Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12. To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR) (13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin

Combination Therapy of Prostate Cancer Utilizing Functionalized Iron Oxide Nanoparticles Carrying TNF-a and Lactonic Sophorolipids

Prostate cancer is one of the most prevalent forms of cancer afflicting men in the United States. In recent years, advances in the field of nanotechnology have allowed for new and innovative ways to treat various types of cancer and various other diseases. Our research focuses on the treatment of the LNCaP line prostate cancer utilizing iron oxide nanoparticles (IONPs) loaded with soluble TNF-a and lactonic sophorolipids (LSLs). TNF-a is a cytokine responsible for apoptosis initiation, while LSLs are naturally-glycolipids shown to alleviate inflammation and improve immune response in certain diseases. We hypothesized that this combination may possess a synergistic effect, displaying greater therapeutic effects than either compound alone. We synthesized polyacrylic acid (PAA)-coated IONPs to serve as a vehicle for these compounds for target-specific delivery. The surface carboxylate groups of the PAA coating can be chemically modified, allowing for binding of ligands to target cell-specific surface receptors or antigens. We conjugated our IONPs with glutamic acid with the aim of targeting the over-expressed glutamate receptors on the surface of the LNCaP cells. This combination therapy showed significant LNCaP cell death within 48 hours of incubation, while healthy cells were unaffected. The therapeutic effects were determined using cytotoxicity, MitoSOX, apoptosis, and migration assays. The results of the combined therapy suggest that these compounds may be a viable alternative to chemotherapeutic drugs in prostate cancer treatment

Activatable MR Prodrug for Targeted Delivery and Treatment of Cancer

In this study, a new multimodal theranostic tool is reported utilizing nanoceria delivery system conjugated with the ICAM1 antibody and a magnetic resonance (MR) probe as a prodrug with both MR and cytotoxic properties. The prodrug was synthesized from doxorubicin and phenyl-amine modified DTPA chelated with gadolinium utilizing dithiobis(succinimidyl propionate) (DSP) as a crosslinker. Nanoceria was synthesized from cerium oxide and polyacrylic acid using a water-based alkali precipitation technique. Doxorubicin and the synthesized prodrug were encapsulated separately within the nanoceria polymer matrix using a solvent diffusion method. The drug/prodrug-encapsulated nanoceria’s carboxylated surface was functionalized with the ICAM1 antibody utilizing EDC/NHS chemistries and the resulting formulations were purified and characterized by DLS, zeta potential, UV/Vis, and MR. The efficacy of this platform was measured by treating MDA-MB-231 breast cancer (TNBC) cells and MCF-7 cells with the drug/prodrug-loaded, ICAM1-conjugated nanoceria and analyzing the results of the treatment. Results were evaluated by cytotoxicity assays (MTT), fluorescence microscopy, reactive oxygen species determination, and comet assays. In all, the results show the nanoceria platform is target-specific to TNBC, and the encapsulated prodrug is able to be activated releasing doxorubicin and initiating apoptosis in an in vivo breast cancer model

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process

Susceptibility profile of blaOXA-23 and metallo-β-lactamases co-harbouring isolates of carbapenem resistant Acinetobacter baumannii (CRAB) against standard drugs and combinations

BackgroundThe rapid emergence of carbapenem resistant Acinetobacter baumannii (CRAB) has resulted in an alarming situation worldwide. Realizing the dearth of literature on susceptibility of CRAB in genetic context in the developing region, this study was performed to determine the susceptibility profile against standard drugs/combinations and the association of in-vitro drug synergy with the prevalent molecular determinants.Methods and findingsA total of 356 clinical isolates of A. baumannii were studied. Confirmation of the isolates was done by amplifying recA and ITS region genes. Susceptibility against standard drugs was tested by Kirby Bauer disc diffusion. Minimum inhibitory concentration (MIC), MIC50 and MIC90 values against imipenem, meropenem, doripenem, ampicillin/sulbactam, minocycline, amikacin, polymyxin B, colistin and tigecycline was tested as per guidelines. Genes encoding enzymes classes A (blaGES, blaIMI/NMC-A, blaSME, blaKPC), B (blaIMP, blaVIM, blaNDM) and D (blaOXA-51,blaOXA-23 and blaOXA-58) were detected by multiplex polymerase chain reaction. Synergy against meropenem-sulbactam and meropenem-colistin combinations was done by checkerboard MIC method. Correlation of drug synergy and carbapenemase encoding genes was statistically analyzed.ResultsOf the total, resistance above 90% was noted against gentamicin, ciprofloxacin, levofloxacin, ceftazidime, cefepime, ceftriaxone, cotrimoxazole and piperacillin/tazobactam. By MIC, resistance rates from highest to lowest was seen against imipenem 89.04% (n=317), amikacin 80.33% (n=286), meropenem 79.49% (n=283), doripenem 77.80% (n=277), ampicillin/sulbactam 71.62% (n=255), tigecycline 55.61% (n=198), minocycline 14.04% (n=50), polymyxin B 10.11% (n=36), and colistin 2.52% (n=9). CRAB was 317 (89.04%), 81.46% (n=290) were multidrug resistant and 13.48% (n=48) were extensively drug resistant. All the CRAB isolates harboured blaOXA-51 gene (100%) and 94% (n=298) blaOXA-23 gene. The blaIMP gene was most prevalent 70.03% (n=222) followed by blaNDM, 59.62% (n=189). Majority (87.69%, 278) were co-producers of classes D and B carbapenemases, blaOXA-23 with blaIMP and blaNDM being the commonest. Synergy with meropenem-sulbactam and meropenem-colistin was 47% and 57% respectively. Reduced synergy (p= <0.0001) was noted for those harbouring blaOXA-51+blaOXA-23with blaNDM gene alone or co-producers.ConclusionPresence of blaNDM gene was a significant cause of synergy loss in meropenem-sulbactam and meropenem-colistin. In blaNDM endemic regions, tigecycline, minocycline and polymyxins could be viable options against CRAB isolates with more than one carbapenemase encoding genes

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 degrees C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 degrees C, exposure to lipid rafts resulted in substantial toxin activity at 37 degrees C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1

Gut Microbiome and Crohn’s Disease: An Enigmatic Crosstalk

Crohn’s disease (CD) is a chronic, recurrent, immune-mediated inflammatory bowel disease that demonstrates a spectrum of intestinal and extra-intestinal manifestations. The pathogenesis of CD is multifactorial and involves a complex interplay between environmental and microbiological factors in a genetically susceptible host. There is robust evidence suggesting the role of gut microbial dysbiosis in the development as well as exacerbation of CD by immune dysregulation and alteration in the immune microbiota crosstalk. Patients with CD show reduced commensal microbial diversity, along with increased numbers of pathogenic Enterobacteriaceae and Proteobacteriaceae. Faecalibacterium prausnitzii, an anti-inflammatory molecule-producing bacteria, is also seen in reduced numbers in patients with CD and is associated with an increased risk of recurrence. There has been a paradigm shift in the management of patients of CD, from controlling symptoms to controlling inflammation and promoting mucosal healing. Current treatment strategies aim to replace, remove, reset, or redesign the gut microbiota for the therapeutic benefits of patients with CD. These include microbial restoration therapies such as dietary modification, the use of pre-, pro-, and postbiotics, and fecal microbiota transfer (FMT). This chapter focuses on the role of gut microbiota in the pathophysiology of CD and the emerging concepts in microbial therapeutics

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB(5) protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

Wormhole supported by dark energy admitting conformal motion

In this article, we study the possibility of sustaining a static and spherically symmetric traversable wormhole geometries admitting conformal motion in Einstein gravity, which presents a more systematic approach to search a relation between matter and geometry. In wormhole physics, the presence of exotic matter is a fundamental ingredient and we show that this exotic source can be dark energy type which support the existence of wormhole spacetimes. In this work we model a wormhole supported by dark energy which admits conformal motion. We also discuss the possibility of detection of wormholes in the outer regions of galactic halos by means of gravitational lensing. The studies of the total gravitational energy for the exotic matter inside a static wormhole configuration are also done.Comment: 9 pages, 2 tables and 4 figures, Accepted for Publication in European Physical Journal