Intoxicated Cells Research Articles

Cocoa beans improve mitochondrial biogenesis via PPARγ/PGC1α dependent signalling pathway in MPP+ intoxicated human neuroblastoma cells (SH-SY5Y)

Polyphenols are shown to protect from or delay the progression of chronic neurodegenerative diseases. Mitochondrial dysfunction plays a key role in the pathogenesis of Parkinson’s disease (PD). This study was aims to gain insight into the role of ahydroalcoholic extract of cocoa (standardised for epicatechin content) on mitochondrial biogenesis in MPP+ intoxicated human neuroblastoma cells (SHSY5Y). The effects of cocoa on PPARγ, PGC1α, Nrf2 and TFAM protein expression and mitochondrial membrane potential were evaluated. A pre-exposure to cocoa extract decreased reactive oxygen species formation and restored mitochondrial membrane potential. The cocoa extract was found to up-regulate the expression of PPARγ and the downstream signalling proteins PGC1α, Nrf2 and TFAM. It increased the expression of the anti-apoptotic protein BCl2 and increased superoxide dismutase activity. Further, the cocoa extract down-regulated the expression of mitochondria fission 1 (Fis1) and up-regulated the expression of mitochondria fusion 2 (Mfn2) proteins, suggesting an improvement in mitochondrial functions in MPP+ intoxicated cells upon treatment with cocoa. Interestingly, cocoa up-regulates the expression of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. No change in the expression of PPARγ on treatment with cocoa extract was observed when the cells were pre-treated with PPARγ antagonist GW9662. This data suggests that cocoa mediates mitochondrial biogenesis via a PPARγ/PGC1α dependent signalling pathway and also has the ability to improve dopaminergic functions by increasing tyrosine hydroxylase expression. Based on our data, we propose that a cocoa bean extract and products thereof could be used as potential nutritional supplements for neuroprotection in PD.

The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells.

ABSTRACTColibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli. Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.

Biofilm Producing Salmonella Typhi: Chronic Colonization and Development of Gallbladder Cancer.

Salmonella enterica subspecies enterica serovar Typhi is the aetiological agent of typhoid or enteric fever. In a subset of individuals, S. Typhi colonizes the gallbladder causing an asymptomatic chronic infection. Nonetheless, these asymptomatic carriers provide a reservoir for further spreading of the disease. Epidemiological studies performed in regions where S. Typhi is endemic, revealed that the majority of chronically infected carriers also harbour gallstones, which in turn, have been indicated as a primary predisposing factor for the onset of gallbladder cancer (GC). It is now well recognised, that S. Typhi produces a typhoid toxin with a carcinogenic potential, that induces DNA damage and cell cycle alterations in intoxicated cells. In addition, biofilm production by S. Typhi may represent a key factor for the promotion of a persistent infection in the gallbladder, thus sustaining a chronic local inflammatory response and exposing the epithelium to repeated damage caused by carcinogenic toxins. This review aims to highlight the putative connection between the chronic colonization by highly pathogenic strains of S. Typhi capable of combining biofilm and toxin production and the onset of GC. Considering the high risk of GC associated with the asymptomatic carrier status, the rapid identification and profiling of biofilm production by S. Typhi strains would be key for effective therapeutic management and cancer prevention.

MIMOSA PUDICA EXERTS NEUROPROTECTION AGAINST MPP+INDUCED NEUROTOXICITY IN SHSY5Y CELL LINES-AN IN VITRO MODEL OF ANTI-PARKINSONISM

<p><strong>Objective: </strong>Parkinson’s disease (PD) is one of the of a neurodegenerative disorder, It’s decreased the dopaminergic neurones, tyrosine hydroxylase (TH) and increased the α-synuclein protein level. This study was conducted to investigate the neuroprotective effect of <em>Mimosa pudica</em> have the abilities to improve TH and DAT proteins expression against MPP<sup>+ </sup>induced neurotoxicity, in <em>in</em><em> vitro</em> model of<em> </em>Parkinson’s disease using SH-SY5Y human neuroblastoma cell lines. <strong></strong></p><p><strong>Methods: </strong><em>Mimosa pudica</em> were pre-treated with various concentration for cell viability assay. Vehicle alone or <em>Mimosa pudica</em> (300µg) for 24 h, and then were co-treated with 1000µM MPP<sup>+ </sup>for 15 min in the continued presence of vehicle or <em>Mimosa pudica</em>. After treatment, cells were collected for protein expression.<strong></strong></p><p><strong>Results: </strong>Cell viability assay confers the inhibitory concentration cell death of <em>Mimosa pudica</em>. MPP<sup>+</sup>significantly down-regulated the protein expression of<em> </em>TH (p<0.01) and DAT (p<0.05)<em>. Mimosa pudica</em> decreased the expression of α synuclein (p<0.01) in MPP<sup>+ </sup>intoxicated cell lines.</p><p><strong>Conclusion: </strong>The present study showed that <em>Mimosa pudica</em> exerts neuroprotection by suppressing α synuclein and the dopaminergic neurdegeneration. <em>Mimosa pudica</em> may be due to quecertin which might be acted via the anti-oxidant mechanism. The above finding suggests that <em>Mimosa pudica</em> may act as a potential target in the management of PD. <strong></strong></p><p> </p>

Congo red dye affects survival and reproduction in the cladoceran Ceriodaphnia dubia. Effects of direct and dietary exposure.

Nearly 7 00000 tons of dyes are produced annually throughout the world. Azo dyes are widely used in the textile and paper industries due to their low cost and ease of application. Their extensive use results in large volumes of wastewater being discharged into aquatic ecosystems. Large volume discharges constitute a health risk since many of these dyes, such as Congo Red, are elaborated with benzidine, a known carcinogenic compound. Information regarding dye toxicity in aquatic ecosystems is limited. Therefore, the aim of the present study was to evaluate the effect of Congo Red on survival and reproduction of Ceriodaphnia dubia. We determined the 48 h median lethal concentration (LC50) and evaluated the effects of sublethal concentrations in subchronic exposures by using as food either fresh algae or algae previously exposed to the dye. LC50 was 13.58 mg L-1. In subchronic assays, survival was reduced to 80 and 55 %, and fertility to 40 and 70 %, as compared to the control, in C. dubia fed with intoxicated cells or with the mix of intoxicated + fresh algae, respectively, so the quantity and type of food had a significant effect. We determined that Congo Red is highly toxic to C. dubia since it inhibits survival and fertility in concentrations exceeding 3 mg L-1. Our results show that this dye produces negative effects at very low concentrations. Furthermore, our findings warn of the risk associated with discharging dyes into aquatic environments. Lastly, the results emphasize the need to regulate the discharge of effluents containing azo dyes.

The Role of ExoS in Dissemination of Pseudomonas aeruginosa during Pneumonia.

Hospital-acquired pneumonia is associated with high rates of morbidity and mortality, and dissemination to the bloodstream is a recognized risk factor for particularly poor outcomes. Yet the mechanism by which bacteria in the lungs gain access to the bloodstream remains poorly understood. In this study, we used a mouse model of Pseudomonas aeruginosa pneumonia to examine this mechanism. P. aeruginosa uses a type III secretion system to deliver effector proteins such as ExoS directly into the cytosol of eukaryotic cells. ExoS, a bi-functional GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT), inhibits phagocytosis during pneumonia but has also been linked to a higher incidence of dissemination to the bloodstream. We used a novel imaging methodology to identify ExoS intoxicated cells during pneumonia and found that ExoS is injected into not only leukocytes but also epithelial cells. Phagocytic cells, primarily neutrophils, were targeted for injection with ExoS early during infection, but type I pneumocytes became increasingly injected at later time points. Interestingly, injection of these pneumocytes did not occur randomly but rather in discrete regions, which we designate ““fields of cell injection” (FOCI). These FOCI increased in size as the infection progressed and contained dead type I pneumocytes. Both of these phenotypes were attenuated in infections caused by bacteria secreting ADPRT-deficient ExoS, indicating that FOCI growth and type I pneumocyte death were dependent on the ADPRT activity of ExoS. During the course of infection, increased FOCI size was associated with enhanced disruption of the pulmonary-vascular barrier and increased bacterial dissemination into the blood, both of which were also dependent on the ADPRT activity of ExoS. We conclude that the ADPRT activity of ExoS acts upon type I pneumocytes to disrupt the pulmonary-vascular barrier during P. aeruginosa pneumonia, leading to bacterial dissemination.

Distinct roles of the repeat-containing regions and effector domains of the Vibrio vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin.

ABSTRACTVibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.

Reactive oxygen species involved in CT26 immunogenic cell death induced by Clostridium difficile toxin B

Immunogenic cell death (ICD) is a new concept appeared in recent years. Despite growing interests of research on ICD, the circumstances that trigger immune responses against dying tumor cells remain largely unknown. It was demonstrated that recombinant Clostridium difficile toxin B (rTcdB) can induce ICD in intoxicated cells, but its mechanism remains unclear. This work aims at exploring whether reactive oxygen species (ROS) involved in rTcdB induced ICD using the chemical agent N-acetyl cysteine (NAC), diphenylene iodonium (DPI) and Antimycin A (Anti.A). The results suggested that ROS involved in rTcdB induced apoptosis and autophagy. DPI and Anti.A successfully inhibited the antitumor immune effect induced by rTcdB. As ICD is determined by a variety of factors, rTcdB is a potential tool for further exploring the circumstances that trigger ICD, which may offer us a good choice for designing the new chemotherapeutic drugs with immunogenic properties.

Clostridium difficile toxin B intoxicated mouse colonic epithelial CT26 cells stimulate the activation of dendritic cells.

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis mainly through two exotoxins TcdA and TcdB that target intestinal epithelial cells. Dendritic cells (DCs) play an important role in regulating intestinal inflammatory responses. In the current study, we explored the interaction of TcdB-intoxicated epithelial cells with mouse bone marrow-derived DCs. TcdB induced cell death and heat shock protein translocation in mouse intestinal epithelial CT26 cells. The intoxicated epithelial cells promoted the phagocytosis and the TNF-α secretion by DCs. Incubation with TcdB-intoxicated CT26 cells stimulated DC maturation. Moreover, TcdB-treated CT26 cells induced DC immigration when they were injected into mice subcutaneously. Taken together, these data demonstrate that TcdB-intoxicated intestinal epithelial cells are able to stimulate DC activation in vitro and attract DCs in vivo, indicating that epithelial cells may be able to regulate DC activation under the exposure of TcdB during C. difficile infection.

Utility of Clostridium difficile toxin B for inducing anti-tumor immunity.

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.

The cytolethal distending toxin effects on Mammalian cells: a DNA damage perspective.

The cytolethal distending toxin (CDT) is produced by many pathogenic Gram-negative bacteria and is considered as a virulence factor. In human cells, CDT exposure leads to a unique cytotoxicity associated with a characteristic cell distension and induces a cell cycle arrest dependent on the DNA damage response (DDR) triggered by DNA double-strand breaks (DSBs). CDT has thus been classified as a cyclomodulin and a genotoxin. Whereas unrepaired damage can lead to cell death, effective, but improper repair may be detrimental. Indeed, improper repair of DNA damage may allow cells to resume the cell cycle and induce genetic instability, a hallmark in cancer. In vivo, CDT has been shown to induce the development of dysplastic nodules and to lead to genetic instability, defining CDT as a potential carcinogen. It is therefore important to characterize the outcome of the CDT-induced DNA damage and the consequences for intoxicated cells and organisms. Here, we review the latest results regarding the host cell response to CDT intoxication and focus on DNA damage characteristics, cell cycle modulation and cell outcomes.

Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

Homeopathic Thuja 30C ameliorates benzo(a)pyrene–induced DNA damage, stress and viability of perfused lung cells of mice in vitro

To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose (2.2 μmol/L) for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C (used against tumor or cancer) or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species (ROS) accumulation, total glutathione (GSH) content, and generations of heat shock protein (hsp)-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4',6-diamidino-2-phenylindole staining was performed. Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP. Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.

Exoenzyme S ADP-Ribosylates Rab5 Effector Sites To Uncouple Intracellular Trafficking

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

Neuroprotective effects of antioxidants, Idebenone and Ferulic Acid, in MPTP/MPP+ intoxicated PC12 cells as a model of Parkinson's Disease

Evidence suggests that α‐synuclein inclusions and COX‐2 upregulation contribute to mitochondrial dysfunction and neuroinflammation in Parkinson's disease (PD). Increased expression of COX‐2 has been reported to cause dopamine oxidation, which in turn triggers oxidative stress and α‐synuclein accumulation. Other studies also suggest that NF‐κB regulates the expression of neuronal COX‐2 at the genetic level. The present study investigated the protective effect of Idebenone, a synthetic analog of CoQ10, and Ferulic acid, a naturally occurring phenolic compound, against MPTP/MPP+‐induced oxidative stress and neurodegeneration in PC12 cells, used as an in‐vitro model of PD. Results showed that both drugs dose dependently decreased the expression of α‐synuclein, along with a decrease in the expressions of COX‐2 and NF‐κB, as compared to the increase induced by MPTP/MPP+. In addition, the ROS levels and Bax/Bcl‐2 ratio for Idebenone and Ferulic acid significantly decreased as compared to the increased levels seen with the toxin. This suggests that the neuroprotection offered by the two drugs may be anti‐oxidative as well as anti‐apoptotic in nature. The research was supported by Long Island University.

Relative efficacies of insulin and poly (lactic-co-glycolic) acid encapsulated nano-insulin in modulating certain significant biomarkers in arsenic intoxicated L6 cells

The present study evaluates relative efficacies of insulin and PLGA-loaded-nano-insulin (NIn) in combating arsenic-induced impairment of glucose uptake, insulin resistance and mitochondrial dysfunction in L6 skeletal muscle cells. L6 cells were treated with 0.5mM sodium arsenite for 30min and then the cells were further treated with either 100nM insulin (standardized dose) or either of the two doses, 50nM and 100nM of nano-insulin. Various biomarkers like pyruvate-kinase and glucokinase, ATP/ADP ratio, mitochondrial membrane potential, cytosolic release of mitochondrial cytochrome c, cell membrane potential and calcium-ion level were studied and analyzed to ascertain the status of mitochondrial functioning in all experimental and control sets of L6 cells. The size, morphology and zeta potential of formulated NIn were determined by using dynamic light scattering, scanning electronic and atomic-force microscopies. Expression of signalling cascades like GLUT4, IRS1, IRS2, UCP2, PI3, and p38 was critically analyzed. Overall results suggested that both insulin and NIn improved mitochondrial functioning in arsenite-intoxicated L6 cells, NIn showing better effects at a much lower dose (at nearly 10-fold decreased dose) than that produced by insulin. Nano-insulin being non-toxic and effective at a much lesser dose, therefore, has potential for therapeutic use in the management of arsenic induced diabetes.

Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV–ACT), but the properties of OMV–ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV–ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV–ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV–ACT can deliver ACT by processes distinct from those of ACT alone.

Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?

Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.

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.