Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

BRCA1 and p53 tumor suppressor molecules in Alzheimer's disease.

Tumor suppressor molecules play a pivotal role in regulating DNA repair, cell proliferation, and cell death, which are also important processes in the pathogenesis of Alzheimer's disease. Alzheimer's disease is the most common neurodegenerative disorder, however, the precise molecular events that control the death of neuronal cells are unclear. Recently, a fundamental role for tumor suppressor molecules in regulating neurons in Alzheimer's disease was highlighted. Generally, onset of neurodegenerative diseases including Alzheimer's disease may be delayed with use of dietary neuro-protective agents against oxidative stresses. Studies suggest that dietary antioxidants are also beneficial for brain health in reducing disease-risk and in slowing down disease-progression. We summarize research advances in dietary regulation for the treatment of Alzheimer's disease with a focus on its modulatory roles in BRCA1 and p53 tumor suppressor expression, in support of further therapeutic research in this field.

Cutting edge: Crohn's disease-associated Nod2 mutation limits production of proinflammatory cytokines to protect the host from Enterococcus faecalis-induced lethality.

Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohn's disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod2(2939iCstop), the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod2(2939iCstop) are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod2(2939iCstop) were associated with reduced production of TNF-α and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-α signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.

Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis.

Caspase-1 is activated by a variety of stimuli after the assembly of the "inflammasome," an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1beta and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin.

Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.

IL-13 attenuates early local CXCL2-dependent neutrophil recruitment for Candida albicans clearance during a severe murine systemic infection.

To investigate the role of IL-13 during a severe systemic Candida albicans infection, BALB/c control and IL-13-/- mice were examined for colony forming units (CFU) in the kidneys and survival days after intravenous infection. Proinflammatory mediators and cell recruitment into the tissue were measured by quantitative real-time PCR, a multiple ELISA system, and morphological cell differentiation. The IL-13-/- group exhibited a lower CFU number in the kidneys at 4 days and survived longer than the control mice, which was accompanied by significantly higher expression of C-X-C motif ligand 2 (CXCL2), IFN-γ, and polymorphonuclear neutrophils (PMNs) in the infected kidneys. By contrast, the expression of transforming growth factor ß (TGF-ß) and IL-17 A on day 10 were significantly higher in the control mice than in the IL-13-/- group. When using an intratracheal infection model, the IL-13-/- group recruited a greater number of PMNs in 6 h, with rapidly increased CXCL2 in the alveolar space. In vitro testing with cultured bone-marrow-derived cells demonstrated rapid CXCL2 mRNA upregulation at 3 h after contact with C. albicans, which decreased with recombinant IL-13 pretreatment, whereas rIL-13 retained TGF-ß upregulation. In a murine model of Candida systemic infection, preexistent IL-13 limits both the rapid CXCL2 elevation and PMN aggregation in the target organ to suppress inflammatory mediators, which also attenuates local pathogen clearance within four days.

The inflammasome in pathogen recognition and inflammation.

The nucleotide-binding oligomerization domain-like receptor (NLR) family of proteins is involved in the regulation of innate immune responses and cell death pathways. Some NLR family members promote the activation of proinflammatory caspases within multiprotein complexes, called inflammasomes. Recent studies analyzing mice deficient in various components of the inflammasome have provided insight into the role of these molecules in host defense against pathogens and in autoinflammatory disorders. Here, we review these studies and propose that membrane disruption leads to activation of the inflammasome.

MDP-induced interleukin-1beta processing requires Nod2 and CIAS1/NALP3.

Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.

NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.

Epigallocatechin gallate inhibits the type III secretion system of Gram-negative enteropathogenic bacteria under model conditions.

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, inhibits the type III secretion system (T3SS) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Salmonella enterica serovar Typhimurium, and Yersinia pseudotuberculosis. The inhibitory effect causes the inhibition of hemolysis, cell invasion, cell adhesion and apoptosis, which are functions of the type III secretion device. In the case of EPEC, EspB accumulates in the cells. RT-PCR showed that the translation of EspB was not blocked. The transcription of escN, which supplies energy for the injection of the effector factor into the host cells, was also not inhibited. EGCG does not suppress the transcription and translation of T3SS constitutive protein in bacterial cells, but it seems to suppress the normal construction or secretion of T3SS. When Luria-Bertani (LB) medium was used to visualize the EGCG-induced inhibition of T3SS, the inhibitory effect disappeared. The inhibition of T3SS was partially canceled when the T3SS inhibitory potency of EGCG was examined by adding yeast extract, which is a component of LB medium, to DMEM. These results suggest that EGCG probably inhibits secretion by suppressing some metabolic mechanisms of T3SS.

NALP3: a key player in caspase-1 activation.

The NLR (NACHT-LRR) family of proteins have been implicated in the regulation of immune responses and cell death pathways. Some NLR family members can form multiprotein complexes, called inflammasomes, involved in the activation of pro-inflammatory caspases. Mutations in the NALP3/CIAS1/cryopyrin gene, a member of the NLR family, are linked to three auto-inflammatory disorders: Muckle-Wells syndrome, familial cold auto-inflammatory syndrome and neonatal-onset multisystem inflammatory disease. NALP3 along with the adaptor molecule ASC activates caspase-1 in response to a wide variety of stimuli. Here we review recent findings on the biology of NALP3 suggesting that it has functions beyond that of pathogen recognition.

Nods: a family of cytosolic proteins that regulate the host response to pathogens.

Genetic variation in Nod2 is associated with susceptibility to Crohn's disease. Nod2 and its homologue, Nod1, are members of a growing family of cytosolic factors related to the apoptosis regulator Apaf-1 and a class of plant disease resistance proteins. Nod1 and Nod2 confer responsiveness to lipopolysaccharides and interact with RICK, a mediator of NF-kappaB activation. Nod1 and Nod2 and related Nods appear to regulate the host response to pathogens, a process that may be faulty in certain inflammatory diseases. Recent studies that suggest that Nods may be involved in the recognition of pathogen components in the cytosol of mammalian cells are reviewed.

Function of α-synuclein and PINK1 in Lewy body dementia (Review).

α-Synuclein (α-syn) is the major protein component of Lewy bodies, a key pathological characteristic of the degenerating brain. The misfolding and aggregation of α-syn is associated with both the idiopathic and familial forms of Parkinson's disease (PD) and Lewy body dementia (LBD). However, the function of α-syn is poorly understood, as it shows both neurotoxic and neuroprotective activities. Mutations in phosphatase and tensin homologue-induced putative kinase 1 (PINK1) also cause recessively inherited PD. Studies support the notion of neuroprotective roles for PINK1, as it protects cells from damage-induced mitochondrial dysfunction, oxidative stress and cell apoptosis. PINK1 plays an essential role in mitochondrial quality control and its homeostasis is maintained through mitochondrial stabilization. The α-syn aggregation is linked to various aspects of mitochondrial dysfunction and PINK1-related mitophagy. Determination of the molecular pathways that lead to α-syn oligomerization and further aggregation may be the basis for the successful design and development of treatments for these neurodegenerative diseases. The present review summarizes the function of PINK1 underlying α-syn aggregation and the mechanisms through which mitochondrial dysfunction plays a role in this process.

PI3K/AKT/PTEN pathway as a target for Crohn's disease therapy (Review).

The pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease, is a subject of increasing interest. Loss-of-function mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) are strong genetic factors linked to Crohn's disease, which eventually leads to an excessive mucosal inflammatory response directed against components of normal gut microbiota. Reactive oxygen species (ROS) play an important role in inflammation processes, as well as in transduction of signals from receptors for several cytokines, such as tumor necrosis factor α (TNFα). ROS activate nuclear factor-κB (NF-κB) via IκB kinase (IKK) through the PI3K/AKT/PTEN pathway. Therefore, this pathway is recognized to play a key role in Crohn's disease. Loss of function has been demonstrated to occur as an early event in a wide variety of diseases. Given this prevalent involvement in a number of diseases, the molecular development that modulates this pathway has been the subject of several studies. In addition, it has been the focus of extensive research and drug discovery activities. A better understanding of the molecular assemblies may reveal novel targets for the therapeutic development against Crohn's disease.

Dietary regulation of PI3K/AKT/GSK-3ß pathway in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the formation of senile plaques and neurofibrillary tangles composed of phosphorylated Tau. Several findings suggest that correcting signal dysregulation for Tau phosphorylation in AD may offer a potential therapeutic approach. The PI3K/AKT/GSK-3ß pathway has been shown to play a pivotal role in neuroprotection, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. This pathway appears to be crucial in AD because it promotes protein hyper-phosphorylation in Tau. Understanding those regulations may provide a better efficacy of new therapeutic approaches. In this review, we summarize advances in the involvement of the PI3K/AKT/GSK-3ß pathways in cell signaling of neuronal cells. We also review recent studies on the features of several diets and the signaling pathway involved in AD.

The tumor suppressor PTEN interacts with p53 in hereditary cancer (Review).

Numerous hereditary syndromes caused by mutations in multiple tumor suppressor genes can cause cancers. Germline mutations in PTEN and p53 tumor suppressor cause Cowden syndrome and Li-Fraumeni syndrome, respectively. There exists some phenotypic overlap in these syndromes, and they are associated with high risks of breast cancer. The tumor suppressor protein PTEN is a dual-specificity phosphatase which has protein phosphatase activity and lipid phosphatase activity that antagonizes PI3K activity. Cells that lack PTEN have constitutively higher levels of PIP3 and activated downstream targets. PTEN gene is recognized as one of the most frequently mutated or mutated in many human cancers. Li-Fraumeni syndrome results from germline mutations of the tumor suppressor p53 gene encoding a transcriptional factor able to regulate cell cycle and apoptosis when DNA damage occurs. The p53 protein cooperates with PTEN and might be an essential blockage in development of mammary tumors. Many findings have demonstrated that PTEN as well as p53 plays a critical role in DNA damage response. This review summarizes the function of PTEN and p53 in carcinogenic cell signaling. In addition, we will discuss the role of PTEN signaling through its interaction with p53 and MDM2 pathways for the potential implications in hereditary cancer prevention and therapeutic intervention.

Connection between Tumor Suppressor BRCA1 and PTEN in Damaged DNA Repair.

Genomic instability finally induces cell death or apoptosis. The tumor suppressor, phosphatase and tensin homolog on chromosome 10 (PTEN), is a dual-specificity phosphatase, which has protein phosphatase activity and lipid phosphatase activity that antagonizes PI3K activity. Cells that lack PTEN have constitutively higher levels of PIP3 and activated downstream PI3K/AKT targets. BRCA1, a well-known breast cancer tumor suppressor, is to associate with breast cancer risk and genetic susceptibility. Many studies have demonstrated that PTEN, as well as BRCA1, plays a critical role in DNA damage responses. The BRCA1 functionally cooperates with PTEN and might be an essential blockage in the development of several tumors. Actually, the PTEN and BRCA1 genes are recognized as one of the most frequently deleted and/or mutated in many human cancers. The PI3K/AKT pathway is constitutively active in BRCA1-defective human cancer cells. Loss or decrease of these PTEN or BRCA1 function, by either mutation or reduced expression, has a role in various tumor developments. This review summarizes recent findings of the function of BRCA1 and PTEN involved in genomic stability and cancer cell signaling.