Search

• Citrobacter rodentium is a key physiological model for studying A/E pathogenesis. • C. rodentium encodes effectors that subvert host cell death signaling. • C. rodentium modulates apoptosis to block neutrophil and Th17 cell recruitment. • RIPK3-dependent necroptosis limits C. rodentium persistence in host colon. • NLRP3 and NLRC4-mediated secretion of IL-18 impairs C. rodentium colonisation. 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. [ABSTRACT FROM AUTHOR]

Pathogenic microbes have evolved countless sophisticated mechanisms to subvert host immune responses and cause disease. Understanding evasion strategies employed by pathogens has led to numerous discoveries on specific host cell processes that are critical for controlling infection. Programmed cell death (PCD) is a key host defence to microbial infection, as well as being critical for organ development and cellular homeostasis in multicellular organisms. Much of our current understanding of PCD as a host response to infection has stemmed from the discovery and study of viral inhibitors of apoptosis, and more recently viral inhibition of the newly characterised from of PCD termed necroptosis, the mechanisms of which are still under intense investigation. Many bacterial pathogens also encode inhibitors of PCD, yet these discoveries are relatively more recent and thus the biological significance of such mechanisms is still under debate. In this viewpoint article, we will argue the concept that necroptosis is merely a 'back-up' mechanism in the event that apoptosis is inhibited, or whether it is a true host innate response to infection that has evolved in response to a growing arsenal of microbial evasion strategies. [ABSTRACT FROM AUTHOR]

In many parts of the world, enteropathogenic Escherichia coli (EPEC) are a leading cause of death in children with diarrhea. Much of what we know about the pathogenesis of EPEC infections is based on the study of one or two prototypic strains that have provided deep insight into the precise mechanisms by which EPEC colonizes the intestine, evades host immunity, and spreads from person to person. In some cases, defining the biochemical activity of the host-interacting effector proteins from these prototypic strains has led to the discovery of novel post-translational protein modifications and new understandings of biology and host-pathogen interactions. However, genomic analysis of recent EPEC isolates has revealed that the EPEC pathotype is more diverse than previously appreciated. Although by definition all strains carry the locus of enterocyte effacement, the effector repertoires of different clonal groups are quite divergent, suggesting that there is still a great deal to learn about the genetic basis of EPEC virulence. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens have the ability to manipulate cellular processes and interfere with host cell function through the translocation of bacterial ‘effector’ proteins. Dedicated protein secretion machines from Gram-negative pathogens, including type III, type IV and type VI secretion systems, inject virulence proteins into infected cells, altering normal cell physiology, including cell structure, metabolism, trafficking and signalling. While effectors were once thought to exert an effect simply by their localization and binding to host cell proteins, increasingly effectors are being recognised as enzymes, in some cases mediating highly novel post-translational modifications on host proteins. Here we highlight some of the more unusual activities of translocated effectors from enteropathogenic Escherichia coli and Legionella pneumophila . [ABSTRACT FROM AUTHOR]

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic E scherichia coli ( EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system ( T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor-induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection. [ABSTRACT FROM AUTHOR]

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. [ABSTRACT FROM AUTHOR]

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP-NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His-NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-κB, to the host cell nucleus. NF-κB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-κB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFa and IL-1β stimulated p65 nuclear translocation and IkB degradation, NleB inhibited the TNFa pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Synopsis: XIAP deficiency causes hemophagocytic lymphohistiocytosis and Crohn's disease. Analysis of XIAP‐deficient patients and cellular studies show that apoptotic caspases and GSDMD act redundantly downstream of caspase‐8 to cause excess cell death and IL‐1β release. XIAP deficiency increases cleaved caspase‐8 and GSDMD in mucosal tissue and cellsCrosstalk in apoptosis and pyroptosis sensitises cells to caspase‐8‐driven death and IL‐1β activation upon XIAP inhibitionPannexin‐1 and GSDMD are dispensable for NLRP3 inflammasome formation downstream of caspase‐8Pannexin‐1 cleavage by caspase‐3 and caspase‐7 is required for efficient NLRP3 activation following intrinsic apoptosis [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents a study which explores that Tankyrase-mediated ADP-ribosylation, a regulator of Tumor necrosis factor (TNF)-induced death . It mentions that findings of the study suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

The article presents a study which explores about the Tankyrase-mediated ADP-ribosylation as a regulator of Tumor necrosis factor (TNF)-induced death. It mentions that TNF, a key component of the innate immune response and suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Abstract: Gastrointestinal bacterial pathogens such as enteropathogenic Escherichia coli, Salmonella and Shigella control inflammatory and apoptotic signaling in human intestinal cells to establish infection, replicate and disseminate to other hosts. These pathogens manipulate host cell signaling through the translocation of virulence effector proteins directly into the host cell cytoplasm, which then target various signaling pathways. Death receptors such as TNFR1, FAS and TRAIL-R induce signaling cascades that are crucial to the clearance of pathogens, and as such are major targets for inhibition by pathogens. This review focuses on what is known about how bacterial gut pathogens inhibit death receptor signaling to suppress inflammation and prevent apoptosis. [Copyright &y& Elsevier]

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. Author summary: Bacterial gut pathogens including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), manipulate host cell function by using a type III secretion system to inject 'effector' proteins directly into the host cell cytoplasm. We and others have shown that many of these effectors are novel enzymes, including NleB1, which transfers a single N-acetylglucosamine (GlcNAc) sugar to arginine residues, mediating Arg-GlcNAc glycosylation. Here, we found that a close homologue of NleB1 that is also present in EPEC and EHEC termed NleB2, uses a different sugar during glycosylation. We demonstrated that in contrast to NleB1, the preferred nucleotide-sugar substrate of NleB2 is UDP-glucose and we identified the amino acid residue within NleB2 that dictates this unique catalytic activity. Substitution of this residue in NleB2 and NleB1 switches the sugar donor usage of these enzymes but does not affect their ability to inhibit host cell signalling. Thus, NleB2 is the first identified bacterial arginine-glucose transferase, an activity which has previously only been described in plants and algae. [ABSTRACT FROM AUTHOR]

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. • MNV infection of murine macrophages induces morphological changes associated with cell death. • Chemical perturbation and cell lines deficient in pyroptosis or necroptosis pathways do not affect MNV replication. • Infection of murine macrophages with MNV results in the activation of pro-apoptotic proteins. • Induction of apoptosis is independent of caspase-8 indicating a role for intrinsic apoptosis. • Inhibition of caspases or depletion of a range of caspases results in prolonged cell survival and reduced MNV production. [ABSTRACT FROM AUTHOR]

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 Citrobacterrodentium 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. [ABSTRACT FROM AUTHOR]

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 (NleBCR 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 Arg117 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/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner. [ABSTRACT FROM AUTHOR]

The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif 49GITR52, was critical for TAB2 and TAB3 binding. NleE mutants lacking 49GITR52 were unable to methylate TAB3, and wild type NleE but not NleE49AAAA52 where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the 49GITR52 motif. Ectopic expression of an N-terminal fragment of NleE (NleE34-52) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the 49GITR52 motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response. [ABSTRACT FROM AUTHOR]

Upon infection of epithelial cells, enteropathogenic E scherichia coli suppresses host cell inflammatory signalling in a type III secretion system ( T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF- κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain ( RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector. [ABSTRACT FROM AUTHOR]

OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between -731 and -412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirB-dependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression. [ABSTRACT FROM AUTHOR]

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. • Mice lacking pyroptosis and apoptosis cannot control Salmonella infection • Macrophages lacking pyroptosis and apoptosis resist Salmonella -induced killing • Caspase-1 kills Salmonella -infected cells by activating GSDMD, BID, or other caspases • Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost The clearance of intracellular pathogens requires the killing of infected cells, but it remains unclear why host cells have so many different means of inducing programmed cell death. Doerflinger et al. demonstrate that interconnectivity between pyroptosis and apoptosis involving flexible deployment of caspases ensures control of Salmonella infection in mice. [ABSTRACT FROM AUTHOR]