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The rising resistance to conventional antimicrobial therapies in veterinary contexts poses a formidable challenge. While various innovative treatment strategies for pathogenic infections have emerged, their success still needs to be improved, warranting continued research. Recent investigations into natural products as potential sources for biologically active therapeutics have gained traction. Phytochemicals present a promising alternative in combating a spectrum of pathogens, including bacteria, fungi and parasites. One such class of phytochemicals with mounting potential is the structurally diverse terpenes. These chemicals contribute to plants' characteristic odour and medicinal effects and have been widely investigated in the scientific literature for their exceptional antibacterial activity. Their efficacy is demonstrated through diverse mechanisms, encompassing damage to bacterial membranes, suppression of virulence factors, and interference with enzymes, toxins, and biofilm formation. This review comprehensively examines terpenes' in vitro and in vivo activity and their derivatives against pathogens, elucidating their potential against antimicrobial resistance (AMR) and the underlying mechanisms specific to each terpene class. The findings underscore the burgeoning potential of terpene therapy as a viable alternative or supplementary approach to conventional antibiotics in addressing bacterial and parasitic infections in livestock and companion animals., Competing Interests: Declarations. Competing Interests: The authors declare no conflict of interest in the present study. The funding sources had no part in study design and conceptualisation, collection, analysis, or interpretation of data, writing the manuscript, or in the decision to publish results. Ethics Approval: Not applicable. Consent for Publication: Not applicable., (© 2025. The Author(s).)

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation of cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Aktepe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1β. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1β activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1β production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL S132P in biological membranes and MLKL S132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, Mlkl S131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease., (© 2023. Springer Nature Limited.)

Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1β maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1β maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency., (© 2023 The Authors.)

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing., Competing Interests: J.M.M. and A.L.S. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma., (© 2022 The Authors.)

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Citrobacter rodentium is a mouse-specific extracellular enteropathogen, commonly used as a small animal model for studying human enteropathogenic Escherichia coli infections. Both pathogens share a core set of virulence factors, including a type III secretion system, which enables translocation of effector proteins into infected cells to subvert host antimicrobial responses. Notably, these bacterial effectors have been reported to specifically target components of the apoptotic, necroptotic and pyroptotic signaling cascades in vivo, resulting in compromised immune cell recruitment and impaired mucosal homeostasis. Identifying the contributions of each cell death modality to bacterial control in a physiological model represents a crucial step in furthering our understanding of host-pathogen evolution and may provide insight into the host evasion strategies utilised by other enteric pathogens., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Shigella is a highly infectious human pathogen, yet mice are naturally resistant to infection. In this issue of Cell Host & Microbe, Luchetti et al. (2021) discuss this species specificity, demonstrating that Shigella directly targets the pore-forming protein Gasdermin D for degradation, thus preventing pyroptosis to enable infection of human cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Salmonella enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-) is a monophasic variant of Salmonella Typhimurium that has emerged as a global cause of multidrug resistant salmonellosis. We used Bayesian phylodynamics, genomic epidemiology, and phenotypic characterization to describe the emergence and evolution of Salmonella 4,[5],12:i:- in Australia. We show that the interruption of the genetic region surrounding the phase II flagellin, FljB, causing a monophasic phenotype, represents a stepwise evolutionary event through the accumulation of mobile resistance elements with minimal impairment to bacterial fitness. We identify three lineages with different population dynamics and discrete antimicrobial resistance profiles emerged, likely reflecting differential antimicrobial selection pressures. Two lineages are associated with travel to South-East Asia and the third lineage is endemic to Australia. Moreover antimicrobial-resistant Salmonella 4,[5],12:i- lineages efficiently infected and survived in host phagocytes and epithelial cells without eliciting significant cellular cytotoxicity, suggesting a suppression of host immune response that may facilitate the persistence of Salmonella 4,[5],12:i:-., (© 2021. The Author(s).)

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: The authors have declared that no competing interests exist.

Interleukin-1β (IL-1β) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1β activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1β is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1β is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1β cleavage by caspase-1. IL-1β K133 is modified by ubiquitin and forms a salt bridge with IL-1β D129. Loss of IL-1β K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1β. Accordingly, Il1b K133R/K133R mice have increased levels of precursor IL-1β upon inflammasome priming and increased production of bioactive IL-1β, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1β activity and safeguard against damaging inflammation.

Objectives: During gastrointestinal infection, dysbiosis can result in decreased production of microbially derived short-chain fatty acids (SCFAs). In response to the presence of intestinal pathogens, we examined whether an engineered acetate- or butyrate-releasing diet can rectify the deficiency of SCFAs and lead to the resolution of enteric infection., Methods: We tested whether a high acetate- or butyrate-producing diet (HAMSA or HAMSB, respectively) condition Citrobacter rodentium infection in mice and assess its impact on host-microbiota interactions. We analysed the adaptive and innate immune responses, changes in gut microbiome function, epithelial barrier function and the molecular mechanism via metabolite sensing G protein-coupled receptor 43 (GPR43) and IL-22 expression., Results: HAMSA diet rectified the deficiency in acetate production and protected against enteric infection. Increased SCFAs affect the expression of pathogen virulence genes. HAMSA diet promoted compositional and functional changes in the gut microbiota during infection similar to healthy microbiota from non-infected mice. Bacterial changes were evidenced by the production of proteins involved in acetate utilisation, starch and sugar degradation, amino acid biosynthesis, carbohydrate transport and metabolism. HAMSA diet also induced changes in host proteins critical in glycolysis, wound healing such as GPX1 and epithelial architecture such as EZR1 and PFN1. Dietary acetate assisted in rapid epithelial repair, as shown by increased colonic Muc-2 , Il-22 , and anti-microbial peptides. We found that acetate increased numbers of colonic IL-22 producing TCRαβ + CD8αβ + and TCRγδ + CD8αα + intraepithelial lymphocytes expressing GPR43., Conclusion: HAMSA diet may be an effective therapeutic approach for fighting inflammation and enteric infections and offer a safe alternative that may impact on human health., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Receptor Interacting Protein Kinases (RIPKs) are cellular signaling molecules that are critical for homeostatic signaling in both communicable and non-communicable disease processes. In particular, RIPK1, RIPK2, RIPK3 and RIPK7 have emerged as key mediators of intracellular signal transduction including inflammation, autophagy and programmed cell death, and are thus essential for the early control of many diverse pathogenic organisms. In this review, we discuss the role of each RIPK in host responses to bacterial and viral pathogens, with a focus on studies that have used pathogen infection models rather than artificial stimulation with purified pathogen associated molecular patterns. We also discuss the intricate mechanisms of host evasion by pathogens that specifically target RIPKs for inactivation, and finally, we will touch on the controversial issue of drug development for kinase inhibitors to treat chronic inflammatory and neurological disorders, and the implications this may have on the outcome of pathogen infections., (Copyright © 2020. Published by Elsevier Ltd.)

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

During infection, Salmonella species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, Salmonella exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the Salmonella- containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of Salmonella infection by SseK3, an arginine N- acetylglucosamine (GlcNAc) transferase effector translocated via the Salmonella pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to Salmonella infection by interfering with the activity of key Rab GTPases., (Copyright © 2020 Gan, Scott, Newson, Wibawa, Wong Fok Lung, Pollock, Ng, van Driel, Pearson, Hartland and Giogha.)

Salmonella enterica subsp. enterica serovar Typhimurium ( S . Typhimurium) is a Gram-negative bacterium with a broad host range that causes non-typhoidal salmonellosis in humans. S . Typhimurium infects epithelial cells and macrophages in the small intestine where it replicates in a specialized intracellular niche called the Salmonella -containing vacuole (SCV) and promotes inflammation of the mucosa to induce typically self-limiting gastroenteritis. Virulence and spread of the bacterium is determined in part by the host individual's ability to limit the infection through innate immune responses at the gastrointestinal mucosa, including programmed cell death. S . Typhimurium however, has evolved a myriad of mechanisms to counteract or exploit host responses through the use of Type III Secretion Systems (T3SS), which allow the translocation of virulence (effector) proteins into the host cell for the benefit of optimal bacterial replication and dissemination. T3SS effectors have been found to interact with apoptotic, necroptotic, and pyroptotic cell death cascades, interfering with both efficient clearance of the bacteria and the recruitment of neutrophils or dendritic cells to the area of infection. The interplay of host inflammation, programmed cell death responses, and bacterial defenses in the context of non-typhoidal Salmonella (NTS) infection is a continuing area of interest within the field, and as such has been reviewed here.

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 Arg 376 and Arg 293 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., (© 2019 Newson et al.)

Gut-focused hypnotherapy is an effective treatment for irritable bowel syndrome but is not widely available. This study assessed whether providing hypnotherapy by Skype might partially overcome this problem. Using a 50-point or more reduction in the IBS Symptom Severity Score as the primary outcome measure, 65% of subjects responded to Skype hypnotherapy with all other outcomes significantly improving. The primary outcome figure for face-to-face hypnotherapy was 76%. When other outcome scores for Skype and face-to-face treatment were compared, the mean changes were these: symptom severity (-94.1 vs. -129.2), noncolonic score (-52.3 vs. -64.8), quality of life (+56.4 vs. +66.2), anxiety (-3.3 vs. -3.0), depression (-1.7 vs. -2.5), and a 30% or more pain reduction (44% vs. 62%). Skype hypnotherapy is effective but slightly less so than face-to-face treatment. However, many patients would have been unable to access treatment without the Skype option.

Background: Posterior tibial nerve stimulation (PTNS) is a novel treatment for patients with faecal incontinence (FI) and may be effective in selected patients; however, its mechanism of action is unknown. We sought to determine the effects of PTNS on anorectal physiological parameters., Methods: Fifty patients with FI underwent 30 min of PTNS treatment, weekly for 12 weeks. High-resolution anorectal manometry, bowel diaries and Vaizey questionnaires were performed before and after treatment. Successful treatment was determined as a greater than 50% reduction in FI episodes., Results: Fifty patients with FI were studied; 39 women, median age 62 years (range 30-82). Compared with pretreatment, there were reductions in episodes of urgency (16.0 versus 11.4, p = 0.006), overall FI (14.5 versus 9.1, p = 0.001), urge FI (5.4 versus 3.2, p = 0.016) and passive FI (9.1 versus 5.9, p = 0.008). Vaizey score was reduced (16.1 versus 14.5, p = 0.002). Rectal sensory volumes (ml) decreased (onset 40.3 versus 32.6, p = 0.014, call 75.7 versus 57.5, p < 0.001, urge 104.1 versus 87.4, p = 0.004). There was no significant change in anal canal pressures (mmHg) (maximum resting pressure 41.4 versus 44.2, p = 0.39, maximum squeeze pressure, 78.7 versus 88.2, p = 0.15, incremental squeeze pressure 37.2 versus 44.1, p = 0.22). Reduction in FI episodes did not correlate with changes in physiological parameters ( p > 0.05). Treatment success of 44% was independent of changes in manometric parameters ( p > 0.05)., Conclusions: PTNS has a measureable physiological effect on rectal sensory volumes without an effect on anal canal pressures. It also reduces FI episodes; however, this effect is independent of changing physiology, suggesting that PTNS has a complex mechanism of action., Competing Interests: Conflict of interest statement: PW has acted as a consultant for, or received research grant support from, the following pharmaceutical companies in the last 5 years: Almirall Pharma, Boehringer–Ingelheim, Chr. Hansen, Danone Research, Ironwood Pharmaceuticals, Salix, Shire UK, Sucampo Pharmaceuticals and Allergan. PW is an associate editor for Therapeutic Advances in Gastroenterology. No other authors report any conflicts of interest.

The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleB CR in C. rodentium ) that modifies conserved arginine residues in death domain-containing host proteins with N -acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection in vitro , or C. rodentium infection in vivo NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg 117 in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleB CR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)