Structure and biophysics of type III secretion in bacteria.

PubMed

Chatterjee, Srirupa; Chaudhury, Sukanya; McShan, Andrew C; Kaur, Kawaljit; De Guzman, Roberto N

2013-04-16

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

PubMed Central

Molina, Judith; Rodriguez-Diaz, Rayner; Fachado, Alberto; Jacques-Silva, M. Caroline

2014-01-01

Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting β-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates β-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to β-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to β-cells to regulate insulin secretion from the human islet. PMID:24658304

Symbiotic implications of type III protein secretion machinery in Rhizobium.

PubMed

Viprey, V; Del Greco, A; Golinowski, W; Broughton, W J; Perret, X

1998-06-01

The symbiotic plasmid of Rhizobium sp. NGR234 carries a cluster of genes that encodes components of a bacterial type III secretion system (TTSS). In both animal and plant pathogens, the TTSS is an essential component of pathogenicity. Here, we show that secretion of at least two proteins (y4xL and NolX) is controlled by the TTSS of NGR234 and occurs after the induction with flavonoids. Polar mutations in two TTSS genes, rhcN and the nod-box controlled regulator of transcription y4xl, block the secretion of both proteins and strongly affect the ability of NGR234 to nodulate a variety of tropical legumes including Pachyrhizus tuberosus and Tephrosia vogelii.

Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234.

PubMed

Marie, Corinne; Deakin, William J; Viprey, Virginie; Kopciñska, Joanna; Golinowski, Wladyslaw; Krishnan, Hari B; Perret, Xavier; Broughton, William J

2003-09-01

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

PubMed Central

Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U.

2015-01-01

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

USDA-ARS?s Scientific Manuscript database

The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kjaerulff, Soren; Jensen, Martin Roland

2005-10-28

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal didmore » not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.« less

Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phan, Jason; Tropea, Joseph E.; Waugh, David S.

2010-11-16

Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure ofmore » a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.« less

Impact of Type III Secretion Effectors and of Phenoxyacetamide Inhibitors of Type III Secretion on Abscess Formation in a Mouse Model of Pseudomonas aeruginosa Infection

PubMed Central

Berube, Bryan J.; Murphy, Katherine R.; Torhan, Matthew C.; Bowlin, Nicholas O.; Williams, John D.; Bowlin, Terry L.; Moir, Donald T.

2017-01-01

ABSTRACT Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro. In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection. PMID:28807906

Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon

DOE PAGES

Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.; ...

2014-12-26

In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less

Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.

In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less

Central Nervous System Neuropeptide Y Signaling Modulates VLDL Triglyceride Secretion

PubMed Central

Stafford, John M.; Yu, Fang; Printz, Richard; Hasty, Alyssa H.; Swift, Larry L.; Niswender, Kevin D.

2014-01-01

OBJECTIVE Elevated triglyceride (TG) is the major plasma lipid abnormality in obese and diabetic patients and contributes to cardiovascular morbidity in these disorders. We sought to identify novel mechanisms leading to hypertriglyceridemia. Resistance to negative feedback signals from adipose tissue in key central nervous system (CNS) energy homeostatic circuits contributes to the development of obesity. Because triglycerides both represent the largest energy depot in the body and are elevated in both the plasma and adipose in obesity and diabetes, we hypothesized that the same neural circuits that regulate energy balance also regulate the secretion of TGs into plasma. RESEARCH DESIGN AND METHODS In normal fasting rats, the TG secretion rate was estimated by serial blood sampling after intravascular tyloxapol pretreatment. Neuropeptide Y (NPY) signaling in the CNS was modulated by intracerebroventricular injection of NPY, receptor antagonist, and receptor agonist. RESULTS A single intracerebroventricular injection of NPY increased TG secretion by 2.5-fold in the absence of food intake, and this was determined to be VLDL by fast performance liquid chromatography (FPLC). This effect was recapitulated by activating NPY signaling in downstream neurons with an NPY-Y5 receptor agonist. An NPY-Y1 receptor antagonist decreased the elevated TGs in the form of VLDL secretion rate by 50% compared with vehicle. Increased TG secretion was due to increased secretion of VLDL particles, rather than secretion of larger particles, because apolipoprotein B100 was elevated in FPLC fractions corresponding to VLDL. CONCLUSIONS We find that a key neuropeptide system involved in energy homeostasis in the CNS exerts control over VLDL-TG secretion into the bloodstream. PMID:18332095

Preprotein mature domains contain translocase targeting signals that are essential for secretion.

PubMed

Chatzi, Katerina E; Sardis, Marios Frantzeskos; Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G; Karamanou, Spyridoula; Economou, Anastassios

2017-05-01

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as pre proteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. © 2017 Chatzi et al.

Preprotein mature domains contain translocase targeting signals that are essential for secretion

PubMed Central

Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G.; Karamanou, Spyridoula

2017-01-01

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. PMID:28404644

Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

PubMed

Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

2015-01-01

Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

Visualization and characterization of individual type III protein secretion machines in live bacteria

PubMed Central

Lara-Tejero, María; Bewersdorf, Jörg; Galán, Jorge E.

2017-01-01

Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine’s assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines. PMID:28533372

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Bile salt receptor complex activates a pathogenic type III secretion system

DOE PAGES

Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; ...

2016-07-05

Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure ofmore » VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.« less

Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

PubMed Central

Espina, Marianela; Ausar, S. Fernando; Middaugh, C. Russell; Baxter, M. Aaron; Picking, William D.; Picking, Wendy L.

2007-01-01

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are α-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins. PMID:17327391

Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

PubMed

Houtz, Jessica; Borden, Philip; Ceasrine, Alexis; Minichiello, Liliana; Kuruvilla, Rejji

2016-11-07

Insulin secretion by pancreatic islet β cells is critical for glucose homeostasis, and a blunted β cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet β cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate β cell dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

What's the point of the type III secretion system needle?

PubMed Central

Blocker, Ariel J.; Deane, Janet E.; Veenendaal, Andreas K. J.; Roversi, Pietro; Hodgkinson, Julie L.; Johnson, Steven; Lea, Susan M.

2008-01-01

Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed. PMID:18458349

Expression of and secretion through the Aeromonas salmonicida type III secretion system.

PubMed

Ebanks, Roger O; Knickle, Leah C; Goguen, Michel; Boyd, Jessica M; Pinto, Devanand M; Reith, Michael; Ross, Neil W

2006-05-01

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

Suppression of bacterial cell-cell signalling, biofilm formation and type III secretion system by citrus flavonoids.

PubMed

Vikram, A; Jayaprakasha, G K; Jesudhasan, P R; Pillai, S D; Patil, B S

2010-08-01

This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell-cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell-cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. The results of the study indicate a potential modulation of bacterial cell-cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell-cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell-cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.

Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC.

PubMed

Akeda, Yukihiro; Kodama, Toshio; Saito, Kazunobu; Iida, Tetsuya; Oishi, Kazunori; Honda, Takeshi

2011-11-01

The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Harnessing Novel Secreted Inhibitors of EGF Receptor Signaling for Breast Cancer Treatment

DTIC Science & Technology

2008-04-01

infected Spodoptera frugiperda Sf9 cells, using the amino-terminal BiP signal sequence to direct 9 secretion of the protein into the medium. The...for crystallization of the Argos/Spitz complex was produced by secretion from Sf9 ( Spodoptera frugiperda ) cells using the Bac- to-Bac baculovirus

A Repulsive Electrostatic Mechanism for Protein Export through the Type III Secretion Apparatus

PubMed Central

Rathinavelan, Thenmalarchelvi; Zhang, Lingling; Picking, Wendy L.; Weis, David D.; De Guzman, Roberto N.; Im, Wonpil

2010-01-01

Abstract Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus, which is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. The trajectories reveal a screwlike rotation motion during the export of nativelike helix-turn-helix conformations. Interestingly, the channel interior with excessive electronegative potential creates an energy barrier for MxiH to enter the channel, whereas the same may facilitate the ejection of the effectors into host cells. Structurally known basal regions and ATPase underneath the basal region also have electronegative interiors. Effector proteins also have considerable electronegative potential patches on their surfaces. From these observations, we propose a repulsive electrostatic mechanism for protein translocation through the type III secretion apparatus. Based on this mechanism, the ATPase activity and/or proton motive force could be used to energize the protein translocation through these nanomachines. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported. PMID:20141759

IL-6-Type Cytokine Signaling in Adipocytes Induces Intestinal GLP-1 Secretion.

PubMed

Wueest, Stephan; Laesser, Céline I; Böni-Schnetzler, Marianne; Item, Flurin; Lucchini, Fabrizio C; Borsigova, Marcela; Müller, Werner; Donath, Marc Y; Konrad, Daniel

2018-01-01

We recently showed that interleukin (IL)-6-type cytokine signaling in adipocytes induces free fatty acid release from visceral adipocytes, thereby promoting obesity-induced hepatic insulin resistance and steatosis. In addition, IL-6-type cytokines may increase the release of leptin from adipocytes and by those means induce glucagon-like peptide 1 (GLP-1) secretion. We thus hypothesized that IL-6-type cytokine signaling in adipocytes may regulate insulin secretion. To this end, mice with adipocyte-specific knockout of gp130, the signal transducer protein of IL-6, were fed a high-fat diet for 12 weeks. Compared with control littermates, knockout mice showed impaired glucose tolerance and circulating leptin, GLP-1, and insulin levels were reduced. In line, leptin release from isolated adipocytes was reduced, and intestinal proprotein convertase subtilisin/kexin type 1 ( Pcsk1 ) expression, the gene encoding PC1/3, which controls GLP-1 production, was decreased in knockout mice. Importantly, treatment with the GLP-1 receptor antagonist exendin 9-39 abolished the observed difference in glucose tolerance between control and knockout mice. Ex vivo, supernatant collected from isolated adipocytes of gp130 knockout mice blunted Pcsk1 expression and GLP-1 release from GLUTag cells. In contrast, glucose- and GLP-1-stimulated insulin secretion was not affected in islets of knockout mice. In conclusion, adipocyte-specific IL-6 signaling induces intestinal GLP-1 release to enhance insulin secretion, thereby counteracting insulin resistance in obesity. © 2017 by the American Diabetes Association.

Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

PubMed

Diepold, Andreas; Kudryashev, Mikhail; Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P

2015-01-01

Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors.

PubMed

Bliska, James B; Wang, Xiaoying; Viboud, Gloria I; Brodsky, Igor E

2013-10-01

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called 'pathogen-associated molecular patterns' (PAMPs). Pathogens use virulence factors to counteract PAMP-directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP-directed responses and are critical for infection. A plasmid-encoded T3SS in the human-pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences. © 2013 John Wiley & Sons Ltd.

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways

PubMed Central

Lippestad, Marit; Hodges, Robin R.; Utheim, Tor P.; Serhan, Charles N.; Dartt, Darlene A.

2017-01-01

Purpose Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens. PMID:28892824

Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton.

PubMed

Veenendaal, Andreas K J; Sundin, Charlotta; Blocker, Ariel J

2009-01-01

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

PubMed Central

2013-01-01

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

PubMed

Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

2013-05-09

Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII

Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma.

PubMed

Bullen, Hayley E; Jia, Yonggen; Yamaryo-Botté, Yoshiki; Bisio, Hugo; Zhang, Ou; Jemelin, Natacha Klages; Marq, Jean-Baptiste; Carruthers, Vern; Botté, Cyrille Y; Soldati-Favre, Dominique

2016-03-09

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Yersinia Type III Secretion System Master Regulator LcrF

PubMed Central

Schwiesow, Leah; Lam, Hanh

2015-01-01

Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion.

PubMed

Wulff-Strobel, Christine R; Williams, Andrew W; Straley, Susan C

2002-01-01

LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ's effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ's effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.

Phylogenetic and functional signals in gymnosperm ovular secretions.

PubMed

Nepi, Massimo; Little, Stefan; Guarnieri, Massimo; Nocentini, Daniele; Prior, Natalie; Gill, Julia; Barry Tomlinson, P; Ickert-Bond, Stefanie M; Pirone, Cary; Pacini, Ettore; von Aderkas, Patrick

2017-11-28

Gymnosperms are either wind-pollinated (anemophilous) or both wind- and insect-pollinated (ambophilous). Regardless of pollination mode, ovular secretions play a key role in pollen capture, germination and growth; they are likely also involved in pollinator reward. Little is known about the broad-scale diversity of ovular secretions across gymnosperms, and how these may relate to various reproductive functions. This study analyses the sugar and amino acid profiles of ovular secretions across a range of ambophilous (cycads and Gnetales) and anemophilous gymnosperms (conifers) to place them in an evolutionary context of their possible functions during reproduction. Ovular secretions from 13 species representing all five main lineages of extant gymnosperms were sampled. High-performance liquid chromatography techniques were used to measure sugar and amino acid content. Multivariate statistics were applied to assess whether there are significant differences in the chemical profiles of anemophilous and ambophilous species. Data were compared with published chemical profiles of angiosperm nectar. Chemical profiles were placed in the context of phylogenetic relationships. Total sugar concentrations were significantly higher in ovular secretions of ambophilous species than wind-pollinated taxa such as Pinaceae and Cupressophyta. Ambophilous species had lower amounts of total amino acids, and a higher proportion of non-protein amino acids compared with anemophilous lineages, and were also comparable to angiosperm nectar. Results suggest that early gymnosperms likely had ovular secretion profiles that were a mosaic of those associated with modern anemophilous and ambophilous species. Ginkgo, thought to be anemophilous, had a profile typical of ambophilous taxa, suggesting that insect pollination either exists in Gingko, but is undocumented, or that its ancestral populations were insect-pollinated. Chemical profiles of ovular secretions of ambophilous gymnosperms show a clear

Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

PubMed

Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

2016-01-01

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Reactive oxygen species as a signal in glucose-stimulated insulin secretion.

PubMed

Pi, Jingbo; Bai, Yushi; Zhang, Qiang; Wong, Victoria; Floering, Lisa M; Daniel, Kiefer; Reece, Jeffrey M; Deeney, Jude T; Andersen, Melvin E; Corkey, Barbara E; Collins, Sheila

2007-07-01

One of the unique features of beta-cells is their relatively low expression of many antioxidant enzymes. This could render beta-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H2O2. In both models, insulin secretion could be stimulated by provision of either exogenous H2O2 or diethyl maleate, which raises intracellular H2O2 levels. Provision of exogenous H2O2 scavengers, including cell permeable catalase and N-acetyl-L-cysteine, inhibited glucose-stimulated H2O2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H2O2, had no effect on GSIS. Because oxidative stress is an important risk factor for beta-cell dysfunction in diabetes, the relationship between glucose-induced H2O2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H2O2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.

Signaling from soybean roots to rhizobium: An ATP-binding cassette-type transporter mediates genistein secretion.

PubMed

Sugiyama, Akifumi; Shitan, Nobukazu; Yazaki, Kazufumi

2008-01-01

Legume plants have a unique ability to fix atmospheric nitrogen via symbiosis with rhizobia. For the establishment of symbiosis, legume plants secrete signaling molecules such as flavonoids from root tissues, leading to the attraction of rhizobia and the induction of rhizobial nod genes. Genistein and daidzein are found in soybean root exudates and function as signal molecules in soybean-Bradyrhizobium japonicum chemical communication. Although it is more than 20 years since these signal flavonoids were identified, almost nothing has been characterized concerning the membrane transport process of these molecules from soybean roots. To elucidate the transport mechanism we performed membrane transport assays with plasma membrane-enriched vesicles and various inhibitors. As a result, we concluded that an ATP-binding cassette-type transporter is involved in the secretion of genistein from soybean roots. The possible involvement of a pleiotropic drug resistance-type ABC transporter in this secretion is also discussed.

Eavesdropping and crosstalk between secreted quorum sensing peptide signals that regulate bacteriocin production in Streptococcus pneumoniae.

PubMed

Miller, Eric L; Kjos, Morten; Abrudan, Monica I; Roberts, Ian S; Veening, Jan-Willem; Rozen, Daniel E

2018-06-13

Quorum sensing (QS), where bacteria secrete and respond to chemical signals to coordinate population-wide behaviors, has revealed that bacteria are highly social. Here, we investigate how diversity in QS signals and receptors can modify social interactions controlled by the QS system regulating bacteriocin secretion in Streptococcus pneumoniae, encoded by the blp operon (bacteriocin-like peptide). Analysis of 4096 pneumococcal genomes detected nine blp QS signals (BlpC) and five QS receptor groups (BlpH). Imperfect concordance between signals and receptors suggested widespread social interactions between cells, specifically eavesdropping (where cells respond to signals that they do not produce) and crosstalk (where cells produce signals that non-clones detect). This was confirmed in vitro by measuring the response of reporter strains containing six different blp QS receptors to cognate and non-cognate peptides. Assays between pneumococcal colonies grown adjacent to one another provided further evidence that crosstalk and eavesdropping occur at endogenous levels of signal secretion. Finally, simulations of QS strains producing bacteriocins revealed that eavesdropping can be evolutionarily beneficial even when the affinity for non-cognate signals is very weak. Our results highlight that social interactions can mediate intraspecific competition among bacteria and reveal that competitive interactions can be modified by polymorphic QS systems.

The glycogen metabolism via Akt signaling is important for the secretion of enamel matrix in tooth development.

PubMed

Ida-Yonemochi, Hiroko; Otsu, Keishi; Ohshima, Hayato; Harada, Hidemitsu

2016-02-01

Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation. Copyright © 2016 Elsevier B.V. All rights reserved.

Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton▿ †

PubMed Central

Veenendaal, Andreas K. J.; Sundin, Charlotta; Blocker, Ariel J.

2009-01-01

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds. PMID:18996990

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

PubMed Central

Sandoval, Alejandro; Duran, Paz; Gandini, María A.; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2018-01-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. PMID:28807144

Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

PubMed

Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

2016-05-17

Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

BDNF-induced nitric oxide signals in cultured rat hippocampal neurons: time course, mechanism of generation, and effect on neurotrophin secretion.

PubMed

Kolarow, Richard; Kuhlmann, Christoph R W; Munsch, Thomas; Zehendner, Christoph; Brigadski, Tanja; Luhmann, Heiko J; Lessmann, Volkmar

2014-01-01

BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous application of the NO donor sodium-nitroprusside markedly inhibited neurotrophin secretion. However, endogenously generated NO in response to depolarization and neurotrophin stimulation, both did not result in a negative feedback on neurotrophin secretion. These results suggest that a negative feedback of NO signaling on synaptic secretion of neurotrophins operates only at high intracellular levels of nitric oxide that are under physiological conditions not reached by depolarization or BDNF signaling.

BDNF-induced nitric oxide signals in cultured rat hippocampal neurons: time course, mechanism of generation, and effect on neurotrophin secretion

PubMed Central

Kolarow, Richard; Kuhlmann, Christoph R. W.; Munsch, Thomas; Zehendner, Christoph; Brigadski, Tanja; Luhmann, Heiko J.; Lessmann, Volkmar

2014-01-01

BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous application of the NO donor sodium-nitroprusside markedly inhibited neurotrophin secretion. However, endogenously generated NO in response to depolarization and neurotrophin stimulation, both did not result in a negative feedback on neurotrophin secretion. These results suggest that a negative feedback of NO signaling on synaptic secretion of neurotrophins operates only at high intracellular levels of nitric oxide that are under physiological conditions not reached by depolarization or BDNF signaling. PMID:25426021

Broadly Protective Shigella Vaccine Based on Type III Secretion Apparatus Proteins

PubMed Central

Martinez-Becerra, Francisco J.; Kissmann, Julian M.; Diaz-McNair, Jovita; Choudhari, Shyamal P.; Quick, Amy M.; Mellado-Sanchez, Gabriela; Clements, John D.

2012-01-01

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine. PMID:22202122

Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion.

PubMed

Veenendaal, Andreas K J; Hodgkinson, Julie L; Schwarzer, Lynn; Stabat, David; Zenk, Sebastian F; Blocker, Ariel J

2007-03-01

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.

Twin-arginine signal peptide of Bacillus subtilis YwbN can direct Tat-dependent secretion of methyl parathion hydrolase.

PubMed

Liu, Ruihua; Zuo, Zhenqiang; Xu, Yingming; Song, Cunjiang; Jiang, Hong; Qiao, Chuanling; Xu, Ping; Zhou, Qixing; Yang, Chao

2014-04-02

The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

PubMed

Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

PubMed

Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T

2017-08-08

Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high

Unique Signatures of Population III Stars in the Global 21-cm Signal

NASA Astrophysics Data System (ADS)

Mirocha, Jordan; Mebane, Richard H.; Furlanetto, Steven R.; Singal, Krishma; Trinh, Donald

2018-05-01

We investigate the effects of Population III stars on the sky-averaged 21-cm background radiation, which traces the collective emission from all sources of ultraviolet and X-ray photons before reionization is complete. While UV photons from Pop III stars can in principle shift the onset of radiative coupling of the 21-cm transition - and potentially reionization - to early times, we find that the remnants of Pop III stars are likely to have a more discernible impact on the 21-cm signal than Pop III stars themselves. The X-rays from such sources preferentially heat the IGM at early times, which elongates the epoch of reheating and results in a more gradual transition from an absorption signal to emission. This gradual heating gives rise to broad, asymmetric wings in the absorption signal, which stand in contrast to the relatively sharp, symmetric signals that arise in models treating Pop II sources only. A stronger signature of Pop III, in which the position of the absorption minimum becomes inconsistent with Pop II-only models, requires extreme star-forming events that may not be physically plausible, lending further credence to predictions of relatively high frequency absorption troughs, νmin ˜ 100 MHz. As a result, though the trough location alone may not be enough to indicate the presence of Pop III, the asymmetric wings should arise even if only a few Pop III stars form in each halo before the transition to Pop II star formation occurs, provided that the Pop III IMF is sufficiently top-heavy and at least some Pop III stars form in binaries.

Prevalence of type III secretion system in effective biocontrol pseudomonads.

PubMed

Almario, Juliana; Gobbin, Davide; Défago, Geneviève; Moënne-Loccoz, Yvan; Rezzonico, Fabio

2014-05-01

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN(+) biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN(-) biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Methylated trivalent arsenicals are potent inhibitors of glucose stimulated insulin secretion by murine pancreatic islets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Douillet, Christelle; Currier, Jenna; Saunders, Jesse

Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAs{sup III}) or its methylated trivalent metabolites, methylarsonite (MAs{sup III}) and dimethylarsinite (DMAs{sup III}), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAs{sup III}, MAs{sup III} ormore » DMAs{sup III} inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs{sup III} and DMAs{sup III} were more potent than iAs{sup III} as GSIS inhibitors with estimated IC{sub 50} ≤ 0.1 μM. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAs{sup III}, MAs{sup III} or DMAs{sup III} could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic β-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes. - Highlights: ► Trivalent arsenicals inhibit glucose stimulated insulin secretion by pancreatic islets. ► MAs{sup III} and DMAs{sup III} are more potent inhibitors than arsenite with IC{sub 50} ∼ 0.1 μM. ► The arsenicals have little or no effects on insulin expression in pancreatic islets. ► The

Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

2012-09-17

The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are requiredmore » for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.« less

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

PubMed Central

Ligtenberg, Katherine Given; Miller, Nathan C.; Mitchell, Anthony; Plano, Gregory V.

2013-01-01

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR+] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV*327). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR− phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV*327 mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR+, LCR−, or dominant negative LCR− phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor. PMID:23222719

Extracellular secretion of recombinant proteins

DOEpatents

Linger, Jeffrey G.; Darzins, Aldis

2014-07-22

Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Lung, Tania Wong Fok; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare V L; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O'Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-09-12

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.

A type III effector antagonises death receptor signalling during bacterial gut infection

PubMed Central

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-01-01

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. 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-GlcNAc transferase activity of NleB1, which specifically modified Arg117 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 (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

PubMed Central

Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

2014-01-01

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

PubMed

Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2017-09-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca V α 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K + . In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V α 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa

PubMed Central

Dong, Yi-Hu; Zhang, Xi-Fen; Zhang, Lian-Hui

2013-01-01

The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources. PMID:23292701

Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

PubMed Central

Hancock, Melissa L.; Canetta, Sarah E.; Role, Lorna W.; Talmage, David A.

2008-01-01

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function. PMID:18458158

Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

PubMed

Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

2008-05-05

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

PubMed

Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

2008-06-01

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meshcheryakov, Vladimir A.; Kitao, Akio; Core Research for Evolutionary Science and Technology, Tokyo 113-0032

2013-05-01

Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, themore » crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.« less

Novel actions of IGFBP-3 on intracellular signaling pathways of insulin-secreting cells

PubMed Central

Chen, Xiaoyan; Ferry, Robert J.

2011-01-01

Understanding mechanisms underlying apoptotic destruction of insulin-secreting cells is critical to validate therapeutic targets for type 1 diabetes mellitus. We recently reported insulin-like growth factor binding protein-3 (IGFBP-3) as a novel mediator of apoptosis in insulin-secreting cells. In light of emerging IGF-independent roles for IGFBP-3, we investigated the mechanisms underlying actions of the novel, recombinant human mutant G56G80G81-IGFBP-3, which lacks intrinsic IGF binding affinity. Using the rat insulinoma RINm5F cell line, we report the first studies in insulin-secreting cells that IGFBP-3 selectively suppresses multiple, key intracellular phosphorelays. By immunoblot, we demonstrate that G56G80G81-IGFBP-3 suppresses phosphorylation of c-raf-MEK-ERK pathway and p38 kinase in time-dependent and dose-dependent manners. SAPK/JNK signaling was unaffected. These data delineate several novel intracellular sites of action for IGFBP-3 in insulin-secreting cells. PMID:16275148

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.

2008-05-03

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonasmore » aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.« less

Structure of the heterotrimeric complex that regulates type III secretion needle formation

PubMed Central

Quinaud, Manuelle; Plé, Sophie; Job, Viviana; Contreras-Martel, Carlos; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa

2007-01-01

Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF55–85-PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus- or flagellum-forming molecules. Disruption of specific PscG–PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials. PMID:17470796

Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

PubMed Central

2010-01-01

Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei. PMID:20540813

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-05-27

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-10-28

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2015-04-14

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2013-10-01

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

PubMed

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism.

PubMed

Rossello, Jessica; Lima, Analía; Gil, Magdalena; Rodríguez Duarte, Jorge; Correa, Agustín; Carvalho, Paulo C; Kierbel, Arlinet; Durán, Rosario

2017-08-31

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle.

PubMed

Sharma, V K; Sacco, R E; Kunkle, R A; Bearson, S M D; Palmquist, D E

2012-04-01

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.

Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma.

PubMed

Müller, Nadja; Michen, Susanne; Tietze, Stefanie; Töpfer, Katrin; Schulte, Alexander; Lamszus, Katrin; Schmitz, Marc; Schackert, Gabriele; Pastan, Ira; Temme, Achim

2015-06-01

Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation

PubMed Central

Koyama, Takashi; Mirth, Christen K.

2016-01-01

In Drosophila, the fat body, functionally equivalent to the mammalian liver and adipocytes, plays a central role in regulating systemic growth in response to nutrition. The fat body senses intracellular amino acids through Target of Rapamycin (TOR) signaling, and produces an unidentified humoral factor(s) to regulate insulin-like peptide (ILP) synthesis and/or secretion in the insulin-producing cells. Here, we find that two peptides, Growth-Blocking Peptide (GBP1) and CG11395 (GBP2), are produced in the fat body in response to amino acids and TOR signaling. Reducing the expression of GBP1 and GBP2 (GBPs) specifically in the fat body results in smaller body size due to reduced growth rate. In addition, we found that GBPs stimulate ILP secretion from the insulin-producing cells, either directly or indirectly, thereby increasing insulin and insulin-like growth factor signaling activity throughout the body. Our findings fill an important gap in our understanding of how the fat body transmits nutritional information to the insulin producing cells to control body size. PMID:26928023

A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78.

PubMed

Chaves, Daniela Fojo Seixas; de Souza, Emanuel Maltempi; Monteiro, Rose Adele; de Oliveira Pedrosa, Fábio

2009-11-02

Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant-bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.

a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

NASA Astrophysics Data System (ADS)

Rathinavelan, Thenmalarchelvi; Im, Wonpil

2010-01-01

Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

PubMed Central

de Carvalho, João Carlos Monteiro; Mayfield, Stephen Patrick

2018-01-01

Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications. PMID:29408937

Potassium transport of Salmonella is important for type III secretion and pathogenesis

PubMed Central

Liu, Yehao; Ho, Katharina Kim; Su, Jing; Gong, Hao; Chang, Alexander C.

2013-01-01

Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K+), sodium ion (Na+) and proton (H+) are involved in nearly all aspects of bacterial growth and survival. K+ is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K+ transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K+ transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K+ transport through deletion of K+ transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K+ transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K+ transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria. PMID:23728623

Loss of mTORC1 signaling alters pancreatic α cell mass and impairs glucagon secretion

PubMed Central

Bozadjieva, Nadejda; Dai, Xiao-Qing; Cummings, Kelsey; Gimeno, Jennifer; Powers, Alvin C.; Gittes, George K.; Rüegg, Markus A.; Hall, Michael N.; MacDonald, Patrick E.

2017-01-01

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic α cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of α cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in α cells (αRaptorKO), we showed that mTORC1 signaling is dispensable for α cell development, but essential for α cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in αRaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In αRaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for α cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling α cell–mass maintenance. PMID:29106387

The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

PubMed Central

Shaulov, Lihi; Gershberg, Jenia; Deng, Wanyin; Finlay, B. Brett

2017-01-01

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. PMID:28049143

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.

PubMed

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M

2007-02-09

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

PubMed

Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

2016-07-01

Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Risk of developing pneumonia is enhanced by the combined traits of fluoroquinolone resistance and type III secretion virulence in respiratory isolates of Pseudomonas aeruginosa.

PubMed

Sullivan, Eva; Bensman, Joyce; Lou, Mimi; Agnello, Melissa; Shriner, Kimberly; Wong-Beringer, Annie

2014-01-01

To determine the differential association of host characteristics, antimicrobial resistance, and type III secretion system virulence of Pseudomonas aeruginosa isolates with respiratory syndromes in hospitalized adult patients. Retrospective, cohort study. Community teaching hospital. Two hundred eighteen consecutive adult patients with respiratory culture positive for P. aeruginosa between January 2005 to January 2010. Medical charts were reviewed to obtain demographic, laboratory, radiographic, and clinical information. Isolates were assayed by polymerase chain reaction for genes encoding the type III secretion system effectors (ExoU, ExoS, and PcrV) and for strain relatedness using randomly amplified polymorphic DNA analysis. Levofloxacin susceptibility was determined by broth microdilution. Patients were grouped by colonization, bronchitis, or pneumonia and were compared for differential risk of developing the clinical syndrome with respect to host and microbial characteristics. Half of the study cohort (54%, 117 of 218) had pneumonia, 32% (70 of 218) had bronchitis, and 14% (31 of 218) had colonization; in-hospital mortality was 35%, 11%, and 0%, respectively. Host factors strongly associated with pneumonia development were residence in long-term care facility, healthcare-associated acquisition of P. aeruginosa, higher Acute Physiology and Chronic Health Evaluation II score, presence of enteral feeding tube, mechanical ventilation, and recent history of pneumonia. Fluoroquinolone-resistant (57% vs 34%, 16%; p < 0.0001) and multidrug-resistant (36% vs 26%, 7%; p = 0.0045) strains were more likely to cause pneumonia than bronchitis or colonization, respectively. Analysis of host and microbial factors in a multivariate regression model yielded the combined traits of fluoroquinolone resistance and gene encoding the type III secretion system ExoU effector in P. aeruginosa as the single most significant predictor of pneumonia development. These results suggest that

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

SEL1L Regulates Adhesion, Proliferation and Secretion of Insulin by Affecting Integrin Signaling

PubMed Central

Diaferia, Giuseppe R.; Cirulli, Vincenzo; Biunno, Ida

2013-01-01

SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function. PMID:24324549

KISS1R signals independently of Gαq/11 and triggers LH secretion via the β-arrestin pathway in the male mouse.

PubMed

Ahow, Maryse; Min, Le; Pampillo, Macarena; Nash, Connor; Wen, Junping; Soltis, Kathleen; Carroll, Rona S; Glidewell-Kenney, Christine A; Mellon, Pamela L; Bhattacharya, Moshmi; Tobet, Stuart A; Kaiser, Ursula B; Babwah, Andy V

2014-11-01

Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a Gαq/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the Gαq/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of Gαq/11 via β-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the Gαq/11- and β-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either β-arrestin-1 or β-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that β-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that Gαq/11-uncoupled KISS1R mutants, like L148S, will display Gαq/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from Gαq/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking β-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is β-arrestin-dependent. Overall, we conclude that KISS1R signals via Gαq/11 and β-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of Gαq/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism.

Virulence of Erwinia amylovora, a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses.

PubMed

Holtappels, Michelle; Noben, Jean-Paul; Valcke, Roland

2016-09-01

Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD

PubMed Central

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.

2007-01-01

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-01-01

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa.

PubMed

Dong, Yi-Hu; Zhang, Xi-Fen; Zhang, Lian-Hui

2013-02-01

The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources. © 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus

PubMed Central

O'Boyle, Nicky; Boyd, Aoife

2013-01-01

Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS) to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signaling proteins, thereby bringing about changes in the regulation of cellular behavior. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favors bacterial colonization and persistence of V. parahaemolyticus in the host. PMID:24455490

Effects of chromium(III) picolinate on cortisol and DHEAs secretion in H295R human adrenocortical cells.

PubMed

Kim, Beob G; Adams, Julye M; Jackson, Brian A; Lindemann, Merlin D

2010-02-01

Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 microM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 microM of CrPic and the lowest at 200 microM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 microM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 microM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 microM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

PubMed Central

Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.

2011-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746

The unconventional secretion of IL-1β: Handling a dangerous weapon to optimize inflammatory responses.

PubMed

Sitia, Roberto; Rubartelli, Anna

2018-04-04

Interleukin 1β (IL-1β) is a major mediator of inflammation, with a causative role in many diseases. Unlike most other cytokines, however, it lacks a secretory signal sequence, raising intriguing mechanistic, functional and evolutionary questions. Despite decades of strenuous efforts in many laboratories, how IL-1β is secreted is still a matter of intense debate. Here, we summarize the different mechanisms and pathways that have been proposed for IL-1β secretion. At least two of them, namely the endolysosomal vesicle-based and gasdermin D-dependent pathways (types III and I in the recent Rabouille's classification of unconventional protein secretion), can be triggered in monocytes, the main source of IL-1β in humans, according to the type and strength of the pro-inflammatory stimuli. As during the escalation of human conflicts, monocytes deploy secretory mechanisms of increasing efficiency and dangerousness, shifting from the specific and controlled type III pathway to the much faster release of type I. Thus, the different mechanisms are activated depending on the severity of the conditions, from the self-limiting type III pathways in response of low pathogen load or small trauma, to the uncontrolled responses that underlie autoinflammatory disorders and sepsis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

PubMed Central

Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

2017-01-01

Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

PubMed

Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

2017-12-01

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini

2012-03-26

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC.more » While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.« less

Genetically engineered T cells to target EGFRvIII expressing glioblastoma.

PubMed

Bullain, Szofia S; Sahin, Ayguen; Szentirmai, Oszkar; Sanchez, Carlos; Lin, Ning; Baratta, Elizabeth; Waterman, Peter; Weissleder, Ralph; Mulligan, Richard C; Carter, Bob S

2009-09-01

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

Calcitonin Gene-Related Peptide Reduces Taste-Evoked ATP Secretion from Mouse Taste Buds.

PubMed

Huang, Anthony Y; Wu, Sandy Y

2015-09-16

Immunoelectron microscopy revealed that peripheral afferent nerve fibers innervating taste buds contain calcitonin gene-related peptide (CGRP), which may be as an efferent transmitter released from peripheral axon terminals. In this report, we determined the targets of CGRP within taste buds and studied what effect CGRP exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura-2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings showed that a subset of Presynaptic (Type III) taste cells (53%) responded to 0.1 μm CGRP with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 0.1 μm CGRP. Using pharmacological tools, the actions of CGRP were probed and elucidated by the CGRP receptor antagonist CGRP(8-37). We demonstrated that this effect of CGRP was dependent on phospholipase C activation and was prevented by the inhibitor U73122. Moreover, applying CGRP caused taste buds to secrete serotonin (5-HT), a Presynaptic (Type III) cell transmitter, but not ATP, a Receptor (Type II) cell transmitter. Further, our previous studies showed that 5-HT released from Presynaptic (Type III) cells provides negative paracrine feedback onto Receptor (Type II) cells by activating 5-HT1A receptors, and reducing ATP secretion. Our data showed that CGRP-evoked 5-HT release reduced taste-evoked ATP secretion. The findings are consistent with a role for CGRP as an inhibitory transmitter that shapes peripheral taste signals via serotonergic signaling during processing gustatory information in taste buds. The taste sensation is initiated with a highly complex set of interactions between a variety of cells located within the taste buds before signal propagation to the brain. Afferent signals from the oral cavity are carried to the brain in chemosensory fibers that contribute to chemesthesis, the general chemical sensitivity of the mucus

An optimization formulation for characterization of pulsatile cortisol secretion.

PubMed

Faghih, Rose T; Dahleh, Munther A; Brown, Emery N

2015-01-01

Cortisol is released to relay information to cells to regulate metabolism and reaction to stress and inflammation. In particular, cortisol is released in the form of pulsatile signals. This low-energy method of signaling seems to be more efficient than continuous signaling. We hypothesize that there is a controller in the anterior pituitary that leads to pulsatile release of cortisol, and propose a mathematical formulation for such controller, which leads to impulse control as opposed to continuous control. We postulate that this controller is minimizing the number of secretory events that result in cortisol secretion, which is a way of minimizing the energy required for cortisol secretion; this controller maintains the blood cortisol levels within a specific circadian range while complying with the first order dynamics underlying cortisol secretion. We use an ℓ0-norm cost function for this controller, and solve a reweighed ℓ1-norm minimization algorithm for obtaining the solution to this optimization problem. We use four examples to illustrate the performance of this approach: (i) a toy problem that achieves impulse control, (ii) two examples that achieve physiologically plausible pulsatile cortisol release, (iii) an example where the number of pulses is not within the physiologically plausible range for healthy subjects while the cortisol levels are within the desired range. This novel approach results in impulse control where the impulses and the obtained blood cortisol levels have a circadian rhythm and an ultradian rhythm that are in agreement with the known physiology of cortisol secretion. The proposed formulation is a first step in developing intermittent controllers for curing cortisol deficiency. This type of bio-inspired pulse controllers can be employed for designing non-continuous controllers in brain-machine interface design for neuroscience applications.

What couples glycolysis to mitochondrial signal generation in glucose-stimulated insulin secretion?

PubMed

Ishihara, H; Wollheim, C B

2000-05-01

Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.

High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

PubMed

Savijoki, K; Kahala, M; Palva, A

1997-02-28

A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins

PubMed Central

Chappell, Alfred E.; Bunz, Michael; Smoll, Eric; Dong, Hui; Lytle, Christian; Barrett, Kim E.; McCole, Declan F.

2018-01-01

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H2O2), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl− secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H2O2 inhibit Ca2+-dependent Cl− secretion across T84 colonic epithelial cells by elevating cytosolic Ca2+, which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca2+-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca2+-dependent stimuli is mediated in part by K+ efflux through basolateral K+ channels and Cl− uptake by the Na+-K+-2Cl− cotransporter, NKCC1. We demonstrate that H2O2 inhibits Ca2+-dependent basolateral K+ efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl− conductance. Thus, we have demonstrated that H2O2 inhibits Ca2+-dependent Cl− secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.—Chappell, A. E., Bunz, M., Smoll, E., Dong, H., Lytle, C., Barrett, K. E., McCole, D. F. Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins. FASEB J. 22, 000–000 (2008) PMID:18211955

The type III secreted effector DspE is required early in Solanum tuberosum leaf infection by Pectobacterium carotovorum to elicit cell death, and requires Wx(3-6)D/E motifs

USDA-ARS?s Scientific Manuscript database

Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effect...

Salmonella-secreted Virulence Factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heffron, Fred; Niemann, George; Yoon, Hyunjin

In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellentmore » reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.« less

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells.

PubMed

Lu, Naisheng; Li, Mengjiao; Lei, Hulong; Jiang, Xueyuan; Tu, Weilong; Lu, Yang; Xia, Dong

2017-09-01

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P 4 ) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P 4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E 2 ) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E 2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P 4 , and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E 2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P 4 and E 2 hormone synthesis in PGCs via the cAMP signaling pathway. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Pseudomonas syringae pv. Tomato DC3000 Type III secretion effector polymutants reveal an interplay between hopAD1 and AvrPtoB

USDA-ARS?s Scientific Manuscript database

The model pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of plants by injecting a complex repertoire of effector proteins into host cells via the type III secretion system. The model effector AvrPtoB has multiple domains and plant protein interactors i...

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

PubMed

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

2016-11-01

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

PubMed Central

Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

2011-01-01

Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

Vesicular secretion of auxin

PubMed Central

Schlicht, Markus; Volkmann, Dieter; Mancuso, Stefano

2008-01-01

The plant hormone auxin is secreted in root apices via phospholipase Dζ2 (PLDζ2) activity which produces specific population of phosphatidic acid that stimulates secretion of vesicles enriched with auxin. These vesicles were reported to be localized at plant synapses which are active in auxin secretion, especially at the transition zone of the root apex. There are several implications of this vesicular secretion of auxin. In root apices, auxin emerges as plant neurotransmitter-like signal molecule which coordinates activities of adjacent cells via electric and chemical signaling. Putative quantal release of auxin after electrical stimulation, if confirmed, would be part of neuronal communication between plant cells. As auxin transport across plant synapses is tightly linked with integrated sensory perception of environment, especially of omnipresent gravity and light, this process is proposed to mediate the plant perception of environment. These neuronal features allow sessile plants to integrate multitude of sensory signals into the adaptive behavior of whole plants and the animal-like exploratory behavior of growing roots. PMID:19704646

C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

PubMed

Fujiwara, Miho; Kato, Shintaro; Niwa, Yuki; Suzuki, Takehiro; Tsuchiya, Miyu; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

2016-08-01

R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling. © 2016 Federation of European Biochemical Societies.

Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

PubMed Central

Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

2011-01-01

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection. PMID:21559274

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

PubMed Central

Intile, Peter J.; Balzer, Grant J.; Wolfgang, Matthew C.

2015-01-01

ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box

Nicotinic acid increases adiponectin secretion from differentiated bovine preadipocytes through G-protein coupled receptor signaling.

PubMed

Kopp, Christina; Hosseini, Afshin; Singh, Shiva P; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

2014-11-18

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

PubMed Central

Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

2014-01-01

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

TRPM channels phosphorylation as a potential bridge between old signals and novel regulatory mechanisms of insulin secretion.

PubMed

Diaz-Garcia, Carlos Manlio; Sanchez-Soto, Carmen; Hiriart, Marcia

2013-03-01

Transient receptor potential channels, especially the members of the melastatin family (TRPM), participate in insulin secretion. Some of them are substrates for protein kinases, which are involved in several neurotransmitter, incretin and hormonal signaling cascades in β cells. The functional relationships between protein kinases and TRPM channels in systems of heterologous expression and native tissues rise issues about novel regulation pathways of pancreatic β-cell excitability. The aim of the present work is to review the evidences about phosphorylation of TRPM channels in β cells and to discuss the perspectives on insulin secretion.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

PubMed Central

Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

2010-01-01

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

PubMed Central

2012-01-01

Summary: Flagellar and translocation-associated type III secretion (T3S) systems are present in most Gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria. PMID:22688814

The SPI-1-like Type III secretion system: more roles than you think

PubMed Central

Egan, Frank; Barret, Matthieu; O’Gara, Fergal

2014-01-01

The type III secretion system (T3SS) is a protein delivery system which is involved in a wide spectrum of interactions, from mutualism to pathogenesis, between Gram negative bacteria and various eukaryotes, including plants, fungi, protozoa and mammals. Various phylogenetic families of the T3SS have been described, including the Salmonella Pathogenicity Island 1 family (SPI-1). The SPI-1 T3SS was initially associated with the virulence of enteric pathogens, but is actually found in a diverse array of bacterial species, where it can play roles in processes as different as symbiotic interactions with insects and colonization of plants. We review the multiple roles of the SPI-1 T3SS and discuss both how these discoveries are changing our perception of the SPI-1 family and what impacts this has on our understanding of the specialization of the T3SS in general. PMID:24575107

The SPI-1-like Type III secretion system: more roles than you think.

PubMed

Egan, Frank; Barret, Matthieu; O'Gara, Fergal

2014-01-01

The type III secretion system (T3SS) is a protein delivery system which is involved in a wide spectrum of interactions, from mutualism to pathogenesis, between Gram negative bacteria and various eukaryotes, including plants, fungi, protozoa and mammals. Various phylogenetic families of the T3SS have been described, including the Salmonella Pathogenicity Island 1 family (SPI-1). The SPI-1 T3SS was initially associated with the virulence of enteric pathogens, but is actually found in a diverse array of bacterial species, where it can play roles in processes as different as symbiotic interactions with insects and colonization of plants. We review the multiple roles of the SPI-1 T3SS and discuss both how these discoveries are changing our perception of the SPI-1 family and what impacts this has on our understanding of the specialization of the T3SS in general.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

PubMed

Romero, Damian G; Zhou, Ming Yi; Yanes, Licy L; Plonczynski, Maria W; Washington, Tanganika R; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P

2007-08-01

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Plant-bacterial pathogen interactions mediated by type III effectors.

PubMed

Feng, Feng; Zhou, Jian-Min

2012-08-01

Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family†

PubMed Central

Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu

2006-01-01

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

PubMed Central

Dickenson, Nicholas E; Choudhari, Shyamal P; Adam, Philip R; Kramer, Ryan M; Joshi, Sangeeta B; Middaugh, C Russell; Picking, Wendy L; Picking, William D

2013-01-01

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligo-oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore. PMID:23456854

Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid secretion in airway epithelia

PubMed Central

Turner, Mark J.; Saint‐Criq, Vinciane; Patel, Waseema; Ibrahim, Salam H.; Verdon, Bernard; Ward, Christopher; Garnett, James P.; Tarran, Robert; Cann, Martin J.

2015-01-01

Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

PubMed Central

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

2016-01-01

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. PMID:27541856

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

Spatial signal correlation from an III-nitride synaptic device

NASA Astrophysics Data System (ADS)

Zhang, Shuai; Zhu, Bingcheng; Shi, Zheng; Yuan, Jialei; Jiang, Yuan; Shen, Xiangfei; Cai, Wei; Yang, Yongchao; Wang, Yongjin

2017-10-01

The mechanism by which the external environment affects the internal nervous system is investigated via the spatial correlation of an III-nitride synaptic device, which combines in-plane and out-of-plane illumination. The InGaN/GaN multiple-quantum-well collector (MQW-collector) demonstrates a simultaneous light emission and light detection mode due to the unique property of the MQW-diode. The MQW-collector absorbs the internal incoming light and the external illumination at the same time to generate an integration of the excitatory postsynaptic voltages (EPSVs). Signal cognition can be distinctly decoded from the integrated EPSVs because the signal differences are maintained, which is in good agreement with the simulation results. These results suggest that the nervous system can simultaneously amplify the EPSV amplitude and achieve signal cognition by spatial EPSV summation, which can be further optimized to explore the connections between the internal nervous system and the external environment.

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

PubMed

Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

2016-01-01

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits

PubMed Central

Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A.; Bősze, Zsuzsanna

2017-01-01

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals. PMID:29077768

Bacterial flagella and Type III secretion: case studies in the evolution of complexity.

PubMed

Pallen, M J; Gophna, U

2007-01-01

Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

Type III Nrg1 back signaling enhances functional TRPV1 along sensory axons contributing to basal and inflammatory thermal pain sensation.

PubMed

Canetta, Sarah E; Luca, Edlira; Pertot, Elyse; Role, Lorna W; Talmage, David A

2011-01-01

Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs). Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can acutely activate phosphatidylinositol-3-kinase (PtdIns3K) signaling, as well as regulate levels of α7* nicotinic acetylcholine receptors, along sensory axons. Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in primary sensory neurons that is necessary for the detection of thermal pain and for the development of thermal hypersensitivity to pain under inflammatory conditions. Cell surface expression of TRPV1 can be enhanced by activation of PtdIns3K, making it a potential target for regulation by Type III Nrg1. We now show that Type III Nrg1 signaling in sensory neurons affects functional axonal TRPV1 in a PtdIns3K-dependent manner. Furthermore, mice heterozygous for Type III Nrg1 have specific deficits in their ability to respond to noxious thermal stimuli and to develop capsaicin-induced thermal hypersensitivity to pain. Cumulatively, these results implicate Type III Nrg1 as a novel regulator of TRPV1 and a molecular mediator of nociceptive function.

Vibrio parahaemolyticus Inhibition of Rho Family GTPase Activation Requires a Functional Chromosome I Type III Secretion System▿

PubMed Central

Casselli, Timothy; Lynch, Tarah; Southward, Carolyn M.; Jones, Bryan W.; DeVinney, Rebekah

2008-01-01

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors. PMID:18347050

Comprehensive assessment and performance improvement of effector protein predictors for bacterial secretion systems III, IV and VI.

PubMed

An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor

2018-01-01

Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.

PubMed

Mendes, Carolina C P; Gomes, Dawidson A; Thompson, Mayerson; Souto, Natalia C; Goes, Tercio S; Goes, Alfredo M; Rodrigues, Michele A; Gomez, Marcus V; Nathanson, Michael H; Leite, M Fatima

2005-12-09

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.

Ce(III, IV)-MOF electrocatalyst as signal-amplifying tag for sensitive electrochemical aptasensing.

PubMed

Yu, Hua; Han, Jing; An, Shangjie; Xie, Gang; Chen, Sanping

2018-06-30

Metal-organic frameworks (MOFs) as a new class of porous materials have attracted increasing attention in the field of biomimetic catalysis. This study firstly reports a mixed valence state Ce-MOF possessing intrinsic catalytic activity towards thionine (Thi), and its application in constructing an amplified electrochemical aptasensor for thrombin detection. As noticed, the novel catalytic process combines the advantages of 3D infinite extension of the Ce(III, IV)-MOF skeleton containing large amounts of catalytic sites and spontaneous recycling of the Ce(III)/Ce(IV) for electrochemical reduction of Thi, thereby presenting amplified electrochemical signals. To further improve the aptasensor performance, the high selectivity of proximity binding-induced DNA strand displacement and high efficiency of exonuclease III-assisted recycling amplification were incorporated into the assay. The aptasensor was employed to detect thrombin in complex serum samples, which shows high sensitivity, specificity, stability and reproducibility. This work offers an opportunity to develop MOF-based electrocatalyst as signal-amplifying tag for versatile bioassays and catalytic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

The downregulation of sweet taste receptor signaling in enteroendocrine L-cells mediates 3-deoxyglucosone-induced attenuation of high glucose-stimulated GLP-1 secretion.

PubMed

Wang, Fei; Song, Xiudao; Zhou, Liang; Liang, Guoqiang; Huang, Fei; Jiang, Guorong; Zhang, Lurong

2017-12-26

Sweet taste receptors (STRs) involve in regulating the release of glucose-stimulated glucagon-like peptide-1 (GLP-1). Our in vivo and in vitro studies found that 3-deoxyglucosone (3DG) inhibited glucose-stimulated GLP-1 secretion. This study investigated the role of STRs in 3DG-induced inhibition of high glucose-stimulated GLP-1 secretion. STC-1 cells were incubated with lactisole or 3DG for 1 h under 25 mM glucose conditions. Western blotting was used to study the expression of STRs signaling molecules and ELISA was used to analyse GLP-1 and cyclic adenosine monophosphate (cAMP) levels. Lactisole inhibited GLP-1 secretion. Exposure to 25 mM glucose increased the expressions of STRs subunits when compared with 5.6 mM glucose. 3DG decreased GLP-1 secretion and STRs subunits expressions, with affecting other components of STRs pathway, including the downregulation of transient receptor potential cation channel subfamily M member 5 (TRPM5) expression and the reduction of intracellular cAMP levels. 3DG attenuates high glucose-stimulated GLP-1 secretion by reducing STR subunit expression and downstream signaling components.

T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

PubMed

Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

2010-10-15

Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of

Further Characterization of a Type III Secretion System (T3SS) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila - Part I

EPA Science Inventory

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular cha...

Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals

PubMed Central

Gagnon, Jules; Lavoie, Mathieu; Catala, Mathieu; Malenfant, Francis; Elela, Sherif Abou

2015-01-01

Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions. PMID:25680180

Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

PubMed Central

Canetta, Sarah E.; Luca, Edlira; Pertot, Elyse; Role, Lorna W.; Talmage, David A.

2011-01-01

Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs) [1]. Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can acutely activate phosphatidylinositol-3-kinase (PtdIns3K) signaling, as well as regulate levels of α7* nicotinic acetylcholine receptors, along sensory axons [2]. Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in primary sensory neurons that is necessary for the detection of thermal pain and for the development of thermal hypersensitivity to pain under inflammatory conditions [3]. Cell surface expression of TRPV1 can be enhanced by activation of PtdIns3K [4], [5], [6], making it a potential target for regulation by Type III Nrg1. We now show that Type III Nrg1 signaling in sensory neurons affects functional axonal TRPV1 in a PtdIns3K-dependent manner. Furthermore, mice heterozygous for Type III Nrg1 have specific deficits in their ability to respond to noxious thermal stimuli and to develop capsaicin-induced thermal hypersensitivity to pain. Cumulatively, these results implicate Type III Nrg1 as a novel regulator of TRPV1 and a molecular mediator of nociceptive function. PMID:21949864

The effect of imiquimod on taste bud calcium transients and transmitter secretion.

PubMed

Huang, Anthony Y; Wu, Sandy Y

2016-11-01

Imiquimod is an immunomodulator approved for the treatment of basal cell carcinoma and has adverse side effects, including taste disturbances. Paracrine transmission, representing cell-cell communication within taste buds, has the potential to shape the final signals that taste buds transmit to the brain. Here, we tested the underlying assumption that imiquimod modifies taste transmitter secretion in taste buds of mice. Taste buds were isolated from C57BL/6J mice. The effects of imiquimod on transmitter release in taste buds were measured using calcium imaging with cellular biosensors, and examining the net effect of imiquimod on taste-evoked ATP secretion from mouse taste buds. Up to 72% of presynaptic (Type III) taste cells responded to 100 μM imiquimod with an increase in intracellular Ca 2+ concentrations. These Ca 2 + responses were inhibited by thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca 2 + -ATPase, and by U73122, a PLC inhibitor, suggesting that the Ca 2 + mobilization elicited by imiquimod was dependent on release from internal Ca 2 + stores. Moreover, combining studies of Ca 2 + imaging with cellular biosensors showed that imiquimod evoked secretion of 5-HT, which then provided negative feedback onto receptor (Type II) cells to reduce taste-evoked ATP secretion. Our results provide evidence that there is a subset of taste cells equipped with a range of intracellular mechanisms that respond to imiquimod. The findings are also consistent with a role of imiquimod as an immune response modifier, which shapes peripheral taste responses via 5-HT signalling. © 2016 The British Pharmacological Society.

Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification.

PubMed

Liu, Haisheng; Ma, Changbei; Zhou, Meijuan; Chen, Hanchun; He, Hailun; Wang, Kemin

2016-11-01

This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). Exo III can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by Exo III. During the digestion of MB2, the 2-aminopurine is separated from the DNA strands and released free in solution, inducing an increase of the fluorescent signal. Owing to the presence of a recessed 3'-terminus in the formed double-stranded DNA, Exo III-assisted recyclable cleavage of MB2 was achieved. Therefore, an amplified fluorescence signal was observed. Under the optimized conditions, Dam MTase can be detected in the range of 0.2-40 units/mL with a limit of detection of 0.2 units/mL and good selectivity. Furthermore, the present assay can be used for screening potential DNA MTase inhibitors. Graphical Abstract A quencher-free fluorescence assay for sensitive detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification is reported.

Sema-1a Reverse Signaling Promotes Midline Crossing in Response to Secreted Semaphorins.

PubMed

Hernandez-Fleming, Melissa; Rohrbach, Ethan W; Bashaw, Greg J

2017-01-03

Commissural axons must cross the midline to form functional midline circuits. In the invertebrate nerve cord and vertebrate spinal cord, midline crossing is mediated in part by Netrin-dependent chemoattraction. Loss of crossing, however, is incomplete in mutants for Netrin or its receptor Frazzled/DCC, suggesting the existence of additional pathways. We identified the transmembrane Semaphorin, Sema-1a, as an important regulator of midline crossing in the Drosophila CNS. We show that in response to the secreted Semaphorins Sema-2a and Sema-2b, Sema-1a functions as a receptor to promote crossing independently of Netrin. In contrast to other examples of reverse signaling where Sema1a triggers repulsion through activation of Rho in response to Plexin binding, in commissural neurons Sema-1a acts independently of Plexins to inhibit Rho to promote attraction to the midline. These findings suggest that Sema-1a reverse signaling can elicit distinct axonal responses depending on differential engagement of distinct ligands and signaling effectors. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

PubMed

Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Type III Interferon-Mediated Signaling Is Critical for Controlling Live Attenuated Yellow Fever Virus Infection In Vivo.

PubMed

Douam, Florian; Soto Albrecht, Yentli E; Hrebikova, Gabriela; Sadimin, Evita; Davidson, Christian; Kotenko, Sergei V; Ploss, Alexander

2017-08-15

Yellow fever virus (YFV) is an arthropod-borne flavivirus, infecting ~200,000 people worldwide annually and causing about 30,000 deaths. The live attenuated vaccine strain, YFV-17D, has significantly contributed in controlling the global burden of yellow fever worldwide. However, the viral and host contributions to YFV-17D attenuation remain elusive. Type I interferon (IFN-α/β) signaling and type II interferon (IFN-γ) signaling have been shown to be mutually supportive in controlling YFV-17D infection despite distinct mechanisms of action in viral infection. However, it remains unclear how type III IFN (IFN-λ) integrates into this antiviral system. Here, we report that while wild-type (WT) and IFN-λ receptor knockout (λR -/- ) mice were largely resistant to YFV-17D, deficiency in type I IFN signaling resulted in robust infection. Although IFN-α/β receptor knockout (α/βR -/- ) mice survived the infection, mice with combined deficiencies in both type I signaling and type III IFN signaling were hypersusceptible to YFV-17D and succumbed to the infection. Mortality was associated with viral neuroinvasion and increased permeability of the blood-brain barrier (BBB). α/βR -/- λR -/- mice also exhibited distinct changes in the frequencies of multiple immune cell lineages, impaired T-cell activation, and severe perturbation of the proinflammatory cytokine balance. Taken together, our data highlight that type III IFN has critical immunomodulatory and neuroprotective functions that prevent viral neuroinvasion during active YFV-17D replication. Type III IFN thus likely represents a safeguard mechanism crucial for controlling YFV-17D infection and contributing to shaping vaccine immunogenicity. IMPORTANCE YFV-17D is a live attenuated flavivirus vaccine strain recognized as one of the most effective vaccines ever developed. However, the host and viral determinants governing YFV-17D attenuation and its potent immunogenicity are still unknown. Here, we analyzed the

Abundant Type III Lipid Transfer Proteins in Arabidopsis Tapetum Are Secreted to the Locule and Become a Constituent of the Pollen Exine1[W][OPEN

PubMed Central

Huang, Ming-Der; Chen, Tung-Ling L.; Huang, Anthony H.C.

2013-01-01

Lipid transfer proteins (LTPs) are small secretory proteins in plants with defined lipid-binding structures for possible lipid exocytosis. Special groups of LTPs unique to the anther tapetum are abundant, but their functions are unclear. We studied a special group of LTPs, type III LTPs, in Arabidopsis (Arabidopsis thaliana). Their transcripts were restricted to the anther tapetum, with levels peaking at the developmental stage of maximal pollen-wall exine synthesis. We constructed an LTP-Green Fluorescent Protein (LTP-GFP) plasmid, transformed it into wild-type plants, and monitored LTP-GFP in developing anthers with confocal laser scanning microscopy. LTP-GFP appeared in the tapetum and was secreted via the endoplasmic reticulum-trans-Golgi network machinery into the locule. It then moved to the microspore surface and remained as a component of exine. Immuno-transmission electron microscopy of native LTP in anthers confirmed the LTP-GFP observations. The in vivo association of LTP-GFP and exine in anthers was not observed with non-type III or structurally modified type III LTPs or in transformed exine-defective mutant plants. RNA interference knockdown of individual type III LTPs produced no observable mutant phenotypes. RNA interference knockdown of two type III LTPs produced microscopy-observable morphologic changes in the intine underneath the exine (presumably as a consequence of changes in the exine not observed by transmission electron microscopy) and pollen susceptible to dehydration damage. Overall, we reveal a novel transfer pathway of LTPs in which LTPs bound or nonbound to exine precursors are secreted from the tapetum to become microspore exine constituents; this pathway explains the need for plentiful LTPs to incorporate into the abundant exine. PMID:24096413

Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis.

PubMed

Christensen, Dan Ploug; Ejlerskov, Patrick; Rasmussen, Izabela; Vilhardt, Frederik

2016-03-08

Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNβ and TGF�

Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

PubMed

Rodriguez, Stéphane; Rudloff, Stefan; Koenig, Katrin Franziska; Karthik, Swapna; Hoogewijs, David; Huynh-Do, Uyen

2016-08-01

Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion.

PubMed

Chan, Pei-Chi; Wang, Ya-Chin; Chen, Yi-Ling; Hsu, Wan-Ning; Tian, Yu-Feng; Hsieh, Po-Shiuan

2017-11-01

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags.

PubMed

Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech; Korpys, Paulina; Nicaud, Jean-Marc

2018-06-01

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

PubMed

Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

2018-05-01

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen.

PubMed

Qin, Wen; Sundaram, Meenakshi; Wang, Yuwei; Zhou, Hu; Zhong, Shumei; Chang, Chia-Ching; Manhas, Sanjay; Yao, Erik F; Parks, Robin J; McFie, Pamela J; Stone, Scot J; Jiang, Zhenghui G; Wang, Congrong; Figeys, Daniel; Jia, Weiping; Yao, Zemin

2011-08-05

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Epidermal growth factor receptor and EGFRvIII in glioblastoma: signaling pathways and targeted therapies. | Office of Cancer Genomics

Cancer.gov

Amplification of epidermal growth factor receptor (EGFR) and its active mutant EGFRvIII occurs frequently in glioblastoma (GBM). While EGFR and EGFRvIII play critical roles in pathogenesis, targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs) or antibodies has only shown limited efficacy in patients. Here we discuss signaling pathways mediated by EGFR/EGFRvIII, current therapeutics, and novel strategies to target EGFR/EGFRvIII-amplified GBM.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

PubMed

Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

2005-01-01

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Construction of a reporter system to study Burkholderia mallei type III secretion and identification of the BopA effector protein function in intracellular survival.

PubMed

Whitlock, Gregory C; Estes, D Mark; Young, Glenn M; Young, Briana; Torres, Alfredo G

2008-12-01

Burkholderia mallei, the aetiological agent of glanders disease, is a Gram-negative facultative intracellular bacterium. Despite numerous studies, the detailed mechanism of its pathogenesis is almost unknown. The presence of a type III secretion system (TTSS) is one of the known mechanisms associated with virulence. An intact TTSS indicates that B. mallei is able to secrete proteins in response to different environmental conditions, which could play an important role in pathogenesis. Therefore, characterization of the TTSS and identification of the secreted proteins associated with bacterial pathogenesis could provide crucial information for the development of a candidate vaccine. In the current study, we used an enzymatic reporter system to establish some of the conditions enabling TTS. Construction of the TTSS bopA mutant revealed that BopA is important for B. mallei invasion and intracellular survival. Overall, our study elucidates how BopA can aid in the optimization of TTS and defines the function of TTS effectors in bacterial intracellular survival and invasion.

The effect of imiquimod on taste bud calcium transients and transmitter secretion

PubMed Central

Wu, Sandy Y

2016-01-01

Background and Purpose Imiquimod is an immunomodulator approved for the treatment of basal cell carcinoma and has adverse side effects, including taste disturbances. Paracrine transmission, representing cell–cell communication within taste buds, has the potential to shape the final signals that taste buds transmit to the brain. Here, we tested the underlying assumption that imiquimod modifies taste transmitter secretion in taste buds of mice. Experimental Approach Taste buds were isolated from C57BL/6J mice. The effects of imiquimod on transmitter release in taste buds were measured using calcium imaging with cellular biosensors, and examining the net effect of imiquimod on taste‐evoked ATP secretion from mouse taste buds. Key Results Up to 72% of presynaptic (Type III) taste cells responded to 100 μM imiquimod with an increase in intracellular Ca2+ concentrations. These Ca2 + responses were inhibited by thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2 +‐ATPase, and by U73122, a PLC inhibitor, suggesting that the Ca2 + mobilization elicited by imiquimod was dependent on release from internal Ca2 + stores. Moreover, combining studies of Ca2 + imaging with cellular biosensors showed that imiquimod evoked secretion of 5‐HT, which then provided negative feedback onto receptor (Type II) cells to reduce taste‐evoked ATP secretion. Conclusion and Implications Our results provide evidence that there is a subset of taste cells equipped with a range of intracellular mechanisms that respond to imiquimod. The findings are also consistent with a role of imiquimod as an immune response modifier, which shapes peripheral taste responses via 5‐HT signalling. PMID:27464850

Epithelial Cell–Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

PubMed Central

Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna C.; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.

2016-01-01

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia. PMID:27064756

Secrets of a secretin.

PubMed

Heinz, Dirk W

2013-12-03

Secretins are major constituents of bacterial type III secretion systems (T3SS). In this issue of Structure, Kowal and colleagues report on the cryo-EM structure of the native YscC secretin from Yersinia, revealing its internal symmetry and mode of length adaptation. Copyright © 2013 Elsevier Ltd. All rights reserved.

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells.

PubMed

Yao, Yufeng; Xie, Yi; Perace, Donna; Zhong, Yi; Lu, Jie; Tao, Jing; Guo, Xiaokui; Kim, Kwang Sik

2009-11-01

Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.

Prostaglandin E2 induces chloride secretion through crosstalk between cAMP and calcium signaling in mouse inner medullary collecting duct cells

PubMed Central

Rajagopal, Madhumitha; Thomas, Sheela V.; Kathpalia, Paru P.; Chen, Yu

2013-01-01

Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na+ channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (IscPGE2). We found that IscPGE2 was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl− secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that IscPGE2 was sensitive to inhibition by BAPTA-AM (Ca2+ chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca2+-activated Cl− channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca2+ to induce Cl− secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca2+ signaling; BAPTA-AM or 2-APB inhibited a component of IscPGE2 that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of IscPGE2 that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca2+ to stimulate Cl− secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake. PMID:24284792

Mechanism of transcription termination by RNA polymerase III utilizes a nontemplate-strand sequence-specific signal element

PubMed Central

Arimbasseri, Aneeshkumar G.; Maraia, Richard J.

2015-01-01

SUMMARY Understanding the mechanism of transcription termination by a eukaryotic RNA polymerase (RNAP) has been limited by lack of a characterizable intermediate that reflects transition from an elongation complex to a true termination event. While other multisubunit RNAPs require multipartite cis-signals and/or ancillary factors to mediate pausing and release of the nascent transcript from the clutches of these enzymes, RNAP III does so with precision and efficiency on a simple oligo(dT) tract, independent of other cis-elements or trans-factors. We report a RNAP III pre-termination complex that reveals termination mechanisms controlled by sequence-specific elements in the non-template strand. Furthermore, the TFIIF-like, RNAP III subunit, C37 is required for this function of the non-template strand signal. The results reveal the RNAP III terminator as an information-rich control element. While the template strand promotes destabilization via a weak oligo(rU:dA) hybrid, the non-template strand provides distinct sequence-specific destabilizing information through interactions with the C37 subunit. PMID:25959395

Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells.

PubMed

Ishihara, Seiichiro; Inman, David R; Li, Wan-Ju; Ponik, Suzanne M; Keely, Patricia J

2017-11-15

In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179-89. ©2017 AACR . ©2017 American Association for Cancer Research.

Type III Interferon-Mediated Signaling Is Critical for Controlling Live Attenuated Yellow Fever Virus Infection In Vivo

PubMed Central

Douam, Florian; Soto Albrecht, Yentli E.; Hrebikova, Gabriela; Sadimin, Evita; Davidson, Christian; Kotenko, Sergei V.

2017-01-01

ABSTRACT Yellow fever virus (YFV) is an arthropod-borne flavivirus, infecting ~200,000 people worldwide annually and causing about 30,000 deaths. The live attenuated vaccine strain, YFV-17D, has significantly contributed in controlling the global burden of yellow fever worldwide. However, the viral and host contributions to YFV-17D attenuation remain elusive. Type I interferon (IFN-α/β) signaling and type II interferon (IFN-γ) signaling have been shown to be mutually supportive in controlling YFV-17D infection despite distinct mechanisms of action in viral infection. However, it remains unclear how type III IFN (IFN-λ) integrates into this antiviral system. Here, we report that while wild-type (WT) and IFN-λ receptor knockout (λR−/−) mice were largely resistant to YFV-17D, deficiency in type I IFN signaling resulted in robust infection. Although IFN-α/β receptor knockout (α/βR−/−) mice survived the infection, mice with combined deficiencies in both type I signaling and type III IFN signaling were hypersusceptible to YFV-17D and succumbed to the infection. Mortality was associated with viral neuroinvasion and increased permeability of the blood-brain barrier (BBB). α/βR−/− λR−/− mice also exhibited distinct changes in the frequencies of multiple immune cell lineages, impaired T-cell activation, and severe perturbation of the proinflammatory cytokine balance. Taken together, our data highlight that type III IFN has critical immunomodulatory and neuroprotective functions that prevent viral neuroinvasion during active YFV-17D replication. Type III IFN thus likely represents a safeguard mechanism crucial for controlling YFV-17D infection and contributing to shaping vaccine immunogenicity. PMID:28811340

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

PubMed Central

Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

Regulation of aldosterone secretion by mineralocorticoid receptor-mediated signaling.

PubMed

Chong, Cherish; Hamid, Anis; Yao, Tham; Garza, Amanda E; Pojoga, Luminita H; Adler, Gail K; Romero, Jose R; Williams, Gordon H

2017-03-01

We posit the existence of a paracrine/autocrine negative feedback loop, mediated by the mineralocorticoid receptor (MR), regulating aldosterone secretion. To assess this hypothesis, we asked whether altering MR activity in zona glomerulosa (ZG) cells affects aldosterone production. To this end, we studied ex vivo ZG cells isolated from male Wistar rats fed chow containing either high (1.6% Na + (HS)) or low (0.03% Na + (LS)) amount of sodium. Western blot analyses demonstrated that MR was present in both the ZG and zona fasciculata/zona reticularis (ZF/ZR/ZR). In ZG cells isolated from rats on LS chow, MR activation by fludrocortisone produced a 20% and 60% reduction in aldosterone secretion basally and in response to angiotensin II (ANGII) stimulation, respectively. Corticosterone secretion was increased in these cells suggesting that aldosterone synthase activity was being reduced by fludrocortisone. In contrast, canrenoic acid, an MR antagonist, enhanced aldosterone production by up to 30% both basally and in response to ANGII. Similar responses were observed in ZG cells from rats fed HS. Modulating glucocorticoid receptor (GR) activity did not alter aldosterone production by ZG cells; however, altering GR activity did modify corticosterone production from ZF/ZR/ZR cells both basally and in response to adrenocorticotropic hormone (ACTH). Additionally, activating the MR in ZF/ZR/ZR cells strikingly reduced corticosterone secretion. In summary, these data support the hypothesis that negative ultra-short feedback loops regulate adrenal steroidogenesis. In the ZG, aldosterone secretion is regulated by the MR, but not the GR, an effect that appears to be secondary to a change in aldosterone synthase activity. © 2017 Society for Endocrinology.

The N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD

PubMed Central

Dickenson, Nicholas E.; Arizmendi, Olivia; Patil, Mrinalini K.; Toth, Ronald T.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

2014-01-01

The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri, providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is comprised of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g. deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. While the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study due to the hydrophobic nature of the IpaB and IpaC translocator proteins. Here we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11–27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation. PMID:24236510

Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

PubMed Central

Mao, Mao; Zhou, Liang

2017-01-01

We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases. PMID:28194184

Global Positioning System III (GPS III)

DTIC Science & Technology

2013-12-01

Galileo satellite navigation system signal, E1. L1C is also compatible with those signals planned for broadcast on Japan’s Quazi-Zenith Satellite...and Galileo constellations, further increasing the accuracy and availability of civil PNT solutions. GPS III December 2013 SAR April 16, 2014...vehicle- level core mate. The overall program continues to make progress on the GPS III Non-Flight Satellite Testbed (GNST), on SV01 development, and

Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

NASA Astrophysics Data System (ADS)

Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

2017-10-01

In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

Regulation of the Yersinia type III secretion system: traffic control

PubMed Central

Dewoody, Rebecca S.; Merritt, Peter M.; Marketon, Melanie M.

2013-01-01

Yersinia species, as well as many other Gram-negative pathogens, use a type III secretion system (T3SS) to translocate effector proteins from the bacterial cytoplasm to the host cytosol. This T3SS resembles a molecular syringe, with a needle-like shaft connected to a basal body structure, which spans the inner and outer bacterial membranes. The basal body of the injectisome shares a high degree of homology with the bacterial flagellum. Extending from the T3SS basal body is the needle, which is a polymer of a single protein, YscF. The distal end of the needle serves as a platform for the assembly of a tip complex composed of LcrV. Though never directly observed, prevailing models assume that LcrV assists in the insertion of the pore-forming proteins YopB and YopD into the host cell membrane. This completes a bridge between the bacterium and host cell to provide a continuous channel through which effectors are delivered. Significant effort has gone into understanding how the T3SS is assembled, how its substrates are recognized and how substrate delivery is controlled. Arguably the latter topic is the least understood; however, recent advances have provided new insight, and therefore, this review will focus primarily on summarizing the current state of knowledge regarding the control of substrate delivery by the T3SS. Specifically, we will discuss the roles of YopK, as well as YopN and YopE, which have long been linked to regulation of translocation. We also propose models whereby the YopK regulator communicates with the basal body of the T3SS to control translocation. PMID:23390616

Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III

PubMed Central

1987-01-01

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42- microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN

PubMed Central

2017-01-01

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. PMID:28619883

Identification and suppression of epidermal growth factor receptor variant III signaling in fibroblast-like synoviocytes from aggressive rheumatoid arthritis by the mimotope.

PubMed

Niu, Jianying; Li, Changhong; Jin, Yinji; Xing, Rui; Sun, Lin; Yu, Ruohan; Jian, Leilei; Liu, Xiangyuan; Yang, Lin

2018-06-01

Epidermal growth factor receptor (EGFR) signaling has been reported to play a vital role in the pathogenesis of rheumatoid arthritis (RA). In current study, we sought to observe whether the active immunization induced by the mimotope could recognize EGFR, inhibit their signaling and disrupt the pathogenic behavior of fibroblast-like synoviocytes (FLS) from RA patients. We prepared a linked EGFR mimotope and performed series of experiments to detect whether the mimotope could induce the desired immune responses. To our surprises, we detected the expression of EGFR variant III (EGFRvIII), but not EGFR in the synovial tissues and FLS from patients with aggressive RA by the linked EGFR mimotope-induced antibodies (LEMIA). Meanwhile, LEMIA could inhibit the signaling caused by the autophosphorylation of EGFRvIII in the FLS. The proliferation, migration, invasion and anti-apoptosis capabilities of the EGFRvIII-expressed FLS were disrupted by LEMIA. These results suggest that EGFRvIII signaling may participate in the malignant behaviors of FLS from aggressive RA. Meanwhile, the linked EGFR mimotope could be used to detect the expression of EGFRvIII and developed to be a potential therapy agent against the aggressive FLS. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets.

PubMed

Kashio, Makiko; Tominaga, Makoto

2015-05-08

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca(2+)-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H2O2, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca(2+)]i increases were likely caused by Ca(2+) influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca(2+). In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H2O2 application at 37 °C induced [Ca(2+)]i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H2O2 on KATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The bacterial alarmone (p)ppGpp activates the type III secretion system in Erwinia amylovora.

PubMed

Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In; Zhao, Youfu

2015-04-01

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp(0)) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp(0) and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp(0) and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation

The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

PubMed Central

Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In

2015-01-01

ABSTRACT The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for

Hoopoes color their eggs with antimicrobial uropygial secretions

NASA Astrophysics Data System (ADS)

Soler, Juan J.; Martín-Vivaldi, M.; Peralta-Sánchez, J. M.; Arco, L.; Juárez-García-Pelayo, N.

2014-09-01

Uropygial gland secretions are used as cosmetics by some species of birds to color and enhance properties of feathers and teguments, which may signal individual quality. Uropygial secretions also reach eggshells during incubation and, therefore, may influence the coloration of birds' eggs, a trait that has attracted the attention of evolutionary biologists for more than one century. The color of hoopoe eggs typically changes along incubation, from bluish-gray to greenish-brown. Here, we test experimentally the hypothesis that dark uropygial secretion of females is responsible for such drastic color change. Moreover, since uropygial secretion of hoopoes has antimicrobial properties, we also explore the association between color and antimicrobial activity of the uropygial secretion of females. We found that eggs stayed bluish-gray in nests where female access to the uropygial secretion was experimentally blocked. Furthermore, experimental eggs that were maintained in incubators and manually smeared with uropygial secretion experienced similar color changes that naturally incubated eggs did, while control eggs that were not in contact with the secretions did not experience such color changes. All these results strongly support the hypothesis that female hoopoes use their uropygial gland secretion to color the eggs. Moreover, saturation of the uropygial secretion was associated with antimicrobial activity against Bacillus licheniformis. Given the known antimicrobial potential of uropygial secretions of birds, this finding opens the possibility that in scenarios of sexual selection, hoopoes in particular and birds in general signal antimicrobial properties of their uropygial secretion by mean of changes in egg coloration along incubation.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

PubMed

Sharma, Shailendra; Sharma, Shiveta; Hirabuchi, Akiko; Yoshida, Kentaro; Fujisaki, Koki; Ito, Akiko; Uemura, Aiko; Terauchi, Ryohei; Kamoun, Sophien; Sohn, Kee Hoon; Jones, Jonathan D G; Saitoh, Hiromasa

2013-05-01

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Role of biphasic calcium phosphate ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs.

PubMed

Wang, Jing; Liu, Dan; Guo, Bo; Yang, Xiao; Chen, Xuening; Zhu, Xiangdong; Fan, Yujiang; Zhang, Xingdong

2017-03-15

The inflammatory reaction initiates fracture healing and could play a role in the osteoinductive effect of calcium phosphate (CaP) ceramics, which has been widely confirmed; however, the underlying mechanism has not been fully elucidated. In this study, various signaling molecules from macrophages under the stimulation of osteoinductive biphasic calcium phosphate (BCP) ceramic and its degradation products were examined and evaluated for their influence on the migration and osteoblastic differentiation of mesenchymal stem cells (MSCs). The results of cellular experiments confirmed that the gene expression of most inflammatory factors (IL-1, IL-6 and MCP-1) and growth factors (VEGF, PDGF and EGF) by macrophages were up-regulated to varying degrees by BCP ceramic and its degradation products. Cell migration tests demonstrated that the conditioned media (CMs), which contained abundant signaling molecules secreted by macrophages cultured on BCP ceramic and its degradation products, promoted the migration of MSCs. qRT-PCR analysis indicated that CMs promoted the gene expression of osteogenic markers (ALP, COL-I, OSX, BSP and OPN) in MSCs. ALP activity and mineralization staining further confirmed that CMs promoted the osteoblastic differentiation of MSCs. The present study confirmed the correlation between the inflammatory reaction and osteoinductive capacity of BCP ceramic. The ceramic itself and its degradation products can induce macrophages to express and secrete various signaling molecules, which then recruit and promote the MSCs to differentiate into osteoblasts. Compared with BCP conditioned media, degradation particles played a more substantial role in this process. Thus, inflammation initiated by BCP ceramic and its degradation products could be necessary for osteoinduction by the ceramic. It is known that the inflammatory reaction initiates fracture healing. The aim of this study was to examine whether osteoinductive BCP ceramics could cause macrophages to

A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

PubMed

Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

2015-11-10

Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

Reactions of green lizards (Lacerta viridis) to major repellent compounds secreted by Graphosoma lineatum (Heteroptera: Pentatomidae).

PubMed

Gregorovičová, Martina; Černíková, Alena

2015-06-01

The chemical defence of Heteroptera is primarily based on repellent secretions which signal the potential toxicity of the bug to its predators. We tested the aversive reactions of green lizards (Lacerta viridis) towards the major compounds of the defensive secretion of Graphosoma lineatum, specifically: (i) a mixture of three aldehydes: (E)-hex-2-enal, (E)-oct-2-enal, (E)-dec-2-enal; (ii) a mixture of these three aldehydes and tridecane; (iii) oxoaldehyde: (E)-4-oxohex-2-enal; (iv) secretion extracted from metathoracic scent glands of G. lineatum adults and (v) hexane as a non-polar solvent. All chemicals were presented on a palatable food (Tenebrio molitor larvae). The aversive reactions of the green lizards towards the mealworms were evaluated by observing the approach latencies, attack latencies and approach-attack intervals. The green lizards exhibited a strong aversive reaction to the mixture of three aldehydes. Tridecane reduced the aversive reaction to the aldehyde mixture. Oxoaldehyde caused the weakest, but still significant, aversive reaction. The secretion from whole metathoracic scent glands also clearly had an aversive effect on the green lizards. Moreover, when a living specimen of G. lineatum or Pyrrhocoris apterus (another aposematic red-and-black prey) was presented to the green lizards before the trials with the aldehyde mixture, the aversive effect of the mixture was enhanced. In conclusion, the mixture of three aldehydes had the strong aversive effect and could signal the potential toxicity of G. lineatum to the green lizards. Copyright © 2015 Elsevier GmbH. All rights reserved.

Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

PubMed Central

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

2008-01-01

Summary The plague-causing bacterium Yersinia pestis utilizes a Type III Secretion System (T3SS) to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as "chaperones" to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa T3SS, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat (TPR) family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal TPR motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the N-terminal 49 residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex. PMID:18281060

Structure of a bacterial type III secretion system in contact with a host membrane in situ.

PubMed

Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R; Hayward, Richard D

2015-12-11

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking 'pump-action' conformational changes that underpin effector injection.

Structure of a bacterial type III secretion system in contact with a host membrane in situ

NASA Astrophysics Data System (ADS)

Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

2015-12-01

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

PubMed

Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile

2015-11-23

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.

Secreted production of Renilla luciferase in Bacillus subtilis.

PubMed

Chiang, Chung-Jen; Chen, Po Ting; Chao, Yun-Peng

2010-01-01

Luciferase (Rluc) from the soft coral Renilla reniformis has been widely used as a bioluminescent reporter, and its secreted production has been solely performed in mammalian cells thus far. To make the production more efficient, a series of approaches was attempted to overproduce Rluc extracellularly in Bacillus subtilis. First, Cys124 in the Rluc gene was substituted with Ala. The mutant gene was subsequently incorporated into a pUB110/R6K-based plasmid, consequently, fusing with the P43 promoter and the sacB signal peptide. With the nitrogen-rich medium, B. subtilis strain bearing the plasmid became able to secret a detectable amount of Rluc. Moreover, the secretion signal for the Rluc gene was replaced by the aprN leader peptide with or without the propeptide. The result led to a more than twofold increase in the secreted Rluc. Finally, by enhancing the transcription of the Rluc gene implementing the P43 and spac tandem promoter, it resulted in the secreted Rluc with a yield of 100 mg/L. Overall, this study illustrates a potential strategy for improving the secretion efficiency of heterologous proteins in B. subtilis.

Functional cloning of Vibrio parahaemolyticus type III secretion system 1 in Escherichia coli K-12 strain as a molecular syringe.

PubMed

Akeda, Yukihiro; Kimura, Tomomi; Yamasaki, Aiko; Kodama, Toshio; Iida, Tetsuya; Honda, Takeshi; Oishi, Kazunori

2012-10-19

The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coli K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coli K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. coli K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications. Copyright © 2012 Elsevier Inc. All rights reserved.

Angiogenic inhibitors delivered by the type III secretion system of tumor-targeting Salmonella typhimurium safely shrink tumors in mice.

PubMed

Shi, Lei; Yu, Bin; Cai, Chun-Hui; Huang, Jian-Dong

2016-12-01

Despite of a growing number of bacterial species that apparently exhibit intrinsic tumor-targeting properties, no bacterium is able to inhibit tumor growth completely in the immunocompetent hosts, due to its poor dissemination inside the tumors. Oxygen and inflammatory reaction form two barriers and restrain the spread of the bacteria inside the tumors. Here, we engineered a Salmonella typhimurium strain named ST8 which is safe and has limited ability to spread beyond the anaerobic regions of tumors. When injected systemically to tumor-bearing immunocompetent mice, ST8 accumulated in tumors at levels at least 100-fold greater than parental obligate anaerobic strain ST4. ST8/pSEndo harboring therapeutic plasmids encoding Endostatin fused with a secreted protein SopA could target vasculature at the tumor periphery, can stably maintain and safely deliver a therapeutic vector, release angiogenic inhibitors through a type III secretion system (T3SS) to interfere with the pro-angiogenic action of growth factors in tumors. Mice with murine CT26 colon cancer that had been injected with ST8/pSEndo showed efficient tumor suppression by inducing more severe necrosis and inhibiting blooding vessel density within tumors. Our findings provide a therapeutic platform for indirectly acting therapeutic strategies such as anti-angiogenesis and immune therapy.

Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

PubMed

Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K

2010-02-05

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Je Min, E-mail: jemin@knu.ac.kr; Department of Horticultural Science, Kyungpook National University, Daegu; Lee, Sang-Jik

Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function inmore » fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.« less

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System*

PubMed Central

Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H.; Wolf-Watz, Magnus

2017-01-01

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. PMID:28039361

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System.

PubMed

Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H; Wolf-Watz, Magnus

2017-02-24

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia , the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscU C binds to the inner rod protein YscI with a dissociation constant ( K d ) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

DOE PAGES

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

2014-12-08

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

Exonuclease III-assisted cascade signal amplification strategy for label-free and ultrasensitive electrochemical detection of nucleic acids.

PubMed

Xiong, Erhu; Yan, Xiaoxia; Zhang, Xiaohua; Liu, Yunqing; Zhou, Jiawan; Chen, Jinhua

2017-01-15

In this work, a simple, signal-on and label-free electrochemical biosensor for ultrasensitive DNA detection is reported on the basis of an autocatalytic and exonuclease III (Exo III)-assisted cascade signal amplification strategy. In the presence of target DNA (T-DNA), the hybridization between the 3'-protruding DNA fragment of hairpin DNA probe (HP1) and T-DNA triggered the Exo III cleavage process, accompanied by the releasing of T-DNA and autonomous generation of new DNA fragment which was used for the successive hybridization with the another hairpin DNA (HP2) on the electrode. After the Exo III cleavage process, numerous quadruplex-forming oligomers which caged in HP2 were liberated on the electrode surface and folded into G-quadruplex-hemin complexes with the help of K + and hemin to give a remarkable electrochemical response. As a result, a low detection limit of 4.83fM with an excellent selectivity toward T-DNA was achieved. The developed electrochemical biosensor should be further extended for the detection of a wide spectrum of analytes and has great potential for the development of ultrasensitive biosensing platform for early diagnosis in gene-related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

PubMed

Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

2018-03-01

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

Structural and regulatory mutations in Vibrio parahaemolyticus type III secretion systems display variable effects on virulence.

PubMed

Calder, Thomas; de Souza Santos, Marcela; Attah, Victoria; Klimko, John; Fernandez, Jessie; Salomon, Dor; Krachler, Anne-Marie; Orth, Kim

2014-12-01

The Gram-negative bacterium, Vibrio parahaemolyticus, is a major cause of seafood-derived food poisoning throughout the world. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two type III secretion systems (T3SS), T3SS1 and T3SS2. Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus of either system, to V. parahaemolyticus CAB strains, which harbor mutations in positive transcriptional regulators of either system. These strains are derived from the clinical RIMD 2210633 strain. We demonstrate that each mutation affects the virulence of the bacterium in a different manner. POR and CAB strains exhibited similar levels of swarming motility and T3SS effector production and secretion, but the CAB3 and CAB4 strains, which harbor a mutation in the T3SS2 master regulator gene, formed reduced biofilm growth under T3SS2 inducing conditions. Additionally, while the cytotoxicity of the POR and CAB strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, creating structural or regulatory mutations in either T3SS1 or T3SS2 causes differential downstream effects on other virulence systems. Understanding the biological differences of strains created from a clinical isolate is critical for interpreting and understanding the pathogenic nature of V. parahaemolyticus. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

PubMed

Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

2017-03-16

Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

Formulation and Immunogenicity studies of Type III Secretion System needle antigens as Vaccine Candidates

PubMed Central

Barrett, Brooke S.; Markham, Aaron P.; Esfandiary, Reza; Picking, Wendy L.; Picking, William D.; Joshi, Sangeeta B.; Middaugh, C. Russell

2013-01-01

Bacterial infections caused by Shigella flexneri, Salmonella typhimurium and Burkholderia pseudomallei are currently difficult to prevent due to the lack of a licensed vaccine. Here we present formulation and immunogenicity studies for the three type III secretion system (TTSS) needle proteins MxiHΔ5, PrgIΔ5 and BsaLΔ5 (each truncated by five residues at its C terminus) as potential candidates for vaccine development. These antigens are found to be thermally stabilized by the presence of carbohydrates and polyols. Additionally, all adsorb readily to aluminum hydroxide apparently through a combination of hydrogen bonds and/or Van der Waals forces. The interaction of these proteins with the aluminum-based adjuvant changes with time to resulting in varying degrees of irreversible binding. Peptide maps of desorbed protein, however, suggest that chemical changes are not responsible for this irreversible association. The ability of MxiHΔ5 and PrgIΔ5 to elicit strong humoral immune responses was tested in a murine model. When administered intramuscularly as monomers, the needle components exhibited dose dependent immunogenic behavior. The polymerized version of MxiH was exceptionally immunogenic even at low doses. The responses of both monomeric and polymerized forms were boosted by adsorption to an aluminum salt adjuvant. PMID:20845448

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia.

PubMed

Norris, J W; Pombo, M; Shirley, E; Blevins, G; Tablin, F

2015-01-01

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. Two affected and 6 clinically normal horses. Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1). Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia. Copyright © The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

PubMed

Almario, R U; Karakas, S E

2015-02-01

Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (p<5×10(-6) as compared to both PCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

Information Theoretic Secret Key Generation: Structured Codes and Tree Packing

ERIC Educational Resources Information Center

Nitinawarat, Sirin

2010-01-01

This dissertation deals with a multiterminal source model for secret key generation by multiple network terminals with prior and privileged access to a set of correlated signals complemented by public discussion among themselves. Emphasis is placed on a characterization of secret key capacity, i.e., the largest rate of an achievable secret key,…

Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors.

PubMed

Sperschneider, Jana; Williams, Angela H; Hane, James K; Singh, Karam B; Taylor, Jennifer M

2015-01-01

The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Minireview: Dopaminergic Regulation of Insulin Secretion from the Pancreatic Islet

PubMed Central

Ustione, Alessandro

2013-01-01

Exogenous dopamine inhibits insulin secretion from pancreatic β-cells, but the lack of dopaminergic neurons in pancreatic islets has led to controversy regarding the importance of this effect. Recent data, however, suggest a plausible physiologic role for dopamine in the regulation of insulin secretion. We review the literature underlying our current understanding of dopaminergic signaling that can down-regulate glucose-stimulated insulin secretion from pancreatic islets. In this negative feedback loop, dopamine is synthesized in the β-cells from circulating l-dopa, serves as an autocrine signal that is cosecreted with insulin, and causes a tonic inhibition on glucose-stimulated insulin secretion. On the whole animal scale, l-dopa is produced by cells in the gastrointestinal tract, and its concentration in the blood plasma increases following a mixed meal. By reviewing the outcome of certain types of bariatric surgery that result in rapid amelioration of glucose tolerance, we hypothesize that dopamine serves as an “antiincretin” signal that counterbalances the stimulatory effect of glucagon-like peptide 1. PMID:23744894

Intracellular Action of a Secreted Peptide Required for Fungal Virulence.

PubMed

Homer, Christina M; Summers, Diana K; Goranov, Alexi I; Clarke, Starlynn C; Wiesner, Darin L; Diedrich, Jolene K; Moresco, James J; Toffaletti, Dena; Upadhya, Rajendra; Caradonna, Ippolito; Petnic, Sarah; Pessino, Veronica; Cuomo, Christina A; Lodge, Jennifer K; Perfect, John; Yates, John R; Nielsen, Kirsten; Craik, Charles S; Madhani, Hiten D

2016-06-08

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence. Copyright © 2016 Elsevier Inc. All rights reserved.

Tumor-secreted PGE2 inhibits CCL5 production in activated macrophages through cAMP/PKA signaling pathway.

PubMed

Qian, Xuesong; Zhang, Jidong; Liu, Jianguo

2011-01-21

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Impaired Processing of Prohormones in Secretogranin III-Null Mice Causes Maladaptation to an Inadequate Diet and Stress.

PubMed

Maeda, Yoshinori; Kudo, Saki; Tsushima, Ken; Sato, Eri; Kubota, Chisato; Kayamori, Aika; Bochimoto, Hiroki; Koga, Daisuke; Torii, Seiji; Gomi, Hiroshi; Watanabe, Tsuyoshi; Hosaka, Masahiro

2018-02-01

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors. Copyright © 2018 Endocrine Society.

β-Hydroxybutyric sodium salt inhibition of growth hormone and prolactin secretion via the cAMP/PKA/CREB and AMPK signaling pathways in dairy cow anterior pituitary cells.

PubMed

Fu, Shou-Peng; Wang, Wei; Liu, Bing-Run; Yang, Huan-Min; Ji, Hong; Yang, Zhan-Qing; Guo, Bin; Liu, Ju-Xiong; Wang, Jian-Fa

2015-02-16

β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows.

Identification of secreted bacterial proteins by noncanonical amino acid tagging

PubMed Central

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

2014-01-01

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

Subversion of plant cellular functions by bacterial type-III effectors: beyond suppression of immunity.

PubMed

Macho, Alberto P

2016-04-01

Most bacterial plant pathogens employ a type-III secretion system to inject type-III effector (T3E) proteins directly inside plant cells. These T3Es manipulate host cellular processes in order to create a permissive niche for bacterial proliferation, allowing development of the disease. An important role of T3Es in plant pathogenic bacteria is the suppression of plant immune responses. However, in recent years, research has uncovered T3E functions different from direct immune suppression, including the modulation of plant hormone signaling, metabolism or organelle function. This insight article discusses T3E functions other than suppression of immunity, which may contribute to the modulation of plant cells in order to promote bacterial survival, nutrient release, and bacterial replication and dissemination. © 2015 The Author. New Phytologist © 2015 New Phytologist Trust.

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

PubMed Central

Xu, Xuefang; McAteer, Sean P.; Tree, Jai J.; Shaw, Darren J.; Wolfson, Eliza B. K.; Beatson, Scott A.; Roe, Andrew J.; Allison, Lesley J.; Chase-Topping, Margo E.; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E. J.; Morabito, Stefano; Gally, David L.

2012-01-01

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and Exonuclease III assisted signal amplification.

PubMed

Huang, Ke-Jing; Shuai, Hong-Lei; Zhang, Ji-Zong

2016-03-15

A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM. Copyright © 2015 Elsevier B.V. All rights reserved.

Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

PubMed Central

Clarke, Douglas N.; Al Ahmad, Abraham; Lee, Boyeon; Parham, Christi; Auckland, Lisa; Fertala, Andrezj; Kahle, Michael; Shaw, Courtney S.; Roberts, Jill; Bix, Gregory J.

2012-01-01

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy. PMID:23028886

A new role for Hedgehogs in juxtacrine signaling.

PubMed

Pettigrew, Christopher A; Asp, Eva; Emerson, Charles P

2014-02-01

The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A cascade signal amplification strategy for sensitive and label-free DNA detection based on Exo III-catalyzed recycling coupled with rolling circle amplification.

PubMed

Liu, Xingti; Xue, Qingwang; Ding, Yongshun; Zhu, Jing; Wang, Lei; Jiang, Wei

2014-06-07

A sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining exonuclease III (Exo III)-catalyzed recycling with rolling circle amplification. In this assay, probe DNA hybridized with template DNA was coupled onto magnetic nanoparticles to prepare a magnetic bead-probe (MNB-probe)-template complex. The complex could hybridize with the target DNA, which transformed the protruding 3' terminus of template DNA into a blunt end. Exo III could then digest template DNA, liberating the MNB-probe and target DNA. The intact target DNA then hybridized with other templates and released more MNB-probes. The liberated MNB-probe captured the primer, circular DNA and then initiated the rolling circle amplification (RCA) reaction, realizing a cascade signal amplification. Using this cascade amplification strategy, a sensitive DNA detection method was developed which was superior to many existing Exo III-based signal amplification methods. Moreover, N-methyl mesoporphyrin IX, which had a pronounced structural selectivity for the G-quadruplex, was used to combine with the G-quadruplex RCA products and generate a fluorescence signal, avoiding the need for any fluorophore-label probes. The spike and recovery experiments in a human serum sample indicated that our assay also had great potential for DNA detection in real biological samples.

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis

PubMed Central

Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A.; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M.; Maitra, Sushmit; Thomas, Stephen G.; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F.; Olszewski, Neil E.; Sun, Tai-ping

2016-01-01

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein–protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors—PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)—that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. PMID:26773002

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

PubMed

Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M; Maitra, Sushmit; Thomas, Stephen G; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E; Sun, Tai-Ping

2016-01-15

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. © 2016 Zentella et al.; Published by Cold Spring Harbor Laboratory Press.

Acquired Type III Secretion System Determines Environmental Fitness of Epidemic Vibrio parahaemolyticus in the Interaction with Bacterivorous Protists

PubMed Central

Matz, Carsten; Nouri, Bianka; McCarter, Linda; Martinez-Urtaza, Jaime

2011-01-01

Genome analyses of marine microbial communities have revealed the widespread occurrence of genomic islands (GIs), many of which encode for protein secretion machineries described in the context of bacteria-eukaryote interactions. Yet experimental support for the specific roles of such GIs in aquatic community interactions remains scarce. Here, we test for the contribution of type III secretion systems (T3SS) to the environmental fitness of epidemic Vibrio parahaemolyticus. Comparisons of V. parahaemolyticus wild types and T3SS-defective mutants demonstrate that the T3SS encoded on genome island VPaI-7 (T3SS-2) promotes survival of V. parahaemolyticus in the interaction with diverse protist taxa. Enhanced persistence was found to be due to T3SS-2 mediated cytotoxicity and facultative parasitism of V. parahaemolyticus on coexisting protists. Growth in the presence of bacterivorous protists and the T3SS-2 genotype showed a strong correlation across environmental and clinical isolates of V. parahaemolyticus. Short-term microcosm experiments provide evidence that protistan hosts facilitate the invasion of T3SS-2 positive V. parahaemolyticus into a coastal plankton community, and that water temperature and productivity further promote enhanced survival of T3SS-2 positive V. parahaemolyticus. This study is the first to describe the fitness advantage of GI-encoded functions in a microbial food web, which may provide a mechanistic explanation for the global spread and the seasonal dynamics of V. parahaemolyticus pathotypes, including the pandemic serotype cluster O3:K6, in aquatic environments. PMID:21629787

Detection of the type III secretion system and its phylogenetic and symbiotic characterization in peanut bradyrhizobia isolated from Guangdong Province, China.

PubMed

Ruan, Huaqin; Hu, Meijuan; Chen, Jingyu; Li, Xue; Li, Ting; Lai, Yongxiu; Wang, En Tao; Gu, Jun

2018-04-08

The distribution of rhcRST and rhcJ-C1 fragments located in different loci of the type III secretion system (T3SS) gene cluster in the peanut-nodulating bradyrhizobia isolated from Guangdong Province, China was investigated by PCR-based sequencing. T3SS was detected in approximately one-third of the peanut bradyrhizobial strains and the T3SS-harboring strains belonging to different Bradyrhizobium genomic species. Diverse T3SS groups corresponding to different symbiotic gene types were defined among the 23 T3SS-harboring strains. The same or similar T3SS genes were detected in different genospecies, indicating that interspecies horizontal transfer of symbiotic genes had occurred in the Bradyrhizobium genus. Copyright © 2018. Published by Elsevier GmbH.

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

PubMed

Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

2015-05-01

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

PubMed Central

Ausmees, Nora; Kobayashi, Hajime; Deakin, William J.; Marie, Corinne; Krishnan, Hari B.; Broughton, William J.; Perret, Xavier

2004-01-01

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect. PMID:15231809

Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.

PubMed

Zenk, Sebastian F; Stabat, David; Hodgkinson, Julie L; Veenendaal, Andreas K J; Johnson, Steven; Blocker, Ariel J

2007-08-01

Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

PubMed

Teper, Doron; Girija, Anil Madhusoodana; Bosis, Eran; Popov, Georgy; Savidor, Alon; Sessa, Guido

2018-01-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

GLP-1 secretion is stimulated by 1,10-phenanthroline via colocalized T2R5 signal transduction in human enteroendocrine L cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jiyoung; Kim, Ki-Suk; Kim, Kang-Hoon

Glucagon-like peptide-1 (GLP-1) hormone is known to regulate blood glucose by an insulinotropic effect and increases proliferation as and also prevents apoptosis of pancreatic β cells. We know that GLP-1 is secreted by nutrients such as fatty acids and sweet compounds but also bitter compounds via stimulation of G-protein coupled receptors (GPCRs) in the gut. Among these, bitter compounds are multiply-contained in phytochemicals or artificial materials and perceived as ligands of various bitter taste receptors. We hypothesized that GLP-1 hormone is secreted through stimulation of a single bitter taste receptor by 1,10-phenanthroline which is known agonist of taste receptor typemore » 2 member 5 (T2R5). To prove this hypothesis, we used the representatively well-known 1,10-phenanthroline as ligand of single receptor and evaluated the existence of T2R5 by double-labeling immunofluorescence and then 1,10-phenanthroline is able to secrete GLP-1 hormone through stimulation of T2R5 in human enteroendocrine cells. Consequently, we verify that GLP-1 hormone is colocalized with T2R5 in the human duodenum and ileum tissue and is secreted by 1,10-phenanthroline via T2R5 signal transduction in differentiated human enteroendocrine L cells. - Highlights: • Taste receptor type 2 member 5 (T2R5) is colocalized with GLP-1 hormone in human enteroendocrine cells. • GLP-1 secretion is stimulated by 1,10-phenanthroline via stimulation of T2R5. • Inhibition of the bitter taste pathway reduce GLP-1 secretion.« less

Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic β-cell line and mouse islets.

PubMed

da Silva Krause, Mauricio; Bittencourt, Aline; Homem de Bittencourt, Paulo Ivo; McClenaghan, Neville H; Flatt, Peter R; Murphy, Colin; Newsholme, Philip

2012-09-01

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24  h) was increased from 128·7±7·3 (no IL6) to 178·4±7·7 (at 100  pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5·5  mM glucose for 2  h, from 0·148 to 0·167±0·003  ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.

Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

PubMed

Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

2016-01-01

Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.

The YopJ superfamily of type III efforts in plant-associated bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...

Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors

PubMed Central

Karlskås, Ingrid Lea; Maudal, Kristina; Axelsson, Lars; Rud, Ida; Eijsink, Vincent G. H.; Mathiesen, Geir

2014-01-01

We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species. PMID:24614815

Magnetic Nature of the CrIII-LnIII Interactions in [CrIII2LnIII3] Clusters with Slow Magnetic Relaxation.

PubMed

Zhao, Xiao-Qing; Xiang, Shuo; Wang, Jin; Bao, Dong-Xu; Li, Yun-Chun

2018-02-01

Two 3 d -4 f hetero-metal pentanuclear complexes with the formula {[Cr III 2 Ln III 3 L 10 (OH) 6 (H 2 O) 2 ]Et 3 NH} [Ln=Tb ( 1 ), Dy ( 2 ); HL=pivalic acid, Et 3 N=triethylamine] have been produced. The metal core of each cluster is made up of a trigonal bipyramid with three Ln III ions (plane) and two Cr III ions (above and below) held together by six μ 3 -OH bridges. Also reported with this series is the diamagnetic Cr III -Y III analogue ( 3 ). Fortunately, we successfully prepared Al III -Ln III analogues with the formula {[Al III 2 Ln III 3 L 10 (OH) 6 (H 2 O) 2 ]Et 3 NH⋅H 2 O} [Ln=Tb ( 4 ), Dy ( 5 )], containing diamagnetic Al III ions, which can be used to evaluate the Cr III -Ln III magnetic nature through a diamagnetic substitution method. Subsequently, static (dc) magnetic susceptibility studies reveal dominant ferromagnetic interactions between Cr III and Ln III ions. Dynamic (ac) magnetic susceptibility studies show frequency-dependent out-of-phase ( χ '') signals for [Cr III 2 Tb III 3 ] ( 1 ), [Cr III 2 Dy III 3 ] ( 2 ), and [Al III 2 Dy III 3 ] ( 5 ), which are derived from the single-ion behavior of Ln III ions and/or the Cr III -Ln III ferromagnetic interactions.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

PubMed

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-12-26

To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling

PubMed Central

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2015-01-01

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed s

A novel gene's role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior-posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos.

PubMed

Khadka, Anita; Martínez-Bartolomé, Marina; Burr, Stephanie D; Range, Ryan C

2018-01-01

The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior-posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8-Fzl5/8-JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8-Fzl5/8-JNK signaling pathway throughout ANE restriction, providing precise

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells.

PubMed

Almaça, Joana; Molina, Judith; Menegaz, Danusa; Pronin, Alexey N; Tamayo, Alejandro; Slepak, Vladlen; Berggren, Per-Olof; Caicedo, Alejandro

2016-12-20

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT 1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT 1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity. Published by Elsevier Inc.

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E.

PubMed

McNally, R Ryan; Zeng, Quan; Sundin, George W

2016-05-20

Many Gram-negative bacterial pathogens mediate host-microbe interactions via utilization of the type III secretion (T3S) system. The T3S system is a complex molecular machine consisting of more than 20 proteins. Collectively, these proteins translocate effectors across extracellular space and into the host cytoplasm. Successful translocation requires timely synthesis and allocation of both structural and secreted T3S proteins. Based on amino acid conservation in animal pathogenic bacteria, HrcU and HrpP were examined for their roles in regulation of T3S hierarchy. Both HrcU and HrpP were shown to be required for disease development in an immature pear infection model and respective mutants were unable to induce a hypersensitive response in tobacco. Using in vitro western blot analyses, both proteins were also shown to be required for the secretion of DspA/E, a type 3 effector and an important pathogenicity factor. Via yeast-two hybridization (Y2H), HrpP and HrcU were revealed to exhibit protein-protein binding. Finally, all HrcU and HrpP phenotypes identified were shown to be dependent on a conserved amino acid motif in the cytoplasmic tail of HrcU. Collectively, these data demonstrate roles for HrcU and HrpP in regulating T3S and represent the first attempt in understanding T3S heirarchy in E. amylovora.

Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition

PubMed Central

Condon, Jennifer C.; Jeyasuria, Pancharatnam; Faust, Julie M.; Mendelson, Carole R.

2004-01-01

Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung. In the mouse, secretion of the major lung surfactant protein, surfactant protein A (SP-A), was first detected in amniotic fluid (AF) at 17 days postcoitum, rising progressively to term (19 days postcoitum). Expression of IL-1β in AF macrophages and activation of NF-κB in the maternal uterus increased with the gestational increase in SP-A. SP-A stimulated IL-1β and NF-κB expression in cultured AF macrophages. Studies using Rosa 26 Lac-Z (B6;129S-Gt(rosa)26Sor) (Lac-Z) mice revealed that fetal AF macrophages migrate to the uterus with the gestational increase in AF SP-A. Intraamniotic (i.a.) injection of SP-A caused preterm delivery of fetuses within 6-24 h. By contrast, injection of an SP-A antibody or NF-κB inhibitor into AF delayed labor by >24 h. We propose that augmented production of SP-A by the fetal lung near term causes activation and migration of fetal AF macrophages to the maternal uterus, where increased production of IL-1β activates NF-κB, leading to labor. We have revealed a response pathway that ties augmented surfactant production by the maturing fetal lung to the initiation of labor. We suggest that SP-A secreted by the fetal lung serves as a hormone of parturition. PMID:15044702

Exosome secretion affects social motility in Trypanosoma brucei

PubMed Central

Shaked, Hadassa; Arvatz, Gil; Tkacz, Itai Dov; Binder, Lior; Waldman Ben-Asher, Hiba; Okalang, Uthman; Chikne, Vaibhav; Cohen-Chalamish, Smadar; Michaeli, Shulamit

2017-01-01

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. PMID:28257521

Type I and Type III Interferons Display Different Dependency on Mitogen-Activated Protein Kinases to Mount an Antiviral State in the Human Gut.

PubMed

Pervolaraki, Kalliopi; Stanifer, Megan L; Münchau, Stephanie; Renn, Lynnsey A; Albrecht, Dorothee; Kurzhals, Stefan; Senís, Elena; Grimm, Dirk; Schröder-Braunstein, Jutta; Rabin, Ronald L; Boulant, Steeve

2017-01-01

Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that

Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis

PubMed Central

Yu, Wenqi; Missiakas, Dominique

2018-01-01

Surface proteins of Staphylococcus aureus are secreted across septal membranes for assembly into the bacterial cross-wall. This localized secretion requires the YSIRK/GXXS motif signal peptide, however the mechanisms supporting precursor trafficking are not known. We show here that the signal peptide of staphylococcal protein A (SpA) is cleaved at the YSIRK/GXXS motif. A SpA signal peptide mutant defective for YSIRK/GXXS cleavage is also impaired for septal secretion and co-purifies with SecA, SecDF and LtaS. SecA depletion blocks precursor targeting to septal membranes, whereas deletion of secDF diminishes SpA secretion into the cross-wall. Depletion of LtaS blocks lipoteichoic acid synthesis and abolishes SpA precursor trafficking to septal membranes. We propose a model whereby SecA directs SpA precursors to lipoteichoic acid-rich septal membranes for YSIRK/GXXS motif cleavage and secretion into the cross-wall. PMID:29757141

Intrinsic optical signal imaging of glucose-stimulated physiological responses in the insulin secreting INS-1 β-cell line

NASA Astrophysics Data System (ADS)

Li, Yi-Chao; Cui, Wan-Xing; Wang, Xu-Jing; Amthor, Franklin; Yao, Xin-Cheng

2011-03-01

Intrinsic optical signal (IOS) imaging has been established for noninvasive monitoring of stimulus-evoked physiological responses in the retina and other neural tissues. Recently, we extended the IOS imaging technology for functional evaluation of insulin secreting INS-1 cells. INS-1 cells provide a popular model for investigating β-cell dysfunction and diabetes. Our experiments indicate that IOS imaging allows simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid image sequences reveal transient optical responses that have time courses comparable to glucose-evoked β-cell electrical activities.

Protein-Phospholipid Interactions in Nonclassical Protein Secretion: Problem and Methods of Study

PubMed Central

Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh; Sterling, Sarah; Neivandt, David

2013-01-01

Extracellular proteins devoid of signal peptides use nonclassical secretion mechanisms for their export. These mechanisms are independent of the endoplasmic reticulum and Golgi. Some nonclassically released proteins, particularly fibroblast growth factors (FGF) 1 and 2, are exported as a result of their direct translocation through the cell membrane. This process requires specific interactions of released proteins with membrane phospholipids. In this review written by a cell biologist, a structural biologist and two membrane engineers, we discuss the following subjects: (i) Phenomenon of nonclassical protein release and its biological significance; (ii) Composition of the FGF1 multiprotein release complex (MRC); (iii) The relationship between FGF1 export and acidic phospholipid externalization; (iv) Interactions of FGF1 MRC components with acidic phospholipids; (v) Methods to study the transmembrane translocation of proteins; (vi) Membrane models to study nonclassical protein release. PMID:23396106

TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

2011-11-18

Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful

NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid β secretion.

PubMed

Massone, Sara; Ciarlo, Eleonora; Vella, Serena; Nizzari, Mario; Florio, Tullio; Russo, Claudio; Cancedda, Ranieri; Pagano, Aldo

2012-07-01

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid β peptide (Aβ) secretion and the concomitant increment of Aβ x-42/Aβ x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aβ formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aβ peptides in the extracellular space, as it may occur in Alzheimer's Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Enhanced secretion of natto phytase by Bacillus subtilis.

PubMed

Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

2015-01-01

Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.

PubMed

Anderson, Gregory G; Yahr, Timothy L; Lovewell, Rustin R; O'Toole, George A

2010-03-01

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes.

The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector

PubMed Central

Habyarimana, Fabien; Sabag-Daigle, Anice

2014-01-01

Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification. PMID:24727228

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

NASA Astrophysics Data System (ADS)

Demers, Jean-Philippe; Habenstein, Birgit; Loquet, Antoine; Kumar Vasa, Suresh; Giller, Karin; Becker, Stefan; Baker, David; Lange, Adam; Sgourakis, Nikolaos G.

2014-09-01

We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion

PubMed Central

Kulkarni, Surashree S.; Zhu, Yongtao; Brendel, Colton J.

2017-01-01

ABSTRACT Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion. IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes. Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh

2012-03-23

Highlights: Black-Right-Pointing-Pointer RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. Black-Right-Pointing-Pointer RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. Black-Right-Pointing-Pointer Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. Black-Right-Pointing-Pointer RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate themore » involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.« less

Optical properties of the uropygial gland secretion: no evidence for UV cosmetics in birds

NASA Astrophysics Data System (ADS)

Delhey, Kaspar; Peters, Anne; Biedermann, Peter H. W.; Kempenaers, Bart

2008-10-01

Ultraviolet (UV) reflectance of the plumage is common in birds and plays an important role in sexual signalling. Recently, it has been proposed that birds are able to modify plumage UV reflectance by the application of uropygial gland secretion. Based on a survey of the optical properties of this secretion from 51 species belonging to 12 avian orders, we show that two main types of uropygial secretions exist, one predominantly found in passerines and one in non-passerines, both reducing relative UV reflectance of a white background (Teflon™ tape). We quantified how each type of secretion (exemplified by blue tit and mallard) affected feather UV reflectance. Both secretions reduced overall brightness and relative UV reflectance of white mallard feathers but hardly affected the reflectance of UV/blue blue tit crown feathers. According to models of avian colour vision, changes in reflectance due to application of the secretion were at or below the discrimination threshold of most birds. We conclude that the uropygial secretion is unlikely to play a major role in modifying plumage UV reflectance. However, the optical properties of the uropygial secretion may have been selected to interfere as little as possible with visual signaling through plumage reflectance.

Unconventional Secretion of Heat Shock Proteins in Cancer

PubMed Central

Santos, Tiago Góss; Martins, Vilma Regina; Hajj, Glaucia Noeli Maroso

2017-01-01

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies. PMID:28468249

Effect of Boron on Thymic Cytokine Expression, Hormone Secretion, Antioxidant Functions, Cell Proliferation, and Apoptosis Potential via the Extracellular Signal-Regulated Kinases 1 and 2 Signaling Pathway.

PubMed

Jin, Erhui; Ren, Man; Liu, Wenwen; Liang, Shuang; Hu, Qianqian; Gu, Youfang; Li, Shenghe

2017-12-27

Boron is an essential trace element in animals. Appropriate boron supplementation can promote thymus development; however, a high dose of boron can lead to adverse effects and cause toxicity. The influencing mechanism of boron on the animal body remains unclear. In this study, we examined the effect of boron on cytokine expression, thymosin and thymopoietin secretion, antioxidant function, cell proliferation and apoptosis, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the thymus of rats. We found that supplementation with 10 and 20 mg/L boron to the drinking water significantly elevated levels of interleukin 2 (IL-2), interferon γ (IFN-γ), interleukin 4 (IL-4), and thymosin α1 in the thymus of rats (p < 0.05), increased the number of positive proliferating cell nuclear antigen (PCNA + ) cells and concentrations of glutathione peroxidase (GSH-Px) and phosphorylated extracellular signal-regulated kinase (p-ERK) (p < 0.05), and promoted mRNA expression of PCNA and ERK1/2 in thymocytes (p < 0.05). However, the number of caspase-3 + cells and the expression level of caspase-3 mRNA were reduced (p < 0.05). Supplementation with 40, 80, and 160 mg/L boron had no apparent effect on many of the above indicators. In contrast, supplementation with 480 and 640 mg/L boron had the opposite effect on the above indicators in rats and elevated levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) (p < 0.05). Our study showed that supplementation of various doses of boron to the drinking water had a U-shaped dose-effect relationship with thymic cytokine expression, hormone secretion, antioxidant function, cell proliferation, and apoptosis. Specifically, supplementation with 10 and 20 mg/L boron promoted thymocyte proliferation and enhanced thymic functions. However, supplementation with 480 and 640 mg/L boron inhibited thymic functions and increased the number of apoptotic

Polydopamine nanotube mediated fluorescent biosensor for Hg(ii) determination through exonuclease III-assisted signal amplification.

PubMed

A, Ravikumar; P, Panneerselvam

2018-05-29

We describe a highly sensitive fluorescence biosensor incorporating polydopamine nanotubes (PDNTs) based on the mechanism of exonuclease III (Exo III) assisted signal amplification for the determination of Hg2+ in aqueous solution. Fluorescent probes of FAM labeled ssDNA (FAM-ssDNA) adsorbed on the PDNTs act as an efficient quencher. In the presence of Hg2+, the FAM-ssDNA can bind to Hg2+ to form double stranded DNA (dsDNA) via the formation of T-Hg2+-T base pairs. Then, the dsDNA was removed from the surface of the PDNTs to restore the fluorescence. The release of the dsDNA was triggered by Exo III digestion. At the same time, the liberated Hg2+ mediates a new cycle of digestion. This assay is ultrasensitive for the selective recognition of Hg2+, and a detection limit as low as 10 pM was achieved. In addition, the fluorescent biosensing system also displays remarkable specificity to Hg2+ in the presence of other possible competing ions. This approach was applied to the determination of Hg2+ in real water samples with good recovery and high efficiency for practical analysis.

Discovery of the type VII ESX-1 secretion needle?

PubMed

Ates, Louis S; Brosch, Roland

2017-01-01

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.

FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms.

PubMed

Mita, Tomohiro; Furuhashi, Masato; Hiramitsu, Shinya; Ishii, Junnichi; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Tanaka, Marenao; Ohno, Kohei; Akasaka, Hiroshi; Ohnishi, Hirofumi; Yoshida, Hideaki; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2015-02-01

Fatty acid-binding protein 4 (FABP4) is expressed in adipocytes, and elevated plasma FABP4 level is associated with obesity-mediated metabolic phenotype. Postprandial regulation and secretory signaling of FABP4 has been investigated. Time courses of FABP4 levels were examined during an oral glucose tolerance test (OGTT; n=53) or a high-fat test meal eating (n=35). Effects of activators and inhibitors of adenyl cyclase (AC)-protein kinase A (PKA) signaling and guanylyl cyclase (GC)-protein kinase G (PKG) signaling on FABP4 secretion from mouse 3T3-L1 adipocytes were investigated. FABP4 level significantly declined after the OGTT or a high-fat meal eating, while insulin level was increased. Treatment with low and high glucose concentration or palmitate for 2 h did not affect FABP4 secretion from 3T3-L1 adipocytes. FABP4 secretion was increased by stimulation of lipolysis using isoproterenol, a β3 -adrenoceptor agonist (CL316243), forskolin, dibutyryl-cAMP and atrial natriuretic peptide, and the induced FABP4 secretion was suppressed by insulin or an inhibitor of PKA (H-89), PKG (KT5823) or hormone sensitive lipase (CAY10499). FABP4 is secreted from adipocytes in association with lipolysis regulated by AC-PKA- and GC-PKG-mediated signal pathways. Plasma FABP4 level declines postprandially, and suppression of FABP4 secretion by insulin-induced anti-lipolytic signaling may be involved in this decline in FABP4 level. © 2014 The Obesity Society.

Evolutionary Interactions Between Visual and Chemical Signals: Chemosignals Compensate for the Loss of a Visual Signal in Male Sceloporus Lizards.

PubMed

Pruett, Jake A; Zúñiga-Vega, J Jaime; Campos, Stephanie M; Soini, Helena A; Novotny, Milos V; Vital-García, Cuauhcihuatl; Martins, Emília P; Hews, Diana K

2016-11-01

Animals rely on multimodal signals to obtain information from conspecifics through alternative sensory systems, and the evolutionary loss of a signal in one modality may lead to compensation through increased use of signals in an alternative modality. We investigated associations between chemical signaling and evolutionary loss of abdominal color patches in males of four species (two plain-bellied and two colorful-bellied) of Sceloporus lizards. We conducted field trials to compare behavioral responses of male lizards to swabs with femoral gland (FG) secretions from conspecific males and control swabs (clean paper). We also analyzed the volatile organic compound (VOC) composition of male FG secretions by stir bar extraction and gas chromatography-mass spectrometry (GC-MS) to test the hypothesis that loss of the visual signal is associated with elaboration of the chemical signal. Males of plain-bellied, but not colorful-bellied species exhibited different rates of visual displays when exposed to swabs of conspecific FG secretions relative to control swabs. The VOC composition of male Sceloporus FG secretions was similar across all four species, and no clear association between relative abundances of VOCs and evolutionary loss of abdominal color patches was observed. The emerging pattern is that behavioral responses to conspecific chemical signals are species- and context-specific in male Sceloporus, and compensatory changes in receivers, but not signalers may be involved in mediating increased responsiveness to chemical signals in males of plain-bellied species.

Identification of the signals for glucose-induced insulin secretion in INS1 (832/13) β-cells using metformin-induced metabolic deceleration as a model.

PubMed

Lamontagne, Julien; Al-Mass, Anfal; Nolan, Christopher J; Corkey, Barbara E; Madiraju, S R Murthy; Joly, Erik; Prentki, Marc

2017-11-24

Metabolic deceleration in pancreatic β-cells is associated with inhibition of glucose-induced insulin secretion (GIIS), but only in the presence of intermediate/submaximal glucose concentrations. Here, we used acute metformin treatment as a tool to induce metabolic deceleration in INS1 (832/13) β-cells, with the goal of identifying key pathways and metabolites involved in GIIS. Metabolites and pathways previously implicated as signals for GIIS were measured in the cells at 2-25 mm glucose, with or without 5 mm metformin. We defined three criteria to identify candidate signals: 1) glucose-responsiveness, 2) sensitivity to metformin-induced inhibition of the glucose effect at intermediate glucose concentrations, and 3) alleviation of metformin inhibition by elevated glucose concentrations. Despite the lack of recovery from metformin-induced impairment of mitochondrial energy metabolism (glucose oxidation, O 2 consumption, and ATP production), insulin secretion was almost completely restored at elevated glucose concentrations. Meeting the criteria for candidates involved in promoting GIIS were the following metabolic indicators and metabolites: cytosolic NAD + /NADH ratio (inferred from the dihydroxyacetone phosphate:glycerol-3-phosphate ratio), mitochondrial membrane potential, ADP, Ca 2+ , 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, H 2 O 2 , reduced glutathione, and 2-monoacylglycerol were not glucose-responsive. Overall these results underscore the significance of mitochondrial energy metabolism-independent signals in GIIS regulation; in particular, the candidate lipid signaling molecules 1-monoacylglycerol, diacylglycerol, and malonyl-CoA; the predominance of K ATP /Ca 2+ signaling control by low ADP·Mg 2+ rather than by high ATP levels; and a role for a more oxidized state (NAD + /NADH) in the cytosol during GIIS that favors high glycolysis rates. © 2017 by

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

PubMed

Wanka, Franziska; Arentshorst, Mark; Cairns, Timothy C; Jørgensen, Thomas; Ram, Arthur F J; Meyer, Vera

2016-08-20

The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency. In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein. Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.

Ghrelin: ghrelin as a regulatory Peptide in growth hormone secretion.

PubMed

Khatib, Nazli; Gaidhane, Shilpa; Gaidhane, Abhay M; Khatib, Mahanaaz; Simkhada, Padam; Gode, Dilip; Zahiruddin, Quazi Syed

2014-08-01

Ghrelin is a type of growth hormone (GH) secretagogue that stimulates the release of GH. It is a first hormone linking gastrointestinal-pituitary axis. This review highlights the interaction of ghrelin with GHRH and somatostatin to regulate the secretion of GH and intends to explore the possible physiological role of the ghrelin-pituitary-GH axis linkage system. Ghrelin is highly conserved among species and is classified into octanoylated (C8:0), decanoylated (C10:0), decenoylated (C10:1) and nonacylated,ghrelin. Acylated ghrelin is the major active form of human ghrelin. The primary production site of ghrelin is the stomach, and it interacts with stomach ghrelin as well as hypothalamic GHRH and somatostatin in the regulation of pituitary GH secretion. Ghrelin stimulate GH release through the GHS receptor to increase intracellular Ca2+ ([Ca2+] levels via IP3 signal transduction pathway. Ghrelin is a specific endogenous ligand for the GHS receptor and provides a definitive proof of the occurance of a GHS-GHS receptor signalling system in the regulation of GH secretion. Studies suggests that ghrelin is a powerful pharmacological agent that exerts a potent, time-dependent stimulation of pulsatile secretion of GH.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

PubMed

Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael

2015-01-01

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokawa, Satoru; School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650; Suzuki, Takahiro

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase frommore » the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.« less

The Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Transcription of the Type III Secretion System▿ †

PubMed Central

Anderson, Gregory G.; Yahr, Timothy L.; Lovewell, Rustin R.; O'Toole, George A.

2010-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes. PMID:20028803

Investment in secreted enzymes during nutrient-limited growth is utility dependent.

PubMed

Cezairliyan, Brent; Ausubel, Frederick M

2017-09-12

Pathogenic bacteria secrete toxins and degradative enzymes that facilitate their growth by liberating nutrients from the environment. To understand bacterial growth under nutrient-limited conditions, we studied resource allocation between cellular and secreted components by the pathogenic bacterium Pseudomonas aeruginosa during growth on a protein substrate that requires extracellular digestion by secreted proteases. We identified a quantitative relationship between the rate of increase of cellular biomass under nutrient-limiting growth conditions and the rate of increase in investment in secreted proteases. Production of secreted proteases is stimulated by secreted signals that convey information about the utility of secreted proteins during nutrient-limited growth. Growth modeling using this relationship recapitulated the observed kinetics of bacterial growth on a protein substrate. The proposed regulatory strategy suggests a rationale for quorum-sensing-dependent stimulation of the production of secreted enzymes whereby investment in secreted enzymes occurs in proportion to the utility they confer. Our model provides a framework that can be applied toward understanding bacterial growth in many environments where growth rate is limited by the availability of nutrients.

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

DOE PAGES

Xu, Qi; Knoshaug, Eric P.; Wang, Wei; ...

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qi; Knoshaug, Eric P.; Wang, Wei

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi.

PubMed

Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel

CytR Homolog of Pectobacterium carotovorum subsp. carotovorum Controls Air-Liquid Biofilm Formation by Regulating Multiple Genes Involved in Cellulose Production, c-di-GMP Signaling, Motility, and Type III Secretion System in Response to Nutritional and Environmental Signals.

PubMed

Haque, M M; Oliver, M M H; Nahar, Kamrun; Alam, Mohammad Z; Hirata, Hisae; Tsuyumu, Shinji

2017-01-01

Pectobacterium carotovorum subsp. carotovorum [Pcc (formerly Erwinia carotovora subsp. carotovora )] PC1 causes soft-rot disease in a wide variety of plant species by secreting multiple pathogenicity-related traits. In this study, regulatory mechanism of a ir- l iquid (AL) biofilm formation was studied using a cytR homolog gene deletion mutant (Δ cytR ) of Pcc PC1. Compared to the wild type (Pcc PC1), the Δ cytR mutant produced fragile and significantly ( P < 0.001) lower amounts of AL biofilm on s alt- o ptimized b roth plus 2% g lycerol (SOBG), yeast peptone dextrose adenine, and also on King's B at 27°C after 72 h incubation in static condition. The wild type also produced significantly higher quantities of AL biofilm on SOBGMg - (magnesium deprived) containing Cupper (Cu 2+ ), Zinc (Zn 2+ ), Manganese (Mn 2+ ), Magnesium (Mg 2+ ), and Calcium (Ca 2+ ) compared to the Δ cytR mutant. Moreover, the wild type was produced higher amounts of biofilms compared to the mutant while responding to pH and osmotic stresses. The Δ fliC (encoding flagellin), flhD ::Tn5 (encoding a master regulator) and Δ motA (a membrane protein essential for flagellar rotation) mutants produced a lighter and more fragile AL biofilm on SOBG compared to their wild counterpart. All these mutants resulted in having weak bonds with the cellulose specific dye (Calcofluor) producing lower quantities of cellulose compared to the wild type. Gene expression analysis using mRNA collected from the AL biofilms showed that Δ cytR mutant significantly ( P < 0.001) reduced the expressions of multiple genes responsible for cellulose production ( bcsA, bcsE , and adrA ), motility ( flhD, fliA, fliC , and motA ) and type III secretion system ( hrpX, hrpL, hrpA , and hrpN ) compared to the wild type. The CytR homolog was therefore, argued to be able to regulate the AL biofilm formation by controlling cellulose production, motility and T3SS in Pcc PC1. In addition, all the mutants exhibited poorer

CytR Homolog of Pectobacterium carotovorum subsp. carotovorum Controls Air-Liquid Biofilm Formation by Regulating Multiple Genes Involved in Cellulose Production, c-di-GMP Signaling, Motility, and Type III Secretion System in Response to Nutritional and Environmental Signals

PubMed Central

Haque, M. M.; Oliver, M. M. H.; Nahar, Kamrun; Alam, Mohammad Z.; Hirata, Hisae; Tsuyumu, Shinji

2017-01-01

Pectobacterium carotovorum subsp. carotovorum [Pcc (formerly Erwinia carotovora subsp. carotovora)] PC1 causes soft-rot disease in a wide variety of plant species by secreting multiple pathogenicity-related traits. In this study, regulatory mechanism of air-liquid (AL) biofilm formation was studied using a cytR homolog gene deletion mutant (ΔcytR) of Pcc PC1. Compared to the wild type (Pcc PC1), the ΔcytR mutant produced fragile and significantly (P < 0.001) lower amounts of AL biofilm on salt-optimized broth plus 2% glycerol (SOBG), yeast peptone dextrose adenine, and also on King’s B at 27°C after 72 h incubation in static condition. The wild type also produced significantly higher quantities of AL biofilm on SOBGMg– (magnesium deprived) containing Cupper (Cu2+), Zinc (Zn2+), Manganese (Mn2+), Magnesium (Mg2+), and Calcium (Ca2+) compared to the ΔcytR mutant. Moreover, the wild type was produced higher amounts of biofilms compared to the mutant while responding to pH and osmotic stresses. The ΔfliC (encoding flagellin), flhD::Tn5 (encoding a master regulator) and ΔmotA (a membrane protein essential for flagellar rotation) mutants produced a lighter and more fragile AL biofilm on SOBG compared to their wild counterpart. All these mutants resulted in having weak bonds with the cellulose specific dye (Calcofluor) producing lower quantities of cellulose compared to the wild type. Gene expression analysis using mRNA collected from the AL biofilms showed that ΔcytR mutant significantly (P < 0.001) reduced the expressions of multiple genes responsible for cellulose production (bcsA, bcsE, and adrA), motility (flhD, fliA, fliC, and motA) and type III secretion system (hrpX, hrpL, hrpA, and hrpN) compared to the wild type. The CytR homolog was therefore, argued to be able to regulate the AL biofilm formation by controlling cellulose production, motility and T3SS in Pcc PC1. In addition, all the mutants exhibited poorer attachment to radish sprouts and AL

Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation.

PubMed Central

Bahrani, F K; Sansonetti, P J; Parsot, C

1997-01-01

The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins. PMID:9316999

Effects of Steaming Time and Frequency for Manufactured Red Liriope platyphylla on the Insulin Secretion Ability and Insulin Receptor Signaling Pathway.

PubMed

Choi, Sun Il; Lee, Hye Ryun; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Young Ju; Prak, So Hae; Lee, Hee Seob; Lee, Jong Sup; Jang, In Surk; Son, Hong Ju; Hwang, Dae Youn

2011-06-01

In oriental medicine, Liriope platyphylla (LP) has long been regarded as a curative herb useful for the treatment of diabetes, asthma, and neurodegenerative disorders. The principal objective of this study was to assess the effects of steaming time and frequency for manufactured Red LP (RLP) on insulin secretion ability and insulin receptor signaling pathway. To achieve our goal, several types of LPs manufactured under different conditions were applied to INS cells and streptozotocin (STZ)-induced diabetic ICR mice, after which alterations in insulin concentrations were detected in the culture supernatants and sera. The optimal concentration for the investigation of insulin secretion ability was found to be 50 ug/mL of LP. At this concentration, maximum insulin secretion was observed in the INS cells treated with LP extract steamed for 3 h (3-SLP) with two repeated steps (3 h steaming and 24 h air-dried) carried out 9 times (9-SALP); no significant changes in viability were detected in any of the treated cells. Additionally, the expression and phosphorylation levels of most components in the insulin receptor signaling pathway were increased significantly in the majority of cells treated with steaming-processed LP as compared to the cells treated with LP prepared without steaming. With regard to glucose transporter (GLUT) expression, alterations of steaming time induced similar responses on the expression levels of GLUT-2 and GLUT-3. However, differences in steaming frequency were also shown to induce dose-dependent responses in the expression level of GLUT-2 only; no significant differences in GLUT-3 expression were detected under these conditions. Furthermore, these responses observed in vitro were similarly detected in STZ-induced diabetic mice. 24-SLP and 9-SALP treatment applied for 14 days induced the down-regulation of glucose concentration and upregulation of insulin concentration. Therefore, these results indicated that the steaming processed LP may

Effects of Steaming Time and Frequency for Manufactured Red Liriope platyphylla on the Insulin Secretion Ability and Insulin Receptor Signaling Pathway

PubMed Central

Choi, Sun Il; Lee, Hye Ryun; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Young Ju; Prak, So Hae; Lee, Hee Seob; Lee, Jong Sup; Jang, In Surk; Son, Hong Ju

2011-01-01

In oriental medicine, Liriope platyphylla (LP) has long been regarded as a curative herb useful for the treatment of diabetes, asthma, and neurodegenerative disorders. The principal objective of this study was to assess the effects of steaming time and frequency for manufactured Red LP (RLP) on insulin secretion ability and insulin receptor signaling pathway. To achieve our goal, several types of LPs manufactured under different conditions were applied to INS cells and streptozotocin (STZ)-induced diabetic ICR mice, after which alterations in insulin concentrations were detected in the culture supernatants and sera. The optimal concentration for the investigation of insulin secretion ability was found to be 50 ug/mL of LP. At this concentration, maximum insulin secretion was observed in the INS cells treated with LP extract steamed for 3 h (3-SLP) with two repeated steps (3 h steaming and 24 h air-dried) carried out 9 times (9-SALP); no significant changes in viability were detected in any of the treated cells. Additionally, the expression and phosphorylation levels of most components in the insulin receptor signaling pathway were increased significantly in the majority of cells treated with steaming-processed LP as compared to the cells treated with LP prepared without steaming. With regard to glucose transporter (GLUT) expression, alterations of steaming time induced similar responses on the expression levels of GLUT-2 and GLUT-3. However, differences in steaming frequency were also shown to induce dose-dependent responses in the expression level of GLUT-2 only; no significant differences in GLUT-3 expression were detected under these conditions. Furthermore, these responses observed in vitro were similarly detected in STZ-induced diabetic mice. 24-SLP and 9-SALP treatment applied for 14 days induced the down-regulation of glucose concentration and upregulation of insulin concentration. Therefore, these results indicated that the steaming processed LP may

Secretion and extracellular space travel of Wnt proteins.

PubMed

Gross, Julia Christina; Boutros, Michael

2013-08-01

Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion. Copyright © 2013 Elsevier Ltd. All rights reserved.

How Shigella Utilizes Ca(2+) Jagged Edge Signals during Invasion of Epithelial Cells.

PubMed

Bonnet, Mariette; Tran Van Nhieu, Guy

2016-01-01

Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca(2+) responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca(2+) increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca(2+) overload. In this review, we will focus on the role of Ca(2+) responses and their regulation by Shigella during the different stages of bacterial infection.

Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreatic α-cell by lowering cAMP

PubMed Central

Elliott, Amicia D.; Ustione, Alessandro

2014-01-01

The dysregulation of glucose-inhibited glucagon secretion from the pancreatic islet α-cell is a critical component of diabetes pathology and metabolic disease. We show a previously uncharacterized [Ca2+]i-independent mechanism of glucagon suppression in human and murine pancreatic islets whereby cAMP and PKA signaling are decreased. This decrease is driven by the combination of somatostatin, which inhibits adenylyl cyclase production of cAMP via the Gαi subunit of the SSTR2, and insulin, which acts via its receptor to activate phosphodiesterase 3B and degrade cytosolic cAMP. Our data indicate that both somatostatin and insulin signaling are required to suppress cAMP/PKA and glucagon secretion from both human and murine α-cells, and the combination of these two signaling mechanisms is sufficient to reduce glucagon secretion from isolated α-cells as well as islets. Thus, we conclude that somatostatin and insulin together are critical paracrine mediators of glucose-inhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose. PMID:25406263

Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle.

PubMed

Deane, Janet E; Cordes, Frank S; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D; Picking, Wendy L; Lea, Susan M; Blocker, Ariel

2006-03-01

A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH(CDelta5) and diffraction data were collected to 1.9 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 A, beta = 96.5 degrees. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 A3 Da(-1) for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants.

PubMed

Xin, Xiu-Fang; He, Sheng Yang

2013-01-01

Since the early 1980s, various strains of the gram-negative bacterial pathogen Pseudomonas syringae have been used as models for understanding plant-bacterial interactions. In 1991, a P. syringae pathovar tomato (Pst) strain, DC3000, was reported to infect not only its natural host tomato but also Arabidopsis in the laboratory, a finding that spurred intensive efforts in the subsequent two decades to characterize the molecular mechanisms by which this strain causes disease in plants. Genomic analysis shows that Pst DC3000 carries a large repertoire of potential virulence factors, including proteinaceous effectors that are secreted through the type III secretion system and a polyketide phytotoxin called coronatine, which structurally mimics the plant hormone jasmonate (JA). Study of Pst DC3000 pathogenesis has not only provided several conceptual advances in understanding how a bacterial pathogen employs type III effectors to suppress plant immune responses and promote disease susceptibility but has also facilitated the discovery of the immune function of stomata and key components of JA signaling in plants. The concepts derived from the study of Pst DC3000 pathogenesis may prove useful in understanding pathogenesis mechanisms of other plant pathogens.

Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins.

PubMed

Pattison, Amanda M; Blomain, Erik S; Merlino, Dante J; Wang, Fang; Crissey, Mary Ann S; Kraft, Crystal L; Rappaport, Jeff A; Snook, Adam E; Lynch, John P; Waldman, Scott A

2016-10-01

Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niemann, George; Brown, Roslyn N.; Gustin, Jean K.

The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced themore » SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.« less

Ghrelin: Ghrelin as a Regulatory Peptide in Growth Hormone Secretion

PubMed Central

Gaidhane, Shilpa; Gaidhane, Abhay M; Khatib, Mahanaaz; Gode, Dilip; Zahiruddin, Quazi Syed

2014-01-01

Background: Ghrelin is a type of growth hormone (GH) secretagogue that stimulates the release of GH. It is a first hormone linking gastrointestinal-pituitary axis. Objective: This review highlights the interaction of ghrelin with GHRH and somatostatin to regulate the secretion of GH and intends to explore the possible physiological role of the ghrelin-pituitary-GH axis linkage system. Observation: Ghrelin is highly conserved among species and is classified into octanoylated (C8:0), decanoylated (C10:0), decenoylated (C10:1) and nonacylated,ghrelin. Acylated ghrelin is the major active form of human ghrelin. The primary production site of ghrelin is the stomach, and it interacts with stomach ghrelin as well as hypothalamic GHRH and somatostatin in the regulation of pituitary GH secretion. Ghrelin stimulate GH release through the GHS receptor to increase intracellular Ca2+ ([Ca2+] levels via IP3 signal transduction pathway. Ghrelin is a specific endogenous ligand for the GHS receptor and provides a definitive proof of the occurance of a GHS–GHS receptor signalling system in the regulation of GH secretion. Conclusion: Studies suggests that ghrelin is a powerful pharmacological agent that exerts a potent, time-dependent stimulation of pulsatile secretion of GH. PMID:25302229

Bile Formation and Secretion

PubMed Central

Boyer, James L.

2014-01-01

Bile is a unique and vital aqueous secretion of the liver that is formed by the hepatocyte and modified down stream by absorptive and secretory properties of the bile duct epithelium. Approximately 5% of bile consists of organic and inorganic solutes of considerable complexity. The bile-secretory unit consists of a canalicular network which is formed by the apical membrane of adjacent hepatocytes and sealed by tight junctions. The bile canaliculi (~1 μm in diameter) conduct the flow of bile countercurrent to the direction of portal blood flow and connect with the canal of Hering and bile ducts which progressively increase in diameter and complexity prior to the entry of bile into the gallbladder, common bile duct, and intestine. Canalicular bile secretion is determined by both bile salt-dependent and independent transport systems which are localized at the apical membrane of the hepatocyte and largely consist of a series of adenosine triphosphate-binding cassette transport proteins that function as export pumps for bile salts and other organic solutes. These transporters create osmotic gradients within the bile canalicular lumen that provide the driving force for movement of fluid into the lumen via aquaporins. Species vary with respect to the relative amounts of bile salt-dependent and independent canalicular flow and cholangiocyte secretion which is highly regulated by hormones, second messengers, and signal transduction pathways. Most determinants of bile secretion are now characterized at the molecular level in animal models and in man. Genetic mutations serve to illuminate many of their functions. PMID:23897680

Secreted and Transmembrane Wnt Inhibitors and Activators

PubMed Central

Cruciat, Cristina-Maria; Niehrs, Christof

2013-01-01

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand–receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

Glucocorticoid hormones are both a major circadian signal and major stress signal: How this shared signal contributes to a dynamic relationship between the circadian and stress systems.

PubMed

Spencer, Robert L; Chun, Lauren E; Hartsock, Matthew J; Woodruff, Elizabeth R

2018-04-01

Glucocorticoid hormones are a powerful mammalian systemic hormonal signal that exerts regulatory effects on almost every cell and system of the body. Glucocorticoids act in a circadian and stress-directed manner to aid in adaptation to an ever-changing environment. Circadian glucocorticoid secretion provides for a daily waxing and waning influence on target cell function. In addition, the daily circadian peak of glucocorticoid secretion serves as a timing signal that helps entrain intrinsic molecular clock phase in tissue cells distributed throughout the body. Stress-induced glucocorticoid secretion also modulates the state of these same cells in response to both physiological and psychological stressors. We review the strong functional interrelationships between glucocorticoids and the circadian system, and discuss how these interactions optimize the appropriate cellular and systems response to stress throughout the day. We also discuss clinical implications of this dual aspect of glucocorticoid signaling, especially for conditions of circadian and HPA axis dysregulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

PubMed Central

Deng, Xin; Liang, Haihua; Chen, Kai; He, Chuan; Lan, Lefu; Tang, Xiaoyan

2014-01-01

Pseudomonas syringae uses the two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanisms and the regulons of RhpRS have yet to be fully elucidated. Here, we show that RhpS functions as a kinase and a phosphatase on RhpR and as an autokinase upon itself. RhpR is phosphorylated by the small phosphodonor acetyl phosphate. A specific RhpR-binding site containing the inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by a DNase I footprint analysis. Electrophoretic mobility shift assay indicated that P-RhpR has a higher binding affinity to the IR motif than RhpR. To identify additional RhpR targets in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) and detected 167 enriched loci including the hrpR promoter, suggesting the direct regulation of T3SS cascade genes by RhpR. A genome-wide microarray analysis showed that, in addition to the T3SS cascade genes, RhpR differentially regulates a large set of genes with various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes by coordinating T3SS expression and many other biological processes. PMID:25249629

Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut▿

PubMed Central

Hemrajani, Cordula; Marches, Olivier; Wiles, Siouxsie; Girard, Francis; Dennis, Alison; Dziva, Francis; Best, Angus; Phillips, Alan D.; Berger, Cedric N.; Mousnier, Aurelie; Crepin, Valerie F.; Kruidenier, Laurens; Woodward, Martin J.; Stevens, Mark P.; La Ragione, Roberto M.; MacDonald, Thomas T.; Frankel, Gad

2008-01-01

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain. PMID:18725419

Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut.

PubMed

Hemrajani, Cordula; Marches, Olivier; Wiles, Siouxsie; Girard, Francis; Dennis, Alison; Dziva, Francis; Best, Angus; Phillips, Alan D; Berger, Cedric N; Mousnier, Aurelie; Crepin, Valerie F; Kruidenier, Laurens; Woodward, Martin J; Stevens, Mark P; La Ragione, Roberto M; MacDonald, Thomas T; Frankel, Gad

2008-11-01

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappaB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC DeltanleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappaB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappaB response elements, we found that NleH causes an increase in NF-kappaB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion.

PubMed

Krtková, Jana; Thomas, Elizabeth B; Alas, Germain C M; Schraner, Elisabeth M; Behjatnia, Habib R; Hehl, Adrian B; Paredez, Alexander R

2016-08-23

Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall

Differential effects of the steaming time and frequency for manufactured red Liriope platyphylla on nerve growth factor secretion ability, nerve growth factor receptor signaling pathway and regulation of calcium concentration.

PubMed

Choi, Sun Il; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Hye Ryun; Lee, Young Ju; Son, Hong Joo; Lee, Hee Seob; Lee, Jong Sup; Kim, Hak Jin; Hwang, Dae Youn

2012-11-01

The herb Liriope platyphylla (LP) has been considered to have curative properties for diabetes, asthma and neurodegenerative disorders. To examine the effects of steaming time and frequency of manufactured red LP (RLP) on the nerve growth factor (NGF) secretion ability and NGF receptor signaling pathway, the NGF concentration, cell differentiation, NGF signaling pathway and calcium concentration were analyzed in neuronal cells treated with several types of LPs manufactured under different conditions. The maximum NGF secretion was observed in B35 cells treated with 50 µg/ml LP extract steamed for 9 h (9-SLP) and with two repeated steps (3 h steaming and 24 h air-dried) carried out 7 times (7-SALP). No significant changes in viability were detected in any of the cells treated with the various LPs, with the exception of 0-SLP and 0-SALP. In addition, PC12 cell differentiation was induced by treatment with the NGF-containing conditional medium (CM) collected from the RLP-treated cells. The levels of TrkA and extracellular signal-regulated kinase (ERK) phosphorylation in the high affinity NGF receptor signaling pathway were significantly higher in the cells treated with 3-SLP or 1-SALP/3-SALP CM compared with those treated with the vehicle CM. In the low affinity NGF receptor pathway, the expression levels of most components were higher in the 9-, 15- and 24-SALP CM-treated cells compared with the vehicle CM-treated cells. However, this level was significantly altered in cells treated with 3-SALP CM. Furthermore, an examination of the RLP function on calcium regulation revealed that only the LP- or RLP-treated cells exhibited changes in intracellular and extracellular calcium levels. RLP induced a significant decrease in the intracellular calcium levels and an increase in the extracellular calcium levels. These results suggest the possibility that steaming-processed LP may aid in the relief of neurodegenerative diseases through the NGF secretion ability and NGF

Dynamic compaction of human mesenchymal stem/precursor cells (MSC) into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6 and STC1)

PubMed Central

Bazhanov, Nikolay; Kuhlman, Jessica; Prockop, Darwin J.

2013-01-01

Human mesenchymal stem/precursor cells (MSC) are similar to some other stem/progenitor cells in that they compact into spheres when cultured in hanging drops or on non-adherent surfaces. Assembly of MSC into spheres alters many of their properties, including enhanced secretion of factors that mediate inflammatory and immune responses. Here we demonstrated that MSC spontaneously aggregated into sphere-like structures after injection into a subcutaneous air pouch or the peritoneum of mice. The structures were similar to MSC spheres formed in cultures demonstrated by the increased expression of genes for inflammation-modulating factors TSG6, STC1, and COX2, a key enzyme in production of PGE2. To identify the signaling pathways involved, hanging drop cultures were used to follow the time-dependent changes in the cells as they compacted into spheres. Among the genes up-regulated were genes for the stress-activated signaling pathway for IL1α/β, and the contact-dependent signaling pathway for Notch. An inhibitor of caspases reduced the up-regulation of IL1A/B expression, and inhibitors of IL1 signaling decreased production of PGE2, TSG6 and STC1. Also, inhibition of IL1A/B expression and secretion of PGE2 negated the anti-inflammatory effects of MSC spheres on stimulated macrophages. Experiments with γ-secretase inhibitors suggested that Notch signaling was also required for production of PGE2 but not TSG6 or STC1. The results indicated that assembly of MSC into spheres triggers caspase-dependent IL1 signaling and the secretion of modulators of inflammation and immunity. Similar aggregation in vivo may account for some of the effects observed with administration of the cells in animal models. PMID:23922312

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

PubMed

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

PubMed Central

2010-01-01

Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between

Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response.

PubMed

Ding, Q; Quah, S Y; Tan, K S

2016-10-01

Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host-microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP-dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP-induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

PubMed Central

Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

2012-01-01

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766

Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD

PubMed Central

Heine, Shannon J.; Diaz-McNair, Jovita; Martinez-Becerra, Francisco J.; Choudhari, Shyamal P.; Clements, John D.; Picking, Wendy L.; Pasetti, Marcela F.

2013-01-01

Shigella spp. are food- and water-borne pathogens that cause shigellosis, a severe diarrheal and dysenteric disease that is associated with a high morbidity and mortality in resource-poor countries. No licensed vaccine is available to prevent shigellosis. We have recently demonstrated that Shigella invasion plasmid antigens (Ipas), IpaB and IpaD, which are components of the bacterial type III secretion system (TTSS), can prevent infection in a mouse model of intranasal immunization and lethal pulmonary challenge. Because they are conserved across Shigella spp. and highly immunogenic, these proteins are excellent candidates for a cross-protective vaccine. Ideally, such a vaccine could be administered to humans orally to induce mucosal and systemic immunity. In this study, we investigated the immunogenicity and protective efficacy of Shigella IpaB and IpaD administered orally with a double mutant of the Escherichia coli heat labile toxin (dmLT) as a mucosal adjuvant. We characterized the immune responses induced by oral vs. intranasal immunization and the protective efficacy using a mouse pulmonary infection model. Serum IgG and fecal IgA against IpaB were induced after oral immunization. These responses, however, were lower than those obtained after intranasal immunization despite a 100-fold dosage increase. The level of protection induced by oral immunization with IpaB and IpaD was 40%, while intranasal immunization resulted in 90% protective efficacy. IpaB- and IpaD-specific IgA antibody-secreting cells in the lungs and spleen and T-cell-derived IL-2, IL-5, IL-17 and IL-10 were associated with protection. These results demonstrate the immunogenicity of orally administered IpaB and IpaD and support further studies in humans. PMID:23644075

Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells.

PubMed

Datta, Amrita; Kim, Hogyoung; Lal, Madhu; McGee, Lauren; Johnson, Adedoyin; Moustafa, Ahmed A; Jones, Jennifer C; Mondal, Debasis; Ferrer, Marc; Abdel-Mageed, Asim B

2017-11-01

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Implication of TLR- but Not of NOD2-Signaling Pathways in Dendritic Cell Activation by Group B Streptococcus Serotypes III and V

PubMed Central

Lemire, Paul; Roy, David; Fittipaldi, Nahuel; Okura, Masatoshi; Takamatsu, Daisuke; Bergman, Eugenia; Segura, Mariela

2014-01-01

Group B Streptococcus (GBS) is an important agent of life-threatening invasive infection. It has been previously shown that encapsulated type III GBS is easily internalized by dendritic cells (DCs), and that this internalization had an impact on cytokine production. The receptors underlying these processes are poorly characterized. Knowledge on the mechanisms used by type V GBS to activate DCs is minimal. In this work, we investigated the role of Toll-like receptor (TLR)/MyD88 signaling pathway, the particular involvement of TLR2, and that of the intracellular sensing receptor NOD2 in the activation of DCs by types III and V GBS. The role of capsular polysaccharide (CPS, one of the most important GBS virulence factors) in bacterial-DC interactions was evaluated using non-encapsulated mutants. Despite differences in the role of CPS between types III and V GBS in bacterial internalization and intracellular survival, no major differences were observed in their capacity to modulate release of cytokines by DC. For both serotypes, CPS had a minor role in this response. Production of cytokines by DCs was shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize GBS and become activated mostly through TLR signaling. Yet, GBS-infected TLR2-/- DCs only showed a partial reduction in the production of IL-6 and CXCL1 compared to control DCs. Surprisingly, CXCL10 release by type III or type V GBS-infected DCs was MyD88-independent. No differences in DC activation were observed between NOD2-/- and control DCs. These results demonstrate the involvement of various receptors and the complexity of the cytokine production pathways activated by GBS upon DC infection. PMID:25436906

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

PubMed Central

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L.; Picking, William D.; Middaugh, C. Russell

2006-01-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions. PMID:16501225

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation*

PubMed Central

Burgess, Jamie L.; Burgess, R. Alan; Morales, Yalemi; Bouvang, Jenna M.; Johnson, Sean J.; Dickenson, Nicholas E.

2016-01-01

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. PMID:27770024

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella.

PubMed

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L; Picking, William D; Middaugh, C Russell

2006-03-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.

Intradermal delivery of Shigella IpaB and IpaD type III secretion proteins: Kinetics of cell recruitment and antigen uptake, mucosal and systemic immunity, and protection across serotypes

PubMed Central

Heine, Shannon J.; Diaz-McNair, Jovita; Andar, Abhay U.; Drachenberg, Cinthia B.; van de Verg, Lillian; Walker, Richard; Picking, Wendy L.; Pasetti, Marcela F.

2014-01-01

Shigella is one of the leading pathogens contributing to the vast pediatric diarrheal disease burden in low-income countries. No licensed vaccine is available and the existing candidates are only partially effective and serotype-specific. Shigella type III secretion system proteins IpaB and IpaD, which are conserved across Shigella spp., are candidates for a broadly protective, subunit-based vaccine. Herein, we investigated the immunogenicity and protective efficacy of IpaB and IpaD administered intradermally (i.d.) with a double-mutant of the E. coli heat-labile enterotoxin (dmLT) adjuvant using microneedles. Different dosage levels of IpaB and IpaD with or without dmLT were tested in mice. Vaccine delivery into the dermis, recruitment of neutrophils, macrophages, dendritic cells (DC) and Langerhans cells (LC), and colocalization of vaccine antigens within skin-activated antigen presenting cells (APC) was demonstrated through histology and immunofluorescence microscopy. Ag-loaded neutrophils, macrophages, DC and LC remained in the tissue at least one week. IpaB, IpaD and dmLT-specific serum IgG and IgG secreting cells were produced following i.d. immunization. The protective efficacy was 70% against S. flexneri and 50% against S. sonnei. Similar results were obtained when the vaccine was administered intranasally, with the i.d. route requiring 25-40 times lower doses. Distinctively, IgG was detected in mucosal secretions; sIgA as well as mucosal and systemic IgA antibody secreting cells (ASC) were seemingly absent. Vaccine-induced T cells produced IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-5 and IL-10. These results demonstrate the potential of i.d. vaccination with IpaB and IpaD to prevent Shigella infection and support further studies in humans. PMID:24453241

Influence of Al(III) on biofilm and its extracellular polymeric substances in sequencing batch biofilm reactors.

PubMed

Hu, Xuewei; Yang, Lei; Lai, Xinke; Yao, Qi; Chen, Kai

2017-10-03

This paper presented the influence of Al(III) on biodegradability, micromorphology, composition and functional groups characteristics of the biofilm extracellular polymeric substances (EPS) during different growth phases. The sequencing batch biofilm reactors were developed to cultivate biofilms under different Al(III) dosages. The results elucidated that Al(III) affected biofilm development adversely at the beginning of biofilm growth, but promoted the biofilm mass and improved the biofilm activity with the growth of the biofilm. The micromorphological observation indicated that Al(III) led to a reduction of the filaments and promotion of the EPS secretion in growth phases of the biofilm, also Al(III) could promote microorganisms to form larger colonies for mature biofilm. Then, the analysis of EPS contents and components suggested that Al(III) could increase the protein (PN) of tightly bound EPS (TB-EPS) which alleviated the metal toxicity inhibition on the biofilm during the initial phases of biofilm growth. The biofilm could gradually adapt to the inhibition caused by Al(III) at the biofilm maturation moment. Finally, through the Fourier transform infrared spectroscopy, it was found that Al(III) was beneficial for the proliferation and secretion of TB-EPS functional groups, especially the functional groups of protein and polysaccharides.

Irisin inhibition of growth hormone secretion in cultured tilapia pituitary cells.

PubMed

Lian, Anji; Li, Xin; Jiang, Quan

2017-01-05

Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, is well-documented to be a regulator of energy metabolism. At present, not much is known about its biological function in non-mammalian species. In this study, a full-length tilapia FDNC5 was cloned and its tissue expression pattern has been confirmed. Based on the sequence obtained, we produced and purified recombinant irisin which could induce uncoupling protein 1 (UCP1) gene expression in tilapia hepatocytes. Further, the rabbit polyclonal irisin antiserum was produced and its specificity was confirmed by antiserum preabsorption. In tilapia pituitary cells, irisin inhibited growth hormone (GH) gene expression and secretion and triggered rapid phosphorylation of Akt, Erk1/2, and p38 MAPK. Furthermore, irisin-inhibited GH mRNA expression could be prevented by inhibiting PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Apparently, fish irisin can act directly at the pituitary level to inhibit GH transcript expression via multiple signaling pathways. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

Effects of the type III secreted pseudomonal toxin ExoS in the yeast Saccharomyces cerevisiae.

PubMed

Stirling, Fiona R; Evans, Tom J

2006-08-01

Pseudomonas aeruginosa secretes a number of toxins by a type III system, and these are important in virulence. One of them, ExoS, is a bifunctional toxin, with a GTPase-activating protein domain, as well as ADP ribosyltransferase (ADPRT) activity. These two domains have numerous potential cellular targets, but the overall mechanism of ExoS action remains unclear. The effects of ExoS in a simple eukaryotic system, the yeast Saccharomyces cerevisiae, using a tetracycline-regulated expression system were studied. This system allowed controlled expression of ExoS in yeast, which was not possible using a galactose-induced system. ExoS was found to be an extremely potent inhibitor of yeast growth, and to be largely dependent on the activity of its ADPRT domain. ExoS produced a dramatic alteration in actin distribution, with the appearance of large aggregates of cortical actin, and thickened disorganized cables, entirely dependent on the ADPRT domain. This phenotype is suggestive of actin stabilization, which was verified by showing that the cortical aggregates of actin induced by ExoS were resistant to treatment with latrunculin A, an agent that prevents actin polymerization. ExoS increased the numbers of mating projections produced following growth arrest with mating pheromone, and prevented subsequent DNA replication, an effect that is again dependent on the ADPRT domain. Following pheromone removal, ExoS produced altered development of the mating projections, which became elongated with a swollen bud-like tip. These results suggest alternative pathways for ExoS action in eukaryotic cells that may result from activation of small GTPases, and this yeast expression system is well suited to explore these pathways.

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

PubMed Central

Lindeberg, Magdalen; Boyd, Carol M.; Keen, Noel T.; Collmer, Alan

1998-01-01

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning. PMID:9515910

Regulation of ghrelin secretion and action.

PubMed

Camiña, Jesus P; Carreira, Marcos C; Micic, Dragan; Pombo, Manuel; Kelestimur, Fahrettin; Dieguez, Carlos; Casanueva, Felipe F

2003-10-01

The pulsatile release of growth hormone (GH) from anterior pituitary gland is regulated by the interplay of at least two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. Furthermore, release of GH in vivo may also be controlled by a third type of receptor, the growth hormone secretagogue receptor, a G-protein-coupled receptor, called GHS receptor type 1a (GHSR1a), which was identified in the pituitary and the hypothalamus in humans using a nonpeptidyl growth hormone secretagogue (MK-0677). Ghrelin, the endogenous ligand for the GHS-R1a, is a 28-amino-acid peptide isolated from human stomach that is modified by a straight chain octanoyl group covalently linked to Ser3, which is essential for its endocrine activity. This hormone, predominantly expressed and secreted by the stomach, has a dual action on GH secretion and food intake, showing interdependency between these actions. The finding that fasting and food intake, respectively, increase and decrease the secretion of ghrelin suggests that this hormone may be the bridge connecting somatic growth and body composition with energy metabolism, and appears to play a role in the alteration of energy homeostasis and body weight in pathophysiological states such as hypothyroidism and hyperthyroidism. Despite this, little is known about the intracellular signaling through which ghrelin exerts its regulatory actions. Activation of intracellular calcium mobilization is one of the earliest known cellular signals elicited by ghrelin. In HEK- 293 cells expressing the GHS-R1a, ghrelin induces a biphasic cytosolic calcium elevation characterized by a spike phase of the response, which reflects Ins(1,4,5)P3- dependent calcium mobilization of intracellular stores, and a sustained phase of the response, which is due to calcium influx across the plasma membrane triggered by aperture of capacitative calcium channels

Characterization of Salmonella enterica Derivatives Harboring Defined aroC and Salmonella Pathogenicity Island 2 Type III Secretion System (ssaV) Mutations by Immunization of Healthy Volunteers

PubMed Central

Hindle, Zoë; Chatfield, Steven N.; Phillimore, Jo; Bentley, Matthew; Johnson, Julie; Cosgrove, Catherine A.; Ghaem-Maghami, Marjan; Sexton, Amy; Khan, Mohammad; Brennan, Frank R.; Everest, Paul; Wu, Tao; Pickard, Derek; Holden, David W.; Dougan, Gordon; Griffin, George E.; House, Deborah; Santangelo, Joseph D.; Khan, Shahid A.; Shea, Jaqueline E.; Feldman, Robert G.; Lewis, David J. M.

2002-01-01

The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi. PMID:12065485

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

PubMed

Michel, Gérard P F; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

2011-07-01

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions

PubMed Central

Guérin, Jeremy; Bigot, Sarah; Schneider, Robert; Buchanan, Susan K.; Jacob-Dubuisson, Françoise

2017-01-01

Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process. PMID:28536673

PI3K, ERK, p38 MAPK and integrins regulate CCR3-mediated secretion of mouse and human eosinophil-associated RNases

PubMed Central

Shamri, Revital; Young, Kristen M.; Weller, Peter F.

2013-01-01

Background Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases. Objective In this study we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation. Methods Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24 and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy. Results We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi-dependent. Both mouse and human eosinophils required the activation of PI3K, ERK and p38 MAPK. In addition, the adhesion molecules β1 and β2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion. Conclusions Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases. PMID:23742707

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

PubMed Central

de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

2016-01-01

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

Secure Communication via a Recycling of Attenuated Classical Signals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, IV, Amos M.

We describe a simple method of interleaving a classical and quantum signal in a secure communication system at a single wavelength. The system transmits data encrypted via a one-time pad on a classical signal and produces a single-photon reflection of the encrypted signal. This attenuated signal can be used to observe eavesdroppers and produce fresh secret bits. The system can be secured against eavesdroppers, detect simple tampering or classical bit errors, produces more secret bits than it consumes, and does not require any entanglement or complex wavelength division multiplexing, thus, making continuous secure two-way communication via one-time pads practical.

Secure Communication via a Recycling of Attenuated Classical Signals

DOE PAGES

Smith, IV, Amos M.

2017-01-12

We describe a simple method of interleaving a classical and quantum signal in a secure communication system at a single wavelength. The system transmits data encrypted via a one-time pad on a classical signal and produces a single-photon reflection of the encrypted signal. This attenuated signal can be used to observe eavesdroppers and produce fresh secret bits. The system can be secured against eavesdroppers, detect simple tampering or classical bit errors, produces more secret bits than it consumes, and does not require any entanglement or complex wavelength division multiplexing, thus, making continuous secure two-way communication via one-time pads practical.

Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

PubMed

Baron, Christian; Coombes, Brian

2007-03-01

Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.

Seasonal prolactin secretion and its role in seasonal reproduction: a review.

PubMed

Curlewis, J D

1992-01-01

The majority of seasonally breeding mammals show a seasonal pattern of prolactin secretion with peak concentrations in spring or summer and a nadir in autumn or winter. Photoperiod influences prolactin secretion via its effects on the secretion of the pineal hormone melatonin. Preliminary evidence suggests that the effects of melatonin on both prolactin and gonadotrophin secretion are via a common target area, possibly within the anterior hypothalamus, and that differences in response to photoperiod may be due to differences in the processing and/or interpretation of the melatonin signal. In contrast to seasonal gonadotrophin secretion, the seasonal changes in prolactin are not due to changes in the sensitivity of a feedback loop and so must be due to direct effects on the hypothalamic pathways that control prolactin secretion. Little else can be said with confidence about the neuroendocrine mechanisms that lead to the seasonal changes in prolactin secretion. Dopamine and noradrenaline turnover in the arcuate nucleus and median eminence decrease under short daylength. If catecholamine turnover in these structures is positively correlated with catecholamine concentrations in the long or short hypophysial portal vessels, it is unlikely that the decrease in prolactin concentration in winter is due to the effects of increased concentrations of dopamine or noradrenaline in the portal vessels. There is, however, evidence for increased pituitary sensitivity to dopamine under short daylength, so increased dopamine concentrations may not be required for suppression of prolactin secretion at this time. In addition to the diminished secretion of prolactin under short daylength, rate of prolactin synthesis and pituitary content of prolactin also decline although the mechanisms that regulate these changes are poorly understood. Although all seasonal breeders show a seasonal change in prolactin secretion, there are continuously breeding species in which prolactin secretion is

Hedgehog Signaling in the Stomach

PubMed Central

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-01-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration and disease. PMID:27750091

Lubiprostone activates Cl- secretion via cAMP signaling and increases membrane CFTR in the human colon carcinoma cell line, T84.

PubMed

Ao, Mei; Venkatasubramanian, Jayashree; Boonkaewwan, Chaiwat; Ganesan, Nivetha; Syed, Asma; Benya, Richard V; Rao, Mrinalini C

2011-02-01

Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains.

PubMed

Li, Shan; Zhang, Li; Yao, Qing; Li, Lin; Dong, Na; Rong, Jie; Gao, Wenqing; Ding, Xiaojun; Sun, Liming; Chen, Xing; Chen, She; Shao, Feng

2013-09-12

The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Chemical constituents of the femoral gland secretions of male tegu lizards (Tupinambis merianae) (Family teiidae).

PubMed

Martín, José; Chamut, Silvia; Manes, Mario E; López, Pilar

2011-01-01

In spite of the importance of chemical signals (pheromones) in the reproductive behaviour of lizards, the chemical compounds secreted by their femoral glands, which may be used as sexual signals, are only known for a few lizard species. Based on mass spectra, obtained by GC-MS, we found 49 lipophilic compounds in femoral gland secretions of male tegu lizards (Tupinambis merianae) (fam. Teiidae), including a very high proportion of carboxylic acids and their esters ranging between n-C8 and n-C20 (mainly octadecanoic and 9,12-octadecadienoic acids), with much less proportions of steroids, tocopherol, aldehydes, and squalene. We discuss the potential function of these compounds in secretions, and compare the compounds found here with those documented for other lizard species.

Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

PubMed Central

Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel

2006-01-01

A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%. PMID:16511329

The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions

PubMed Central

Bartho, Joseph D.; Bellini, Dom; Wuerges, Jochen; Demitri, Nicola; Toccafondi, Mirco; Schmitt, Armin O.; Zhao, Youfu; Walsh, Martin A.

2017-01-01

AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family. PMID:28426806

The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions.

PubMed

Bartho, Joseph D; Bellini, Dom; Wuerges, Jochen; Demitri, Nicola; Toccafondi, Mirco; Schmitt, Armin O; Zhao, Youfu; Walsh, Martin A; Benini, Stefano

2017-01-01

AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.

How Shigella Utilizes Ca2+ Jagged Edge Signals during Invasion of Epithelial Cells

PubMed Central

Bonnet, Mariette; Tran Van Nhieu, Guy

2016-01-01

Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca2+ overload. In this review, we will focus on the role of Ca2+ responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-10-20

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp. Copyright © 2011 Elsevier B.V. All rights reserved.

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-β1 in mouse cardiac fibroblasts.

PubMed

Liu, Jin-Cheng; Wang, Feng; Xie, Mei-Lin; Cheng, Zong-Qi; Qin, Qiong; Chen, Lin; Chen, Rong

2017-02-01

Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1. The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Multimodal sexual signals in male ocellated lizards Lacerta lepida: vitamin E in scent and green coloration may signal male quality in different sensory channels

NASA Astrophysics Data System (ADS)

Martín, José; López, Pilar

2010-06-01

The mechanisms that confer honesty to multiple sexual signals of vertebrates remain less known. We suggest that vitamin E in the scent (femoral secretions) and carotenoids in coloration of Lacerta lepida lizards might advertise quality because they are relevant in metabolism as major antioxidants and have a dietary origin. Results showed that there may be similar trade-offs between immunity and both chemical and visual signaling, as males with more vitamin E in secretions and those with more greenish, darker, and saturated carotenoid-dependent colorations had a higher immune response. This suggests that only high-quality males can divert from metabolism and allocate enough vitamin E to secretions and enough carotenoids to coloration. Therefore, information provided by visual and chemical signals is congruent. The use of multiple sensory channels may allow signaling a male quality under different circumstances or reinforce the reliability of the signal when both signals are perceived simultaneously. However, we also found that characteristics of carotenoid-dependent coloration mirror the amounts of vitamin E in secretions. This might support that carotenoids, which are not true antioxidants in the organism, would just reflect and “inform” on the actual contents of metabolic antioxidant vitamin E, which is part of the true system that prevent oxidation. We suggest that the metabolic roles of vitamins might be the actual basis of honesty of many sexual displays, and that carotenoid-based signals might be just the visible part of the whole system.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling

PubMed Central

Jahani-Asl, Arezu; Yin, Hang; Soleimani, Vahab D; Haque, Takrima; Luchman, H Artee; Chang, Natasha C; Sincennes, Marie-Claude; Puram, Sidharth V; Scott, Andrew M; Lorimer, Ian A J; Perkins, Theodore J; Ligon, Keith L; Weiss, Samuel; Rudnicki, Michael A; Bonni, Azad

2016-01-01

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of those mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis. PMID:27110918

Magnetic Nature of the CrIII–LnIII Interactions in [CrIII 2LnIII 3] Clusters with Slow Magnetic Relaxation

PubMed Central

Xiang, Shuo; Wang, Jin; Bao, Dong‐Xu; Li, Yun‐Chun

2018-01-01

Abstract Two 3d‐4f hetero‐metal pentanuclear complexes with the formula {[CrIII 2LnIII 3L10(OH)6(H2O)2]Et3NH} [Ln=Tb (1), Dy (2); HL=pivalic acid, Et3N=triethylamine] have been produced. The metal core of each cluster is made up of a trigonal bipyramid with three LnIII ions (plane) and two CrIII ions (above and below) held together by six μ 3‐OH bridges. Also reported with this series is the diamagnetic CrIII–YIII analogue (3). Fortunately, we successfully prepared AlIII–LnIII analogues with the formula {[AlIII 2LnIII 3L10(OH)6(H2O)2]Et3NH⋅H2O} [Ln=Tb (4), Dy (5)], containing diamagnetic AlIII ions, which can be used to evaluate the CrIII–LnIII magnetic nature through a diamagnetic substitution method. Subsequently, static (dc) magnetic susceptibility studies reveal dominant ferromagnetic interactions between CrIII and LnIII ions. Dynamic (ac) magnetic susceptibility studies show frequency‐dependent out‐of‐phase (χ′′) signals for [CrIII 2TbIII 3] (1), [CrIII 2DyIII 3] (2), and [AlIII 2DyIII 3] (5), which are derived from the single‐ion behavior of LnIII ions and/or the CrIII–LnIII ferromagnetic interactions. PMID:29435404

Measurement of gastric meal and secretion volumes using magnetic resonance imaging

PubMed Central

Hoad, C.L.; Parker, H.; Hudders, N.; Costigan, C.; Cox, E.F.; Perkins, A.C.; Blackshaw, P.E.; Marciani, L.; Spiller, R.C.; Fox, M.R.; Gowland, P.A.

2015-01-01

MRI can assess multiple gastric functions without ionizing radiation. However, time consuming image acquisition and analysis of gastric volume data, plus confounding of gastric emptying measurements by gastric secretions mixed with the test meal have limited its use to research centres. This study presents an MRI acquisition protocol and analysis algorithm suitable for the clinical measurement of gastric volume and secretion volume. Reproducibility of gastric volume measurements was assessed using data from 10 healthy volunteers following a liquid test meal with rapid MRI acquisition within one breath-hold and semi-automated analysis. Dilution of the ingested meal with gastric secretion was estimated using a respiratory-triggered T1 mapping protocol. Accuracy of the secretion volume measurements was assessed using data from 24 healthy volunteers following a mixed (liquid/solid) test meal with MRI meal volumes compared to data acquired using gamma scintigraphy (GS) on the same subjects studied on a separate study day. The mean (SD) coefficient of variance between 3 observers for both total gastric contents (including meal, secretions and air) and just the gastric contents (meal and secretion only) was 3 (2) % at large gastric volumes (> 200 ml). Mean (SD) secretion volumes post meal ingestion were 64 (51) ml and 110 (40) ml at 15 and 75 minutes respectively. Comparison with GS meal volumes, showed that MRI meal only volume (after correction for secretion volume) were similar to GS, with a linear regression gradient (std err) of 1.06 (0.10) and intercept −11 (24) ml. In conclusion, (i) rapid acquisition removed the requirement to image during prolonged breath-hold (ii) semi-automatic analysis greatly reduced time required to derive measurements and (iii) correction for secretion volumes provides accurate assessment of gastric meal volumes and emptying. Together these features provide the scientific basis of a protocol which would be suitable in clinical practice

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

PubMed Central

Lountos, George T; Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S

2009-01-01

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a β-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU. PMID:19165725

Uncoupling of Secretion From Growth in Some Hormone Secretory Tissues

PubMed Central

2014-01-01

Context: Most syndromes with benign primary excess of a hormone show positive coupling of hormone secretion to size or proliferation in the affected hormone secretory tissue. Syndromes that lack this coupling seem rare and have not been examined for unifying features among each other. Evidence Acquisition: Selected clinical and basic features were analyzed from original reports and reviews. We examined indices of excess secretion of a hormone and indices of size of secretory tissue within the following three syndromes, each suggestive of uncoupling between these two indices: familial hypocalciuric hypercalcemia, congenital diazoxide-resistant hyperinsulinism, and congenital primary hyperaldosteronism type III (with G151E mutation of the KCNJ5 gene). Evidence Synthesis: Some unifying features among the three syndromes were different from features present among common tumors secreting the same hormone. The unifying and distinguishing features included: 1) expression of hormone excess as early as the first days of life; 2) normal size of tissue that oversecretes a hormone; 3) diffuse histologic expression in the hormonal tissue; 4) resistance to treatment by subtotal ablation of the hormone-secreting tissue; 5) causation by a germline mutation; 6) low potential of the same mutation to cause a tumor by somatic mutation; and 7) expression of the mutated molecule in a pathway between sensing of a serum metabolite and secretion of hormone regulating that metabolite. Conclusion: Some shared clinical and basic features of uncoupling of secretion from size in a hormonal tissue characterize three uncommon states of hormone excess. These features differ importantly from features of common hormonal neoplasm of that tissue. PMID:25004249

Regulatory RNAs and the HptB/RetS signalling pathways fine-tune Pseudomonas aeruginosa pathogenesis

PubMed Central

Bordi, Christophe; Lamy, Marie-Cécile; Ventre, Isabelle; Termine, Elise; Hachani, Abderrahman; Fillet, Sandy; Roche, Béatrice; Bleves, Sophie; Méjean, Vincent; Lazdunski, Andrée; Filloux, Alain

2010-01-01

Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-σ factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression. PMID:20398205

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

PubMed

Burgess, Jamie L; Burgess, R Alan; Morales, Yalemi; Bouvang, Jenna M; Johnson, Sean J; Dickenson, Nicholas E

2016-12-09

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Hedgehog signaling in the stomach.

PubMed

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-12-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease, and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration, and disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein Secretion Is Required for Pregnancy-Associated Plasma Protein-A to Promote Lung Cancer Growth In Vivo

PubMed Central

Pan, Hong; Hanada, Sayaka; Zhao, Jun; Mao, Li; Ma, Mark Zhi-Qing

2012-01-01

Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression. PMID:23152806

Interpopulational Variations in Sexual Chemical Signals of Iberian Wall Lizards May Allow Maximizing Signal Efficiency under Different Climatic Conditions.

PubMed

Martín, José; Ortega, Jesús; López, Pilar

2015-01-01

Sexual signals used in intraspecific communication are expected to evolve to maximize efficacy under a given climatic condition. Thus, chemical secretions of lizards might evolve in the evolutionary time to ensure that signals are perfectly tuned to local humidity and temperature conditions affecting their volatility and therefore their persistence and transmission through the environment. We tested experimentally whether interpopulational altitudinal differences in chemical composition of femoral gland secretions of male Iberian wall lizards (Podarcis hispanicus) have evolved to maximize efficacy of chemical signals in different environmental conditions. Chemical analyses first showed that the characteristics of chemical signals of male lizards differed between two populations inhabiting environments with different climatic conditions in spite of the fact that these two populations are closely related genetically. We also examined experimentally whether the temporal attenuation of the chemical stimuli depended on simulated climatic conditions. Thus, we used tongue-flick essays to test whether female lizards were able to detect male scent marks maintained under different conditions of temperature and humidity by chemosensory cues alone. Chemosensory tests showed that chemical signals of males had a lower efficacy (i.e. detectability and persistence) when temperature and dryness increase, but that these effects were more detrimental for signals of the highest elevation population, which occupies naturally colder and more humid environments. We suggest that the abiotic environment may cause a selective pressure on the form and expression of sexual chemical signals. Therefore, interpopulational differences in chemical profiles of femoral secretions of male P. hispanicus lizards may reflect adaptation to maximize the efficacy of the chemical signal in different climates.

Role of obestatin on growth hormone secretion: An in vitro approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pazos, Yolanda, E-mail: yolanda.pazos@usc.es; CIBER Fisiopatologia de la Obesidad y Nutricion; Alvarez, Carlos J.P.

Obestatin, the ghrelin-associated peptide, showed to activate MAPK signaling with no effect on Akt nor cell proliferating activity in rat tumor somatotroph cells (growth cells, GC). A sequential analysis of the obestatin transmembrane signaling pathway indicated a route involving the consecutive activation of G{sub i}, PI3k, novel PKC{epsilon}, and Src for ERK1/2 activation. Furthermore, obestatin treatment triggers growth hormone (GH) release in the first 30 min, being more acute at 15 min. At 1 h, obestatin treated cells showed the same levels in GH secretion than controls. Added to this functionality, obestatin was secreted by GC cells. Based on themore » capacity to stimulate GH release from somatotroph cells, obestatin may act directly in the pituitary through an autocrine/paracrine mechanism.« less

Effects of FGF-23-mediated ERK/MAPK signaling pathway on parathyroid hormone secretion of parathyroid cells in rats with secondary hyperparathyroidism.

PubMed

Chen, Xiao-Jun; Chen, Xiong; Wu, Wen-Jun; Zhou, Qi; Gong, Xiao-Hua; Shi, Bi-Min

2018-04-10

This study is supposed to investigate the effect of FGF-23 on parathyroid hormone (PTH) secretion through ERK/MAPK signaling pathway in secondary hyperparathyroidism (SHPT) rat model. Thirty rats were equally served as the normal and SHPT groups. After transfection, parathyroid cells was assigned into blank, NC, pcDNA3.1-FGF-23, siRNA-FGF-23, U0126, and siRNA-FGF-23 + U0126 groups. The serum levels of Calcium (Ca), Phosphorus (P), alkaline phosphatase (ALP), and PTH were detected. HE and immunohistochemical (IHC) staining were used for the histopathological changes and the FGF-23, EKR1/2, and pEKR1/2 expressions. qRT-PCR and Western blotting were performed to determine the mRNA and protein expression of FGF-23, PTH, MAPK, EKR1/2, and Klotho. The proliferation, apoptosis, and cell cycle were all measured for parathyroid cells by CCK-8 assay, TUNEL staining and Flow cytometry. Compared with the normal group, the SHPT group showed increased serum levels PTH, P, ALP, and FGF-23 and mRNA and protein expressions of FGF-23 and PTH, whereas declined Ca and p-ERK1/2 expression, mRNA and protein expression of Klotho, cell apoptosis rate was reduced. Furthermore, compared to the blank and NC groups, the pcDNA3.1-FGF-23 and U0126 groups had a decreased mRNA expression of Klotho, protein expression of EKR1/2 and Klotho, and cell apoptosis rate was down-regulated, whereas the RNA and protein expressions of FGF-23 and PTH were up-regulated, and cell proliferation was elevated. The opposite results were observed in the siRNA-FGF-23 group. Our study demonstrated that FGF-23 could inhibit signaling transduction of ERK/MAPK pathway and accelerate the secretion of PTH in rats with SHPT. © 2018 Wiley Periodicals, Inc.

The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling

PubMed Central

Zhou, Bangjun; Zeng, Lirong

2018-01-01

In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI) and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2) with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum) homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

Interleukin-6 amplifies glucagon secretion: coordinated control via the brain and pancreas

PubMed Central

Barnes, Tammy M.; Otero, Yolanda F.; Elliott, Amicia D.; Locke, Alicia D.; Malabanan, Carlo M.; Coldren, Anastasia G.; Brissova, Marcela; Piston, David W.

2014-01-01

Inappropriate glucagon secretion contributes to hyperglycemia in inflammatory disease. Previous work implicates the proinflammatory cytokine interleukin-6 (IL-6) in glucagon secretion. IL-6-KO mice have a blunted glucagon response to lipopolysaccharide (LPS) that is restored by intravenous replacement of IL-6. Given that IL-6 has previously been demonstrated to have a transcriptional (i.e., slow) effect on glucagon secretion from islets, we hypothesized that the rapid increase in glucagon following LPS occurred by a faster mechanism, such as by action within the brain. Using chronically catheterized conscious mice, we have demonstrated that central IL-6 stimulates glucagon secretion uniquely in the presence of an accompanying stressor (hypoglycemia or LPS). Contrary to our hypothesis, however, we found that IL-6 amplifies glucagon secretion in two ways; IL-6 not only stimulates glucagon secretion via the brain but also by direct action on islets. Interestingly, IL-6 augments glucagon secretion from both sites only in the presence of an accompanying stressor (such as epinephrine). Given that both adrenergic tone and plasma IL-6 are elevated in multiple inflammatory diseases, the interactions of the IL-6 and catecholaminergic signaling pathways in regulating GCG secretion may contribute to our present understanding of these diseases. PMID:25205821

Pulsatile Hormonal Signaling to Extracellular Signal-regulated Kinase

PubMed Central

Perrett, Rebecca M.; Voliotis, Margaritis; Armstrong, Stephen P.; Fowkes, Robert C.; Pope, George R.; Tsaneva-Atanasova, Krasimira; McArdle, Craig A.

2014-01-01

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses that stimulate synthesis and secretion of pituitary gonadotropin hormones and thereby mediate control of reproduction. It acts via G-protein-coupled receptors to stimulate effectors, including ERK. Information could be encoded in GnRH pulse frequency, width, amplitude, or other features of pulse shape, but the relative importance of these features is unknown. Here we examine this using automated fluorescence microscopy and mathematical modeling, focusing on ERK signaling. The simplest scenario is one in which the system is linear, and response dynamics are relatively fast (compared with the signal dynamics). In this case integrated system output (ERK activation or ERK-driven transcription) will be roughly proportional to integrated input, but we find that this is not the case. Notably, we find that relatively slow response kinetics lead to ERK activity beyond the GnRH pulse, and this reduces sensitivity to pulse width. More generally, we show that the slowing of response kinetics through the signaling cascade creates a system that is robust to pulse width. We, therefore, show how various levels of response kinetics synergize to dictate system sensitivity to different features of pulsatile hormone input. We reveal the mathematical and biochemical basis of a dynamic GnRH signaling system that is robust to changes in pulse amplitude and width but is sensitive to changes in receptor occupancy and frequency, precisely the features that are tightly regulated and exploited to exert physiological control in vivo. PMID:24482225

β-Cell–Specific Protein Kinase A Activation Enhances the Efficiency of Glucose Control by Increasing Acute-Phase Insulin Secretion

PubMed Central

Kaihara, Kelly A.; Dickson, Lorna M.; Jacobson, David A.; Tamarina, Natalia; Roe, Michael W.; Philipson, Louis H.; Wicksteed, Barton

2013-01-01

Acute insulin secretion determines the efficiency of glucose clearance. Moreover, impaired acute insulin release is characteristic of reduced glucose control in the prediabetic state. Incretin hormones, which increase β-cell cAMP, restore acute-phase insulin secretion and improve glucose control. To determine the physiological role of the cAMP-dependent protein kinase (PKA), a mouse model was developed to increase PKA activity specifically in the pancreatic β-cells. In response to sustained hyperglycemia, PKA activity potentiated both acute and sustained insulin release. In contrast, a glucose bolus enhanced acute-phase insulin secretion alone. Acute-phase insulin secretion was increased 3.5-fold, reducing circulating glucose to 58% of levels in controls. Exendin-4 increased acute-phase insulin release to a similar degree as PKA activation. However, incretins did not augment the effects of PKA on acute-phase insulin secretion, consistent with incretins acting primarily via PKA to potentiate acute-phase insulin secretion. Intracellular calcium signaling was unaffected by PKA activation, suggesting that the effects of PKA on acute-phase insulin secretion are mediated by the phosphorylation of proteins involved in β-cell exocytosis. Thus, β-cell PKA activity transduces the cAMP signal to dramatically increase acute-phase insulin secretion, thereby enhancing the efficiency of insulin to control circulating glucose. PMID:23349500

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.

PubMed

Suzuki, Michitaka; Sugimoto, Yuko; Ohsaki, Yuki; Ueno, Makoto; Kato, Shinsuke; Kitamura, Yukisato; Hosokawa, Hiroshi; Davies, Joanna P; Ioannou, Yiannis A; Vanier, Marie T; Ohno, Kousaku; Ninomiya, Haruaki

2007-02-21

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

PubMed

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A; Schmid, Michael C; Schröder, Gunnar; Vergunst, Annette C; Carena, Ilaria; Dehio, Christoph

2005-01-18

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells

PubMed Central

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A.; Schmid, Michael C.; Schröder, Gunnar; Vergunst, Annette C.; Carena, Ilaria; Dehio, Christoph

2005-01-01

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells. PMID:15642951

ClpXP-Dependent RpoS Degradation Enables Full Activation of Type III Secretion System, Amylovoran Production, and Motility in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Zhao, Youfu

2017-11-01

Erwinia amylovora, the causal agent of fire blight disease of apple and pear, employs intracellular proteases, including Lon and ClpXP, for posttranslational regulation of various cellular proteins. It has been shown that Lon plays a critical role in E. amylovora virulence by directly targeting type III secretion system (T3SS) proteins and the Rcs phosphorelay system. In this study, we genetically examined the role of ClpXP and its potential interaction with Lon in E. amylovora. Mutation in clpXP diminished the expression of the T3SS, reduced exopolysaccharide amylovoran production and motility, and resulted in delayed disease progress. Western blot analyses showed highly accumulated RpoS proteins in the clpXP mutant. Moreover, mutation of rpoS in the clpXP mutant background rescued the expression of the T3SS and amylovoran production, suggesting that ClpXP-dependent RpoS degradation positively affects virulence traits. Interestingly, lack of both ClpXP and Lon resulted in significantly reduced virulence but increased expression of the T3SS and amylovoran production. However, this phenomenon was independent of RpoS accumulation, suggesting that ClpXP and Lon are indispensable for full virulence in E. amylovora.

FluG affects secretion in colonies of Aspergillus niger.

PubMed

Wang, Fengfeng; Krijgsheld, Pauline; Hulsman, Marc; de Bekker, Charissa; Müller, Wally H; Reinders, Marcel; de Vries, Ronald P; Wösten, Han A B

2015-01-01

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion.

Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2011-08-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

Mechanisms of Estradiol-Induced Insulin Secretion by the G Protein-Coupled Estrogen Receptor GPR30/GPER in Pancreatic β-Cells

PubMed Central

Sharma, Geetanjali

2011-01-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes. PMID:21673097

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

PubMed

Redhai, Siamak; Hellberg, Josephine E E U; Wainwright, Mark; Perera, Sumeth W; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C I; Wilson, Clive

2016-10-01

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

PubMed Central

Redhai, Siamak; Hellberg, Josephine E. E. U.; Wainwright, Mark; Perera, Sumeth W.; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C. I.

2016-01-01

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion. PMID:27727275

Genetic Polymorphism in Extracellular Regulators of Wnt Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Seo, Eun-Min; Nam, Ju-Suk

2015-01-01

The Wnt signaling pathway is mediated by a family of secreted glycoproteins through canonical and noncanonical mechanism. The signaling pathways are regulated by various modulators, which are classified into two classes on the basis of their interaction with either Wnt or its receptors. Secreted frizzled-related proteins (sFRPs) are the member of class that binds to Wnt protein and antagonizes Wnt signaling pathway. The other class consists of Dickkopf (DKK) proteins family that binds to Wnt receptor complex. The present review discusses the disease related association of various polymorphisms in Wnt signaling modulators. Furthermore, this review also highlights that some of the sFRPs and DKKs are unable to act as an antagonist for Wnt signaling pathway and thus their function needs to be explored more extensively. PMID:25945348

Measurement of gastric meal and secretion volumes using magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Hoad, C. L.; Parker, H.; Hudders, N.; Costigan, C.; Cox, E. F.; Perkins, A. C.; Blackshaw, P. E.; Marciani, L.; Spiller, R. C.; Fox, M. R.; Gowland, P. A.

2015-02-01

MRI can assess multiple gastric functions without ionizing radiation. However, time consuming image acquisition and analysis of gastric volume data, plus confounding of gastric emptying measurements by gastric secretions mixed with the test meal have limited its use to research centres. This study presents an MRI acquisition protocol and analysis algorithm suitable for the clinical measurement of gastric volume and secretion volume. Reproducibility of gastric volume measurements was assessed using data from 10 healthy volunteers following a liquid test meal with rapid MRI acquisition within one breath-hold and semi-automated analysis. Dilution of the ingested meal with gastric secretion was estimated using a respiratory-triggered T1 mapping protocol. Accuracy of the secretion volume measurements was assessed using data from 24 healthy volunteers following a mixed (liquid/solid) test meal with MRI meal volumes compared to data acquired using gamma scintigraphy (GS) on the same subjects studied on a separate study day. The mean ± SD coefficient of variance between 3 observers for both total gastric contents (including meal, secretions and air) and just the gastric contents (meal and secretion only) was 3  ±  2% at large gastric volumes (>200 ml). Mean ± SD secretion volumes post meal ingestion were 64  ±  51 ml and 110  ±  40 ml at 15 and 75 min, respectively. Comparison with GS meal volumes, showed that MRI meal only volume (after correction for secretion volume) were similar to GS, with a linear regression gradient ± std err of 1.06  ±  0.10 and intercept -11  ±  24 ml. In conclusion, (i) rapid volume acquisition and respiratory triggered T1 mapping removed the requirement to image during prolonged breath-holds (ii) semi-automatic analysis greatly reduced the time required to derive measurements and (iii) correction for secretion volumes provided accurate assessment of gastric meal volumes and emptying. Together these features

Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola

PubMed Central

Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

2014-01-01

The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism. PMID:24675748

Identification of 17 HrpX-regulated proteins including two novel type III effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola.

PubMed

Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

2014-01-01

The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9 ≤ X ≤ 25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.

Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

PubMed Central

Pan, Qing; Zhang, Qiang; Chu, Jun; Pais, Roshan; Liu, Shanshan; He, Cheng; Eko, Francis O.

2017-01-01

The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine

Important role of heparan sulfate in postnatal islet growth and insulin secretion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Iwao; Noguchi, Naoya; Nata, Koji

2009-05-22

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet {beta}-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in {beta}-cells. These mice exhibited abnormal islet morphology with reduced {beta}-cell proliferation after 1 week of age and glucosemore » intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.« less

Interpopulational Variations in Sexual Chemical Signals of Iberian Wall Lizards May Allow Maximizing Signal Efficiency under Different Climatic Conditions

PubMed Central

2015-01-01

Sexual signals used in intraspecific communication are expected to evolve to maximize efficacy under a given climatic condition. Thus, chemical secretions of lizards might evolve in the evolutionary time to ensure that signals are perfectly tuned to local humidity and temperature conditions affecting their volatility and therefore their persistence and transmission through the environment. We tested experimentally whether interpopulational altitudinal differences in chemical composition of femoral gland secretions of male Iberian wall lizards (Podarcis hispanicus) have evolved to maximize efficacy of chemical signals in different environmental conditions. Chemical analyses first showed that the characteristics of chemical signals of male lizards differed between two populations inhabiting environments with different climatic conditions in spite of the fact that these two populations are closely related genetically. We also examined experimentally whether the temporal attenuation of the chemical stimuli depended on simulated climatic conditions. Thus, we used tongue-flick essays to test whether female lizards were able to detect male scent marks maintained under different conditions of temperature and humidity by chemosensory cues alone. Chemosensory tests showed that chemical signals of males had a lower efficacy (i.e. detectability and persistence) when temperature and dryness increase, but that these effects were more detrimental for signals of the highest elevation population, which occupies naturally colder and more humid environments. We suggest that the abiotic environment may cause a selective pressure on the form and expression of sexual chemical signals. Therefore, interpopulational differences in chemical profiles of femoral secretions of male P. hispanicus lizards may reflect adaptation to maximize the efficacy of the chemical signal in different climates. PMID:26121693

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

PubMed Central

Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

2013-01-01

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400

Verifiable Secret Redistribution

DTIC Science & Technology

2001-10-01

but they are not trusted with secret. Thus, we require a protocol for redistribution without reconstruction of the secret . We also require...verification that the new shareholders have valid shares (ones that can be used to reconstruct the secret ). We present a new protocol to perform non...secret to shareholders in Shamir’s (m,n) threshold scheme (one in which we require m of n shares to reconstruct the secret ), and wish to redistribute the

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System*

PubMed Central

Fritsch, Maximilian J.; Trunk, Katharina; Diniz, Juliana Alcoforado; Guo, Manman; Trost, Matthias; Coulthurst, Sarah J.

2013-01-01

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial

Droplet based microfluidics for highthroughput screening of antibody secreting cells

NASA Astrophysics Data System (ADS)

Cai, Liheng; Heyman, John; Mazutis, Linas; Ung, Lloyd; Guerra, Rodrigo; Aubrecht, Donald; Weitz, David

2014-03-01

We present a droplet based microfluidic platform that allows highthroughput screening of antibody secreting cells. We coencapsulate single cells, fluorescent probes, and detection beads into emulsion droplets with diameter of 40 micron. The beads capture antibodies secreted by cells, resulting in a pronounced fluorescent signal that activates dielectrophoresis sorting at rate about 500 droplets per second. Moreover, we demonstrate that Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be successfully applied to the cell encapsulated in a single sorted droplet. Our work highlights the potential of droplet based microfluidics as a platform to generate recombinant antibodies.

Pancreatic signals controlling food intake; insulin, glucagon and amylin

PubMed Central

Woods, Stephen C; Lutz, Thomas A; Geary, Nori; Langhans, Wolfgang

2006-01-01

The control of food intake and body weight by the brain relies upon the detection and integration of signals reflecting energy stores and fluxes, and their interaction with many different inputs related to food palatability and gastrointestinal handling as well as social, emotional, circadian, habitual and other situational factors. This review focuses upon the role of hormones secreted by the endocrine pancreas: hormones, which individually and collectively influence food intake, with an emphasis upon insulin, glucagon and amylin. Insulin and amylin are co-secreted by B-cells and provide a signal that reflects both circulating energy in the form of glucose and stored energy in the form of visceral adipose tissue. Insulin acts directly at the liver to suppress the synthesis and secretion of glucose, and some plasma insulin is transported into the brain and especially the mediobasal hypothalamus where it elicits a net catabolic response, particularly reduced food intake and loss of body weight. Amylin reduces meal size by stimulating neurons in the hindbrain, and there is evidence that amylin additionally functions as an adiposity signal controlling body weight as well as meal size. Glucagon is secreted from A-cells and increases glucose secretion from the liver. Glucagon acts in the liver to reduce meal size, the signal being relayed to the brain via the vagus nerves. To summarize, hormones of the endocrine pancreas are collectively at the crossroads of many aspects of energy homeostasis. Glucagon and amylin act in the short term to reduce meal size, and insulin sensitizes the brain to short-term meal-generated satiety signals; and insulin and perhaps amylin as well act over longer intervals to modulate the amount of fat maintained and defended by the brain. Hormones of the endocrine pancreas interact with receptors at many points along the gut–brain axis, from the liver to the sensory vagus nerve to the hindbrain to the hypothalamus; and their signals are

Unitary reconstruction of secret for stabilizer-based quantum secret sharing

NASA Astrophysics Data System (ADS)

Matsumoto, Ryutaroh

2017-08-01

We propose a unitary procedure to reconstruct quantum secret for a quantum secret sharing scheme constructed from stabilizer quantum error-correcting codes. Erasure correcting procedures for stabilizer codes need to add missing shares for reconstruction of quantum secret, while unitary reconstruction procedures for certain class of quantum secret sharing are known to work without adding missing shares. The proposed procedure also works without adding missing shares.

Authentication Without Secrets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierson, Lyndon G.; Robertson, Perry J.

This work examines a new approach to authentication, which is the most fundamental security primitive that underpins all cyber security protections. Current Internet authentication techniques require the protection of one or more secret keys along with the integrity protection of the algorithms/computations designed to prove possession of the secret without actually revealing it. Protecting a secret requires physical barriers or encryption with yet another secret key. The reason to strive for "Authentication without Secret Keys" is that protecting secrets (even small ones only kept in a small corner of a component or device) is much harder than protecting the integritymore » of information that is not secret. Promising methods are examined for authentication of components, data, programs, network transactions, and/or individuals. The successful development of authentication without secret keys will enable far more tractable system security engineering for high exposure, high consequence systems by eliminating the need for brittle protection mechanisms to protect secret keys (such as are now protected in smart cards, etc.). This paper is a re-release of SAND2009-7032 with new figures numerous edits.« less

Mechanism involved in Danshen-induced fluid secretion in salivary glands

PubMed Central

Wei, Fei; Wei, Mu-Xin; Murakami, Masataka

2015-01-01

and NKCC1 to maintain Cl- release and K+ release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca2+ from the calcium stores. The elevation of [Ca2+]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca2+]i is essential for the activation of the luminal Cl- and basolateral K+ channels. The nominal removal of extracellular Ca2+ totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 μL/g-min vs 101.9 ± 17.2 μL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca2+ in the activation of these channels. Therefore, IP3-store Ca2+ release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process. CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs. PMID:25663764

Secreting and sensing the same molecule allows cells to achieve versatile social behaviors

PubMed Central

Youk, Hyun; Lim, Wendell A.

2014-01-01

Cells that secrete and sense the same signaling molecule are ubiquitous. To uncover the functional capabilities of the core ‘secrete-and-sense’ circuit motif shared by these cells, we engineered yeast to secrete and sense the mating pheromone. Perturbing each circuit element revealed parameters that control the degree to which the cell communicated with itself versus with its neighbors. This tunable interplay of self- and neighbor-communication enables cells to span a diverse repertoire of cellular behaviors. These include a cell being asocial by responding only to itself, social through quorum sensing and an isogenic population of cells splitting into social and asocial subpopulations. A mathematical model explained these behaviors. The versatility of the secrete-and-sense circuit motif may explain its recurrence across species. PMID:24503857

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii.

PubMed

Brown, Kevin M; Lourido, Sebastian; Sibley, L David

2016-04-29

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.) © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium.

PubMed

Huber, Robert J

2017-07-01

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3 - cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3 - cells displayed delayed streaming and aggregation, and interestingly, cln3 - cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3 - CM, of which 3 were absent in cln3 - CM. Moreover, 12 proteins that were present in

Bottleneck in secretion of α-amylase in Bacillus subtilis.

PubMed

Yan, Shaomin; Wu, Guang

2017-07-19

Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.

Harnessing Novel Secreted Inhibitors of EGF Receptor Signaling for Breast Cancer Treatment

DTIC Science & Technology

2007-04-01

the original proposal, we described approaches for displaying the basic Argos and Dkk scaffolds as pIII fusions on M13 phage , so that we could...deal of effort into displaying Argos and other relevant proteins as pIII fusions on M13 phage (Task 2a). This has been technically very challenging... display function Argos on the surface of phage M13 , requiring a change in experimental strategy • To replace the phage display , we have established

Ca(2+) signaling mechanisms in bovine adrenal chromaffin cells.

PubMed

Weiss, Jamie L

2012-01-01

Calcium (Ca(2+)) is a crucial intracellular messenger in physiological aspects of cell signaling. Adrenal chromaffin cells are the secretory cells from the adrenal gland medulla that secrete catecholamines, which include epinephrine and norepinephrine important in the 'fight or flight' response. Bovine adrenal chromaffin cells have long been used as an important model for secretion -(exocytosis) not only due to their importance in the short-term stress response, but also as a neuroendocrine model of neurotransmtter release, as they have all the same exocytotic proteins as neurons but are easier to prepare, culture and use in functional assays. The components of the Ca(2+) signal transduction cascade and it role in secretion has been extensively characterized in bovine adrenal chromaffin cells. The Ca(2+) sources, signaling molecules and how this relates to the short-term stress response are reviewed in this book chapter in an endeavor to generally -overview these mechanisms in a concise and uncomplicated manner.

Genetic selection for temperament affects behaviour and the secretion of adrenal and reproductive hormones in sheep subjected to stress.

PubMed

Hawken, P A R; Luckins, N; Tilbrook, A; Fiol, C; Martin, G B; Blache, D

2013-01-01

We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.

[Effect of different nutritional support on pancreatic secretion in acute pancreatitis].