scholarly journals Molecular determinants of enterotoxigenicEscherichia coliheat-stable toxin secretion and delivery

2018 ◽  
Author(s):  
Yuehui Zhu ◽  
Qingwei Luo ◽  
Sierra M. Davis ◽  
Chase Westra ◽  
Tim J. Vickers ◽  
...  
Keyword(s):  
Peptide Sequence ◽  
T84 Cells ◽  
Developing Regions ◽  
Flag Epitope ◽  
Heat Stable ◽  
Molecular Determinants ◽  
Genes Encoding ◽  
Recombinant Peptides ◽  
Toxin Secretion ◽  
Causes Of Mortality

AbstractEnterotoxigenicEscherichia coli(ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, remain major causes of mortality among children in developing regions, and cause substantial morbidity in individuals living in or traveling to endemic areas. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We therefore examined fundamental aspects of ETEC ST biology using ETEC H10407, which carriesestHandestPgenes encoding ST-H and ST-P, respectively, in addition toeltABgenes responsible for LT. In this background, we found that deletion ofestHsignificantly diminished cGMP activation in target epithelia, while deletion ofestPhad a surprisingly modest impact, and a dualestH/estPmutant was not appreciably different than theestHmutant. Nevertheless, either ST-H or ST-P recombinant peptides stimulated cGMP production. We also found that the TolC efflux protein was essential for both toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for optimal delivery of ST and that antibodies against either the adhesin or ST-H significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the ST-H pro-peptide sequence is secreted by the bacteria, potentially providing additional targets for antibody neutralization. These studies collectively extend our understanding of ETEC pathogenesis and potentially inform additional avenues to mitigate disease by these common diarrheal pathogens.

scholarly journals Molecular Determinants of EnterotoxigenicEscherichia coliHeat-Stable Toxin Secretion and Delivery

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Yuehui Zhu ◽  
Qingwei Luo ◽  
Sierra M. Davis ◽  
Chase Westra ◽  
Tim J. Vickers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
T84 Cells ◽  
Flag Epitope ◽  
Content Type ◽  
Heat Stable ◽  
Molecular Determinants ◽  
Genes Encoding ◽  
Recombinant Peptides ◽  
Toxin Secretion ◽  
Substantial Morbidity

ABSTRACTEnterotoxigenicEscherichia coli(ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, causes substantial morbidity among young children in the developing world. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We examined fundamental aspects of ST biology using ETEC strain H10407, which carriesestHandestPgenes encoding STh and STp, respectively, in addition toeltABgenes responsible for LT. Here, we found that deletion ofestHsignificantly diminished cyclic GMP (cGMP) activation in target epithelia, while deletion ofestPhad a surprisingly modest impact, and a dualestH estPmutant was not appreciably different from theestHmutant. However, we noted that either STh or STp recombinant peptides stimulated cGMP production and that the loss ofestPwas compensated by enhancedestHtranscription. We also found that the TolC efflux protein was essential for toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for optimal delivery of ST and that antibodies against either the adhesin or STh significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the STh propeptide sequence is secreted by ETEC, potentially providing additional epitopes for antibody neutralization. These studies collectively extend our understanding of ETEC pathogenesis and potentially inform additional avenues to mitigate disease by these common diarrheal pathogens.

Role of Cytochrome P450 in Prostate Cancer and its Therapy

2020 ◽  
Vol 16 (1) ◽  
pp. 63-73 ◽  
Author(s):  
Rishabh Kaushik ◽  
Sheeza Khan ◽  
Meesha Sharma ◽  
Srinivasan Hemalatha ◽  
Zeba Mueed ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cytochrome P450 ◽  
Drug Discovery ◽  
Cholesterol Metabolism ◽  
Molecular Targets ◽  
Health Concern ◽  
Metabolic Functions ◽  
Novel Drugs ◽  
Genes Encoding ◽  
Causes Of Mortality

Prostate cancer has become a global health concern as it is one of the leading causes of mortality in males. With the emerging drug resistance to conventional therapies, it is imperative to unravel new molecular targets for disease prevention. Cytochrome P450 (P450s or CYPs) represents a unique class of mixed-function oxidases which catalyses a wide array of biosynthetic and metabolic functions including steroidogenesis and cholesterol metabolism. Several studies have reported the overexpression of the genes encoding CYPs in prostate cancer cells and how they can be used as molecular targets for drug discovery. But due to functional redundancy and overlapping expression of CYPs in several other metabolic pathways there are several impediments in the clinical efficacy of the novel drugs reported till now. Here we review the most crucial P450 enzymes which are involved in prostate cancer and how they can be used as molecular targets for drug discovery along with the clinical limitations of the currently existing CYP inhibitors.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187

2001 ◽  
Vol 281 (3) ◽  
pp. G816-G822 ◽  
Author(s):  
Roxana Toriano ◽  
Arlinet Kierbel ◽  
Marco Antonio Ramirez ◽  
Gerhard Malnic ◽  
Mario Parisi
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Rt Pcr ◽  
T84 Cells ◽  
Pharmacological Agents ◽  
Heat Stable ◽  
A 23187 ◽  
Water Secretion ◽  
Secretory Apparatus

The regulated Cl− secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca2+, cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux ( J w = −0.16 ± 0.02 μl · min−1 · cm−2) in parallel with moderate short-circuit current values ( I sc = 1.55 ± 0.23 μA/cm2). The secretory J wreversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I sc, but, unexpectedly, J w was not affected. Bumetanide, an inhibitor of basolateral Na+-K+-2Cl−cotransporter, completely blocked secretory J wwith no change in I sc. Conversely, serosal forskolin increased I sc, but J w switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J w and I sc. No difference between the absorptive and secretory unidirectional Cl−fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl− flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J w values cannot be shown by I sc and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

scholarly journals The Exon 46-Encoded Sequence Is Essential for Stability of Human Erythroid α-Spectrin and Heterodimer Formation

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4188-4196 ◽  
Author(s):  
Rick Wilmotte ◽  
Sandra L. Harper ◽  
Jeanine A. Ursitti ◽  
Joëlle Maréchal ◽  
Jean Delaunay ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Cell Membranes ◽  
Peptide Sequence ◽  
Α Subunit ◽  
Recombinant Peptide ◽  
Alternative Mrna Splicing ◽  
Recombinant Peptides ◽  
Red Blood Cell Membranes

Abstract Human erythroid α-spectrin alleles responsible for hereditary elliptocytosis (αHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism αLELY (LELY: Low Expression LYon) occurs in trans. The αLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA → GTA, Leu → Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the α-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of α-chains from the αLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif α subunit peptides containing the dimer initiation region. The two forms of α spectrin produced by alternative mRNA splicing of the αLELY allele were represented by α18-211857, a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and α18-211857-Δ46, a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with α18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the αIV and αV domains that was the characteristic feature initially used to identify the αLELY (SpαV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the α21 motif, because this region of the α18-211857-Δ46 peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an αLELY homozygote and the two αLELY recombinant peptides strongly suggests that little, if any, of the 50% of the α chains from the αLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant αLELY peptides, the inability of detectable amounts of exon 46− α chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.

scholarly journals CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

2003 ◽  
Vol 285 (6) ◽  
pp. G1300-G1309 ◽  
Author(s):  
Kala M. Kaspar ◽  
Diana D. H. Thomas ◽  
William B. Taft ◽  
Eriko Takeshita ◽  
Ning Weng ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Apical Membrane ◽  
Digestive Enzyme ◽  
Pancreatic Acinar Cells ◽  
Cellular Mechanisms ◽  
T84 Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Phosphopeptide Mapping

Ca2+-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.

T84 cell receptor binding and guanyl cyclase activation by Escherichia coli heat-stable toxin

1987 ◽  
Vol 253 (6) ◽  
pp. G775-G780 ◽  
Author(s):  
A. Guarino ◽  
M. Cohen ◽  
M. Thompson ◽  
K. Dharmsathaphorn ◽  
R. Giannella
Keyword(s):  
Escherichia Coli ◽  
Guanylate Cyclase ◽  
Intestinal Secretion ◽  
Cell Receptor ◽  
Chloride Secretion ◽  
Cell Number ◽  
Cellular Level ◽  
T84 Cells ◽  
Cgmp Production ◽  
Heat Stable

Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system. The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level. To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84. Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible. ST may also be internalized by these cells. Addition of unlabeled STa competitively inhibited binding of 125I-STa. These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations. STa stimulated guanylate cyclase and cGMP production in a dose-related manner. The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors. These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM)

Parasitology ◽  
1998 ◽  
Vol 117 (4) ◽  
pp. 321-330 ◽  
Author(s):  
R. A. SKILTON ◽  
R. P. BISHOP ◽  
C. W. WELLS ◽  
P. R. SPOONER ◽  
E. GOBRIGHT ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Signal Sequence ◽  
Peptide Sequence ◽  
Theileria Parva ◽  
Repeat Region ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Immunodominant Antigens ◽  
Proline Rich Region

To identify the genes encoding novel immunodominant antigens of Theileria parva a λgt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host–sporozoite interaction.

scholarly journals Strength of Envelope Protein Interaction Modulates Cytopathicity of Measles Virus

2002 ◽  
Vol 76 (10) ◽  
pp. 5051-5061 ◽  
Author(s):  
Richard K. Plemper ◽  
Anthea L. Hammond ◽  
Denis Gerlier ◽  
Adele K. Fielding ◽  
Roberto Cattaneo
Keyword(s):  
Measles Virus ◽  
Protein Composition ◽  
Stable Complex ◽  
Flag Epitope ◽  
Epitope Tag ◽  
Molecular Determinants ◽  
Efficient Replication ◽  
Epitope Sequence ◽  
Extensive Cell ◽  
Infected Cells

ABSTRACT To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.

Fenofibrate inhibits intestinal Cl− secretion by blocking basolateral KCNQ1 K+ channels

2007 ◽  
Vol 293 (6) ◽  
pp. G1288-G1299 ◽  
Author(s):  
Poonam J. Bajwa ◽  
Abderrahmane Alioua ◽  
Jimmy W. Lee ◽  
Daniel S. Straus ◽  
Ligia Toro ◽  
...  
Keyword(s):  
Short Circuit ◽  
Ectopic Expression ◽  
Short Circuit Current ◽  
Selective Inhibition ◽  
Flux Analysis ◽  
Peroxisome Proliferator ◽  
Channel Activity ◽  
T84 Cells ◽  
Peroxisome Proliferator Activated Receptor ◽  
Heat Stable

Fibrates are peroxisome proliferator-activated receptor-α (PPARα) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current ( Isc) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl− secretion by isolated jejunum and colon without affecting electroneutral fluxes of 22Na+ or 86Rb+ (K+) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 μM fenofibrate inhibited cAMP-stimulated Isc within 5 min. In T84 cells, fenofibrate rapidly inhibited ∼80% the Cl− secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca2+). Both fenofibrate and clofibrate inhibited cAMP-stimulated Isc with an IC50 ∼1 μM, whereas other PPARα activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K+ channels (putatively KCNQ1/KCNE3) without affecting Ca2+-stimulated K+ channel activity, whereas clofibrate inhibits both K+ pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl− channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 μM fenofibrate rapidly inhibits K+ currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 β-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl− secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.

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