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Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular metabolism and serves as a substrate in enzymatic processes. NAD+ is produced by de novo synthesis or salvage pathways in nearly all bacterial species. Haemophilus influenzae lacks the capacity for de novo synthesis, so it is dependent on import of NAD+ from the external environment or salvage biosynthetic pathways for recycling of NAD+ precursors and breakdown products. However, the actual sources of NAD+ utilized by H. influenzae in the respiratory tract are not well defined. In this study, we found that a variety of bacteria, including species found in the upper airway of humans, released NAD+ that was readily detectable in extracellular culture fluid, and which supported growth of H. influenzae in vitro. By contrast, certain strains of Streptococcus pyogenes (group A streptococcus or GAS) inhibited growth of H. influenzae in vitro by secreting NAD+-glycohydrolase (NADase), which degraded extracellular NAD+. Conversely, GAS strains that lacked enzymatically active NADase released extracellular NAD+, which could support H. influenzae growth. Our results suggest that many bacterial species, including normal flora of the upper airway, release NAD+ into the environment. GAS is distinctive in its ability to both release and degrade NAD+. Thus, colonization of the airway with H. influenzae may be promoted or restricted by co-colonization with GAS in a strain-specific manner that depends, respectively, on release of NAD+ or secretion of active NADase. We suggest that, in addition to its role as a cytotoxin for host cells, NADase may serve a separate function by restricting growth of H. influenzae in the human respiratory tract., Competing Interests: The authors have declared that no competing interests exist.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor., Competing Interests: The authors have declared that no competing interests exist.

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism., Competing Interests: The authors have declared that no competing interests exist.

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum., Competing Interests: The authors have declared that no competing interests exist.

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea: Liviidae) is an important pest of citrus species worldwide because it transmits Candidatus Liberibacter spp. (Alphaproteobacteria), the causative agents of an incurable citrus disease known as huanglongbing or greening disease. Diaphorina citri possesses a vertically-transmitted intracellular symbiont, Candidatus Profftella armatura (Betaproteobacteria), which produces diaphorin, a polyketide that is significantly toxic to mammalian cells. Diaphorin is an analog of pederin, a defensive polyketide in the body fluid of Paederus rove beetles (Coleoptera: Staphylinidae) that deters predators. In the present study, as a first step to assess the possibility that diaphorin is toxic to biological control agents, we assayed diaphorin activities against insects and fungi. The target cells and organisms were (a) the Sf9 cell line derived from the fall armyworm moth Spodoptera frugiperda (Lepidoptera: Noctuidae), (b) the pea aphid Acyrthosiphon pisum (Hemiptera: Sternorrhyncha: Aphidoidea: Aphididae), a phloem sap-sucking insect that is closely related to psyllids, (c) the Asian lady beetle Harmonia axyridis (Coleoptera: Coccinellidae), one of the major predators of D. citri, and (d) the budding yeast Saccharomyces cerevisiae (Ascomycota: Saccharomycetes) as a model of fungal pathogens. For a comparison, we also evaluated pederin activities. The results of our analyses revealed the following: (1) Diaphorin and pederin are significantly toxic to the tested insects and yeast; (2) Their toxicities vary widely among the target cells and organisms; (3) Diaphorin is generally less toxic than pederin; (4) The toxicities of diaphorin and pederin are considerably different in the Sf9 insect cell line and S. cerevisiae, but similar in A. pisum and H. axyridis; and (5) The amount of diaphorin contained in D. citri is toxic to all of the tested cells and organisms, suggesting that this polyketide is potentially harmful for biological control agents., Competing Interests: The authors have declared that no competing interests exist.

Background: The Opuntia spp. have been used in traditional medicine for many centuries. It is used in the management of diseases that involves oxidative stress, especially diabetes, obesity and cancer. Opuntia stricta (Haw) is one of the relatively unknown species in South Africa where it is regarded more as a weed. Because of this, not much is known about its chemical composition., Aim: To determine the chemical composition, antioxidant, anti-inflammatory, and cytotoxic activities of Opuntia stricta cladodes., Methods: The phytochemical composition of acetone, aqueous and ethanol extract of cladodes of Opuntia stricta (Haw), as well as the vitamins A, C and E of its dried weight cladodes and the antioxidant activities, were evaluated using standard in vitro methods. The anti-inflammatory and cytotoxic activities were evaluated using cell-based assays. The phytochemical composition and vitamins were determined spectrophotometrically, while the antioxidant activities were determined by DPPH, nitric oxide, hydrogen peroxide scavenging activity and phosphomolybdenum (total) antioxidant activity. Anti-inflammatory activity was determined using RAW 264.7 cells, while cytotoxicity was determined using U937 cells., Results: The phytochemical composition showed a significant difference in the various extracts. The total phenolics were higher than other phytochemicals in all the extracts used. All the extracts displayed antioxidant activity, while most of the extracts showed anti-inflammatory activity. Only one extract showed cytotoxicity, and it was mild., Conclusion: The results show that the Opuntia stricta is rich in polyphenolic compounds and has good antioxidant activity as well as anti-inflammatory activities., Competing Interests: The authors have declared that no competing interests exist.

Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens.

Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo experiments.

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.

Mutations in the gene coding for superoxide dismutase 1 (SOD1) are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+) site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+). This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

Extracellular adenosine (ADE) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR) are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65) was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.

Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTX(Vv)) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTX(Vv) and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans.

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.

To understand how symmetric structures of many proteins are formed from asymmetric sequences, the proteins with two repeated beta-trefoil domains in Plant Cytotoxin B-chain family and all presently known beta-trefoil proteins are analyzed by structure-based multi-sequence alignments. The results show that all these proteins have similar key structural residues that are distributed symmetrically in their structures. These symmetric key structural residues are further analyzed in terms of inter-residues interaction numbers and B-factors. It is found that they can be distinguished from other residues and have significant propensities for structural framework. This indicates that these key structural residues may conduct the formation of symmetric structures although the sequences are asymmetric.