Toxin Internalization Research Articles

AIP56, an AB toxin secreted by Photobacterium damselae subsp. piscicida, has tropism for myeloid cells

IntroductionThe AB-type toxin AIP56 is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), inducing apoptosis in fish immune cells. The discovery of AIP56-like and AIP56-related toxins in diverse organisms, including human-associated Vibrio strains, highlights the evolutionary conservation of this toxin family, suggesting that AIP56 and its homologs may share conserved receptors across species. These toxins have potential for biotechnological applications, such as therapeutic protein delivery and immune modulation.MethodsHerein, the cell specificity of AIP56 for immune cells was characterized. The tropism of AIP56 for cells of the sea bass, mouse and human immune system was analyzed by following toxin internalization by flow cytometry and arrival of the toxin in the cytosol by evaluating the cleavage of NF-kB p65 by western blotting.ResultsOnly a small population of sea bass neutrophils internalized AIP56, indicating that most of the neutrophilic destruction during Phdp infection and/or AIP56 intoxication does not result from the direct action of the toxin. Moreover, the cellular tropism of AIP56 for myeloid cells was observed in the three species, including its preference for macrophages. Further, mouse and human M0 and M2-like macrophages internalized more toxin than M1-like macrophages. Despite the limited interaction of lymphoid cells with AIP56, mouse B1-cells were able to internalize the toxin, possibly due to its myeloid features.ConclusionAIP56 has tropism for sea bass, mouse and human myeloid cells, with greater affinity for macrophages. This points to an evolutionary conservation of its receptor(s) and mechanism of action across species, raising the possibility that AIP56-like and -related toxins may also play a role in pathogenesis. These findings are relevant for both pathogenicity and biomedical contexts.

Abstract CT191: First-in-human, dose escalation and expansion study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing relapsed/refractory advanced solid tumors: Interim data

Abstract MT-6402 is a PD-L1-targeted ETB capable of directly killing PD-L1 expressing cells by internalization of a de-immunized Shiga-like toxin A subunit (SLTA), resulting in ribosomal destruction. Targeting PD-L1 expressing tumor cells may directly drive tumor regression, whereas targeting PD-L1 expressing immune cells may release immunosuppression and drive tumor immune recognition. MT-6402 also delivers an HLA-A*02 restricted cytomegalovirus (CMV) class I antigen into PD-L1 expressing cells (antigen seeding) that can be recognized by existing CMV-specific cytotoxic T cells. A first-in-human dose escalation (Part A) and expansion (Part B) study with a weekly intravenous administration of MT-6402 in 4-week cycles was initiated in 2021. Safety and tolerability are primary objectives. Efficacy, pharmacokinetics, and pharmacodynamic data are also collected. As of 10Dec2023, 53 total patients (pts) with PD-L1-expressing advanced solid tumors received ≥ 1 dose of MT-6402: 48 pts in Part A ranging in dose 16 to 100 µg/kg and 5 pts in Part B at 63 µg/kg, just below the maximum tolerated dose (MTD) of 83 µg/kg. MT-6402 was well tolerated in Part A, completed Sept2023. 5 dose limiting toxicities have been reported: a Grade (G)3 maculopapular rash at 24 µg/kg, a G3 infusion related reaction at 63 µg/kg, a G1 cardiac troponin T increase leading to dosing hold for ≥2 weeks at 83 µg/kg, a G3 maculopapular rash at 100 µg/kg, and a G1 cardiac troponin T increase at 100 µg/kg. There were no G4 or G5 drug-related adverse events. In Part B, pts with PD-L1 scores of TPS ≥ 50% will be treated at MTD or lower and 5 pts have been treated at 63 µg/kg. Signs of antitumor activity have been reported including a long-lasting PR with 70% tumor reduction have been observed in a pt with CPI-refractory nasopharyngeal carcinoma on treatment 14+ cycles at 63 µg/kg. This pt has a PD-L1 TPS score of 2% indicating immune cell clearance may have driven the response. The most common cancer in Part A is Squamous Cell Carcinoma of the Head and Neck (10 pts). Pharmacodynamic effects include a significant reduction in peripheral PD-L1+ immune cells, indicating a PD-L1 target-specific effect. Substantial alterations in serum cytokines associated with the tumor microenvironment were noted after each dose in most pts in a dose dependent manner. Most pts display increased CD8/CD4 ratios with IL-2 and TNFα cytokine release, indicative of immune activation due to elimination of immune cells. CMV-specific CD8 T-cells exhibited a pattern of extravasation from the periphery in HLA-A*02+/CMV+ pts suggesting antigen seeding. These results describe a novel approach to checkpoint modulation by MT-6402 with targeted cell killing and altered tumor immunophenotype, particularly in HLA-A*02/CMV+ pts. These results provide rationale for continued development of MT-6402. Citation Format: Brian A. Van Tine, Eugene Ahn, Rebecca A. Redman, Minal A. Barve, William J. Edenfield, Julio Peguero, Victoria M. Villaflor, Drew W. Rasco, Steven F. Powell, Admasu T. Mamuye, Chris B. Moore, Joshua M. Pelham, Soratree Charoenthongtrakul, David R. Spigel. First-in-human, dose escalation and expansion study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing relapsed/refractory advanced solid tumors: Interim data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT191.

Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.

TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.

Case of necrotic enteritis associated with campylobacteriosis and coccidiosis in an adult Indian peacock (Pavo cristatus)

BackgroundTo date, Campylobacter jejuni has not been found to be pathogenic to peafowl. The available publications show that out of a total of 44 samples tested from peafowl, this bacterium was isolated only in two cases. Eimeria pavonina infestations in the peafowl have been described, but no fatal cases have been reported yet.Case presentationThe four-year-old peacock was presented with chronic diarrhea, emaciation and weakness. Post mortem examination revealed enlarged and pale kidneys, small intestinal mucosal necrosis and thickening of intestinal wall, and pericardial effusion. The histopathological examination revealed necrotic enteritis with marked mononuclear cells infiltration associated with the presence of coccidia, additionally there was histological evidence of septicemia in liver and kidneys. Bacteria identification was based on light microscopy of the small intestine sample, culture, and biochemical tests. Further identification was based on PCR. Antimicrobial susceptibility profile was created by determination of minimal inhibitory concentration (MIC) values for 6 antimicrobial agents from 5 different classes. PCR assays were performed to detect virulence factors genes responsible for motility, cytolethal distending toxin production, adhesion and internalization. Bacteriology of the small intestine sample showed abundant growth almost exclusively of Campylobacter jejuni, resistant to ciprofloxacin, gentamycin and ampicillin. Bacteria was sensitive to Amoxicillin + clavulanic acid, tetracycline, and erythromycin. All tested virulence factors genes have been detected. The parasitological examination was performed by microscopic examination of fresh faeces and intestinal content, and revealed the moderate number of Eimeria pavonina, Histomonas meleagridis, single Capillaria spp. eggs as well Heterakis spp. like parasites.ConclusionThe above case shows that a virulent isolate of Campylobacter jejuni in combination with a parasitic invasion may cause chronic enteritis in peafowl, which most likely led to extreme exhaustion of the host organism and death.

Mosquito-larvicidal Binary (BinA/B) proteins for mosquito control programs -advancements, challenges, and possibilities.

The increasing global burden of mosquito-borne diseases require targeted, environmentally friendly, and sustainable approaches for effective vector control without endangering the non-target beneficial insect population. Biological interventions such as biopesticides, Wolbachia-mediated biological controls, or sterile insect techniques are used worldwide. Here we review Binary or BinAB toxin-the mosquito-larvicidal component of WHO-recognized Lysinibacillus sphaericus bacterium employed in mosquito control programs. Binary (BinAB) toxin is primarily responsible for the larvicidal effect of the bacterium. BinAB is a single-receptor-specific toxin and is effective against larvae of Culex and Anopheles, but not against Aedes aegypti. The receptor in Culex, the Cqm1 protein, has been extensively studied. It is a GPI-anchored amylomaltase and is located apically in the lipid rafts of the larval-midgut epithelium. The interaction of the toxin components with the receptor is crucial for the mosquito larvicidal activity of the BinAB toxin. Here we extend support for the pore formation model of BinAB toxin internalization and the role of toxin-glycan interactions in the endoplasmic reticulum in mediating larval death. BinAB is phylogenetically safe for humans, as Cqm1-like protein is not expected in the human proteome. This review aims to initiate targeted R&D efforts, such as applying fusion technologies (chimera of BinA, chemical modification of BinA), for efficient mosquito control interventions. In addition, the review also examines other areas such as bioremediation and cancer therapeutics, in which L. sphaericus is proving useful and showing potential for further development.

Diphtheria Toxin Based Bivalent Anti-cMPL Immunotoxin Effectively Depletes Human Hematopoietic Stem and Progenitor Cells

Patient conditioning is a critical initial step in hematopoietic stem and progenitor cell (HSPC) transplantation procedures to enable marrow engraftment of infused cells. Preparative regimens have traditionally been achieved by delivering cytotoxic doses of chemotherapeutic agents, with or without radiation. However, these regimens impair host immune function and are associated with significant morbidity. The use of monoclonal antibodies, either alone or conjugated to an internalizing toxin, to target specific antigens on hematopoietic cells has been proposed as a tractable alternative, especially in contexts, such as ex vivo autologous gene therapy, where preservation of immunity is desired. Efficient clearance of marrow has been demonstrated in preclinical models using CD45- or CD117-targeting antibodies conjugated to the plant toxin Saporin. However, this approach still awaits demonstration of long-term safety and efficacy in humans. In this study, we investigated whether toxin-conjugated antibodies targeting the cMPL receptor on HSPCs can provide the basis for a conditioning regimen prior to transplant. Thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSPC self-renewal and survival. The TPO:cMPL axis also regulates megakaryopoiesis and platelet production but, unlike CD45 and CD117 proteins, cMPL is otherwise not expressed in other blood cell types or in non-hematopoietic tissues. Hence, this approach may uniquely allow effective and specific depletion of host HSCs while sparing most hematopoietic progenitors and mature blood cells.To investigate cMPL as an antigen for targeted depletion of human HSPCs, we produced a recombinant bivalent anti-cMPL single-chain variable fragment (sc(FV) 2) (Orita et al. Blood 2004) fused with diphtheria toxin truncated at residue 390 (DT390) to prevent toxin internalization in off-target cells. We first confirmed the cMPL receptor-dependent cytotoxic effects of the anti-cMPL-DT390 conjugate in a HEK293A cell line engineered to express the human cMPL receptor. We observed marked cellular killing in vitro (IC50 = 21 pM) compared to the cMPL-negative control HEK293A cell line (Fig. A). Next, we assessed anti-cMPL-DT390 for its ability to inhibit growth of human CD34+ cells in vitro. G-CSF mobilized peripheral blood (PB) CD34+ cells were obtained from five healthy individuals. Surface expression of cMPL was compared by flow cytometry in subsets increasingly enriched in cells with long-term repopulating activity, including bulk CD34+, CD34+CD38- and CD34+CD38-CD90+CD45RA-CD49f+ cells. Levels of cMPL expression increased congruently with levels of HSC purity (Fig. B). Consistent with a cMPL dependent cytotoxic effect, increased cellular death was measured in populations expressing higher densities of cMPL receptors (IC50 = 104 nM), suggesting preferential targeting of the most primitive hematopoietic compartment (Fig. C). We then assessed whether anti-cMPL-DT390 could safely target and deplete human HSPCs in vivo in humanized NBSGW immunodeficient mice. At 12 weeks post-transplantation, engrafted animals (mean 19.8% CD45+ cells in PB) received a single maximum tolerated dose of 1.2 mg/kg anti-cMPL-DT390 (n=7) or vehicle control solution (n=7) by tail vein injection. HSPC depletion was assayed by measuring human myeloid (CD45+CD13+) chimerism in the mouse PB after antibody administration. We observed a gradual decline in HSPC activity, as represented by the decreased production of human myeloid cells following administration of anti-cMPL-DT390, peaking at 6 weeks with a 2.6-fold reduction in frequency of human CD45+CD13+ cells compared to untreated animals (p = 0.003) (Fig. D).Overall, our study provides proof-of-concept that bivalent anti-cMPL immunotoxin can effectively target and deplete human HSPCs, and may thus provide a novel nontoxic preparative approach to improve HSPC engraftment in transplantation for genetic and other nonmalignant disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Дослідження функціонального стану ліпідних рафтів у цитоплазматичній мембрані проростків Pisum Sativum при дії кліностатування

The results of cytological, biochemical, and molecular biological studies clearly demonstrate the significant influence of microgravity on the main processes of plant development, revealing the mechanisms which underlie the reactions of plant responses to microgravity and ensure their adaptation to this factor. The key role in cell function belongs to the cytoplasmic membrane (CM). First of all, by its qualities and functions, it can play an important role in the adaptation of plants to microgravity. However, data about the effect of altered gravity on the physicochemical properties of CM are limited. Recently, much attention has been paid to the study of functional microdomains of the lipid bilayer that have a specific localization and content of lipids and proteins - lipid rafts. They are defined as a dynamic nanosized, sterol- and sphingolipid-enriched ordered association of specific proteins, in which a metastable resting state can be activated by a combination of specific lipid-lipid, protein-lipid, and protein-protein interactions. There are more and more experimental data that confirm their participation in such live processes as endocytosis, apoptosis, internalization of toxins, bacteria, viruses, etc., as well as their important role in the protective mechanisms of the cell under different stressors. In this work, a fluorescent marker of sterols – filipin and a fluorescent probe laurdan were used to confirm the presence of cholesterol and sphingolipid-enriched lipid ranges in CM. Our results regarding the increase in generalized polarization in the clinorotation conditions correlate with the data obtained in previous studies and indicate the sensitivity of the CM to a longer action of the clinorotation.

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

Impact of the Escherichia coli Heat-Stable Enterotoxin b (STb) on Gut Health and Function.

Enterotoxigenic Escherichia coli (ETEC) produces the heat-stable enterotoxin b (STb), which is responsible for secretory diarrhea in humans and animals. This toxin is secreted within the intestinal lumen of animals and humans following ETEC colonization, becoming active on enterocytes and altering fluid homeostasis. Several studies have outlined the nature of this toxin and its effects on gut health and the integrity of the intestinal epithelium. This review summarizes the mechanisms of how STb alters the gastrointestinal tract. These include the manipulation of mucosal tight junction protein integrity, the formation of enterocyte cellular pores and toxin internalization and the stimulation of programmed cell death. We conclude with insights into the potential link between STb intoxication and altered gut hormone regulation, and downstream physiology.

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Liposome-Based Study Provides Insight into Cellular Internalization Mechanism of Mosquito-Larvicidal BinAB Toxin.

Glycosylphosphatidylinositols (GPIs) anchored proteins are commonly localized onto lipid rafts. These extracellular proteins participate in a variety of cellular functions, including as receptors for viruses and toxins. Intracellular trafficking of World Health Organization recognized mosquito-larvicidal BinAB toxin is mediated via GPI-anchored Cqm1 receptor protein in Culex mosquitoes. We confirmed conformational change in Cqm1 dimer on interaction with BinA/BinB proteins by dynamic light scattering, modelling of hydrodynamic parameters using the atomic structures, and synchrotron Small Angle solution X-ray scattering (SAXS). A reliable model of the receptor-BinB complex was also constructed from joint SAXS/SANS refinement. We confirmed electrostatic interactions of the Cqm1 ectodomain with lipid rafts reconstituted in model membranes and report receptor-dependent impairment of model liposomes by BinA/B proteins. Liposomal disruption was toxin concentration-dependent as monitored by the release of encapsulated carboxyfluorescein dye. Interestingly, BinA alone, without BinB, showed efficient efflux of the fluorescent dye in agreement with the reported high larvicidal activity of BinA variants. The study provides insight into BinA/B toxin internalization mechanism in the membrane model that is toxin internalization is mediated via receptor-dependent pore formation mechanism. It also suggests a tangible and environmentally safe strategy for control of mosquito population.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups: Distribution in Phase-Separated Giant Unilamellar Vesicles.

The receptor lipid Gb3 is responsible for the specific internalization of Shiga toxin (STx) into cells. The head group of Gb3 defines the specificity of STx binding, and the backbone with different fatty acids is expected to influence its localization within membranes impacting membrane organization and protein internalization. To investigate this influence, a set of Gb3 glycosphingolipids labeled with a BODIPY fluorophore attached to the head group was synthesized. C24 fatty acids, saturated, unsaturated, α‐hydroxylated derivatives, and a combination thereof, were attached to the sphingosine backbone. The synthetic Gb3 glycosphingolipids were reconstituted into coexisting liquid‐ordered (l o)/liquid‐disordered (l d) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb3 with the C24:0 fatty acid partitioned mostly in the l o phase, while the unsaturated C24:1 fatty acid distributes more into the l d phase. The α‐hydroxylation does not influence its partitioning.

Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups: Distribution in Phase‐Separated Giant Unilamellar Vesicles

AbstractThe receptor lipid Gb3 is responsible for the specific internalization of Shiga toxin (STx) into cells. The head group of Gb3 defines the specificity of STx binding, and the backbone with different fatty acids is expected to influence its localization within membranes impacting membrane organization and protein internalization. To investigate this influence, a set of Gb3 glycosphingolipids labeled with a BODIPY fluorophore attached to the head group was synthesized. C24 fatty acids, saturated, unsaturated, α‐hydroxylated derivatives, and a combination thereof, were attached to the sphingosine backbone. The synthetic Gb3 glycosphingolipids were reconstituted into coexisting liquid‐ordered (lo)/liquid‐disordered (ld) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb3 with the C24:0 fatty acid partitioned mostly in the lo phase, while the unsaturated C24:1 fatty acid distributes more into the ld phase. The α‐hydroxylation does not influence its partitioning.

Abstract 2635: ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma

Abstract TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a “lineage-survival” oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling in both ROR1 kinase activity-dependent and -independent manner. It is interesting to note that while alterations in EGFR signaling are involved as a “driver” in lung adenocarcinoma pathogenesis, ROR1 appears to play a “sustainer role” for the EGFR-mediated signaling. Previously, we have also found an unanticipated function of this ROR1 receptor tyrosine kinase (RTK) as a scaffold of CAVIN1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of CAVIN1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and pro-survival signaling towards AKT through multiple RTKs, including EGFR, MET, and IGF-IR are consequently sustained. Therefore, ROR1 is an attractive target for overcoming EGFT-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Caveolae function in vesicular and cholesterol trafficking, and have roles in internalization of cholera toxins and the SV40 virus, as well as glycosylphosphatidylinositol (GPI)-anchored proteins and various RTKs. Endocytosis supports various cellular functions, such as nutrient uptake, intracellular signaling, morphogenesis, and defense against pathogens, and is tightly regulated, while it also ensures an appropriate amplitude of growth factor signaling by recycling RTKs back to the cell surface or sorting them to lysosomes for degradation. Here we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer. Citation Format: Tomoya Yamaguchi, Miyu Hayashi, Lisa Ida, Masatoshi Yamamoto, Can Lu, Taisuke Kajino, Jinglei Cheng, Masahiro Nakatochi, Hisanori Isomura, Motoshi Suzuki, Toyoshi Fujimoto, Takashi Takahashi. ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2635.

Neutralization of cholera toxin by Rosaceae family plant extracts

BackgroundCholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin).MethodsHot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts.ResultsThe antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process.ConclusionsThe traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

Effect of Late Endosomal Dobmp Lipid and Traditional Model Lipids of Electrophysiology on the Anthrax Toxin Channel Activity

Abstract Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.

Particular Features of Diphtheria Toxin Internalization by Resistant and Sensitive Mammalian Cells

Particular Features of Diphtheria Toxin Internalization by Resistant and Sensitive Mammalian Cells

Effect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the anthrax toxin channel activity

Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.