto-GlcNAcylation sites by mass spectrometry. Mutation of the auto-modification sites of NleB, SseK1, and SseK3 abolished or attenuated the capability of enzyme activity toward their death domain targets during infection. Loss of this ability led to the increased susceptibility of the cells to TNF- or TRAIL-induced cell death during bacterial infection. Overall, our study reveals that the auto-GlcNAcylation of NleB, SseK1, and SseK3 is crucial for their biological activity during infection.

Bacteria-Catalyzed Arginine Glycosylation in Pathogens and Host.

In recent years, protein glycosylation in pathogenic bacteria has attracted more and more attention, and accumulating evidence indicated that this type of posttranslational modification is involved in many physiological processes. The NleB from several enteropathogenic bacteria species as well as SseK from Salmonella enterica are type III secretion system effectors, which have an atypical N-acetylglucosamine (N-GlcNAc) transferase activity that specifically modified a conserved arginine in TRADD, FADD, and RIPK1. NleB/SseKs GlcNAcylation of death domain proteins abrogates homotypic and heterotypic death receptors/adaptors interactions, thereby blocking an important antimicrobial host response. Interestingly, NleB/SseKs could also GlcNAcylate themselves, and self-GlcNAcylation of NleB, SseK1, and SseK3 are crucial for their biological activity during infection. In addition, EarP (EF-P specific arginine rhamnosyl transferase for Posttranslational activation) catalyzes arginine rhamnosylation of translation elongation factor P (EF-P). Importantly, this kind of N-linked protein glycosylation is not only important for EF-P dependent rescue of polyproline stalled ribosomes but also for pathogenicity in Pseudomonas aeruginosa and other clinically relevant bacteria. Glycosylation of arginine is unique because the guanidine group of arginine has a high acid dissociation constant value and representing an extremely poor nucleophile. Recently, the crystal structures of NleB, SseKs, EarP, arginine GlcNAcylated death domain-containing proteins, NleB/FADD-DD, and EarP/EF-P/dTDP-ß-L-rhamnose were solved by our group and other groups, revealing the unique catalytic mechanisms. In this review, we provide detailed information about the currently known arginine glycosyltransferases and their potential catalytic mechanisms.

Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica.

Salmonella enterica expresses two virulence-related type III secretion systems (T3SSs) encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, in spite of not having a role in invasion or intracellular proliferation in cultured mammalian cells. In vitro, expression of sseK1 was higher in media mimicking intracellular conditions, when SPI2 was induced, but it was also significant under SPI1 inducing conditions. A detailed analysis of translocation of SseK1 into host cells unveiled that it was a substrate of both, T3SS1 and T3SS2, although with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages, or fibroblasts). The regulation of the expression of sseK1 was examined using lacZ and bioluminescent lux fusions. The two-component system PhoQ/PhoP is a positive regulator of this gene. A combination of sequence analysis, directed mutagenesis and electrophoretic mobility shift assays showed that phosphorylated PhoP binds directly to the promoter region of sseK1 and revealed a PhoP binding site located upstream of the predicted -35 hexamer of this promoter.