Chihiro Sasakawa

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1β processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial–host interactions.

Grb2 Is a Key Mediator of Helicobacter pylori CagA Protein Activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.

Listeria monocytogenes ActA-mediated escape from autophagic recognition

Autophagy degrades unnecessary organelles and misfolded protein aggregates, as well as cytoplasm-invading bacteria. Nevertheless, the bacteria Listeria monocytogenes efficiently escapes autophagy. We show here that recruitment of the Arp2/3 complex and Ena/VASP, via the bacterial ActA protein, to the bacterial surface disguises the bacteria from autophagic recognition, an activity that is independent of the ability to mediate bacterial motility. L. monocytogenes expressing ActA mutants that lack the ability to recruit the host proteins initially underwent ubiquitylation, followed by recruitment of p62 (also known as SQSTM1) and LC3, before finally undergoing autophagy. The ability of ActA to mediate protection from ubiquitylation was further demonstrated by generating aggregate-prone GFP–ActA–Q79C and GFP–ActA–170* chimaeras, consisting of GFP (green fluorescent protein), the ActA protein and segments of polyQ or Golgi membrane protein GCP170 (ref. 6). GFP–ActA–Q79C and GFP–ActA–170* formed aggregates in the host cell cytoplasm, however, these ActA-containing aggregates were not targeted for association with ubiquitin and p62. Our findings indicate that ActA-mediated host protein recruitment is a unique bacterial disguise tactic to escape from autophagy.

Innate lymphoid cells regulate intestinal epithelial cell glycosylation

INTRODUCTION The combination of food intake and the resident gut microbiota exposes the gastrointestinal (GI) tract to numerous antigens. Intestinal epithelial cells (ECs) compose a physical barrier separating the internal organs from the gut microbiota and other pathogenic microorganisms entering the GI tract. Although anatomically contained, the gut microbiota is essential for developing appropriate host immunity. Thus, the mucosal immune system must simultaneously maintain homeostasis with the gut microbiota and protect against infection by pathogens. Maintenance of the gut microbiota requires epithelial cell-surface glycosylation, with fucose residues in particular. Epithelial fucosylation is mediated by the enzyme fucosyltransferase 2 (Fut2). Polymorphisms in the FUT2 gene are associated with the onset of multiple infectious and inflammatory diseases and metabolic syndrome in humans. ILC3s regulate epithelial glycosylation. Commensal bacteria, including segmented filamentous bactiera (SFB), induce IL-22 production by ILC3. LT is produced by ILC3 in a commensal bacteria–independent manner. ILC3-derived IL-22 and LT cooperatively induce the production of Fut2 and subsequent epithelial fucosylation, which protects the host against Salmonella typhimurium infection. RATIONALE Despite its importance, the mechanisms underlying epithelial fucosylation in the GI tract is not well understood. In particular, although commensals such as Bacteroides thetaiotaomicron induce epithelial fucosylation, how mucosal immune cells participate in this process is unknown. We used a combination of bacteriological, gnotobiological, and immunological techniques to elucidate the cellular and molecular basis of epithelial fucosylation by mucosal immune cells in mice, especially innate lymphoid cells (ILCs). To explore the role of ILCs in the induction and maintenance of epithelial fucosylation, we used genetically engineered mice lacking genes associated with the development and function of ILCs. To investigate the physiological functions of ILC-induced epithelial fucosylation, we used a Fut2-deficient mouse model of S. typhimurium infection. RESULTS The induction and maintenance of Fut2 expression and subsequent epithelial fucosylation in the GI tract required type 3 ILCs (ILC3s) that express the transcription factor RORγt and the cytokines interleukin-22 (IL-22) and lymphotoxin (LT). Commensal bacteria, including segmented filamentous bacteria (SFB), induced fucosylation of intestinal columnar ECs and goblet cells. Expression of IL-22 by ILC3 required commensal bacteria, whereas LT was expressed in a commensal-independent manner. Ablation of IL-22 or LT in ILC3 resulted in a marked reduction in epithelial fucosylation, demonstrating that both cytokines are critical for the induction and regulation of epithelial fucosylation. Fucosylation of ECs in response to the intestinal pathogen S. typhimurium was also mediated by ILC3. Compared with control mice, Fut2-deficient mice were more susceptible to pathogenic inflammation as a result of S. typhimurium infection, suggesting that epithelial fucosylation contributes to host defense against S. typhimurium infection. CONCLUSION We demonstrate the critical role of the cytokines IL-22– and/or LT-producing ILC3 in the induction and regulation of intestinal epithelial fucosylation. We also show that ILC3-mediated epithelial fucosylation protects the host from invasion of S. typhimurium into the intestine. Our results provide important details of the glycosylation system and homeostatic responses created by the trilateral ILC3–EC–commensal axis in the intestine. Modulation of mucosal immune cell–mediated epithelial glycosylation may provide novel targets for the treatment or prevention of infectious diseases in humans. Immune cells and bugs make a sugary coat Epithelial cells line the intestinal tract and help to keep the peace between our immune system and our trillions of gut microbes. Such peacekeeping requires glycosylated proteins (proteins with attached carbohydrate chains) present on the epithelial cell surface, but how glycosylation occurs is unclear. Goto et al. find that fucosylation (a type of glycosylation) of gut epithelial cells in mice requires gut microbes (see the Perspective by Hooper). This process also requires innate lymphoid cells there, which produce the cytokines interleukin-22 and lymphotoxin, presumably in response to microbial signals. These cytokines signal epithelial cells to add fucose to membrane proteins, which allows the détente between microbes and immune cells to continue. Science, this issue 10.1126/science.1254009; see also p. 1248 Glycosylation of gut epithelial cells requires gut microbes, innate lymphoid cells, and cytokines. [Also see Perspective by Hooper] Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host–microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria–dependent and –independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

Helicobacter pylori CagA Phosphorylation-Independent Function in Epithelial Proliferation and Inflammation

CagA, a major virulence factor of Helicobacter pylori (Hp), is delivered into gastric epithelial cells and exists in phosphorylated and nonphosphorylated forms. The biological activity of the phosphorylated form is well established; however, function(s) of the nonphosphorylated form remain elusive. Here, we report that a conserved motif in the C-terminal region of CagA, which is distinct from the EPIYA motifs used for phosphorylation and which we designate CRPIA (conserved repeat responsible for phosphorylation-independent activity), plays pivotal roles in Hp pathogenesis. The CRPIA motif in nonphosphorylated CagA was involved in interacting with activated Met, the hepatocyte growth factor receptor, leading to the sustained activation of phosphatidylinositol 3-kinase/Akt signaling in response to Hp infection. This in turn led to the activation of beta-catenin and NF-kappaB signaling, which promote proliferation and inflammation, respectively. Thus, nonphosphorylated CagA activity contributes to the epithelial proliferative and proinflammatory responses associated with development of chronic gastritis and gastric cancer.

EspG, a Novel Type III System-Secreted Protein from Enteropathogenic Escherichia coli with Similarities to VirA of Shigella flexneri

ABSTRACT The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenicEscherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous withShigella flexneri protein VirA, and the clonedespG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPECespG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The clonedorf3 gene could also rescue invasion in aShigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

Molecular mechanisms regulating NLRP3 inflammasome activation

Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome.

Bacteria and host interactions in the gut epithelial barrier

The gut mucosa acts as a barrier against microbial invaders, whereas resident commensal and foreign invading bacteria interact intimately with the gut epithelium and influence the host cellular and immune systems. The epithelial barrier serves as an infectious foothold for many bacterial pathogens and as an entry port for pathogens to disseminate into deeper tissues. Enteric bacterial pathogens can efficiently infect the gut mucosa using highly sophisticated virulence mechanisms that allow bacteria to circumvent the defense barriers in the gut. We provide an overview of the components of the mucosal barrier and discuss the bacterial stratagems that circumvent these barriers with particular emphasis on the roles of bacterial effector proteins.

The versatility of Shigella effectors

When Shigella infect the intestinal epithelium, they deliver several effectors through the type III secretion system (T3SS) into the surrounding space and directly into the host-cell cytoplasm, where they can mimic and usurp host cellular functions or subvert host-cell signalling pathways and the immune response. Although bacterial strategies and mechanisms of infection vary greatly, recent studies of Shigella effectors have revealed that Shigella possess a highly evolved strategy for infection.

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1.

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule‐bound GEF‐H1, a RhoA‐specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG‐ and Orf3‐induced stress fiber formation was inhibited by the expression of dominant‐negative forms of GEF‐H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA–ROCK signaling pathway via GEF‐H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF‐H1.