Epsilon toxin: a fascinating pore-forming toxin

Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.     

Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure

Epsilon toxin (ETX) is a 33‐kDa pore‐forming toxin produced by type B and D strains of Clostridium perfringens. We previously found that ETX caused haemolysis of human red blood cells, but not of erythrocytes from other species. The cellular and molecular mechanisms of ETX‐mediated haemolysis are not well understood. Here, we investigated the effects of ETX on erythrocyte volume and the role of the putative myelin and lymphocyte (MAL) receptors in ETX‐mediated haemolysis. We observed that ETX initially decreased erythrocyte size, followed by a gradual increase in volume until lysis. Moreover, ETX triggered phosphatidylserine (PS) exposure and enhanced ceramide abundance in erythrocytes. Cell shrinkage, PS exposure and enhanced ceramide abundance were preceded by increases in intracellular Ca²⁺ concentration. Interestingly, lentivirus‐mediated RNA interference studies in the human erythroleukaemia cell line (HEL) cells confirmed that MAL contributes to ETX‐induced cytotoxicity. Additionally, ETX was shown to bind to MAL in vitro. The results of this study recommend that ETX‐mediated haemolysis is associated with MAL receptor activation in human erythrocytes. These data imply that interventions affecting local MAL‐mediated autocrine and paracrine signalling may prevent ETX‐mediated erythrocyte damage.     

Extracellular matrix‐associated proteome changes during non‐host resistance in citrus–Xanthomonas interactions

Non‐host resistance (NHR) is a most durable broad‐spectrum resistance employed by the plants to restrict majority of pathogens. Plant extracellular matrix (ECM) is a critical defense barrier. Understanding ECM responses during interaction with non‐host pathogen will provide insights into molecular events of NHR. In this study, the ECM‐associated proteome was compared during interaction of citrus with pathogen Xanthomonas axonopodis pv. citri (Xac) and non‐host pathogen Xanthomonas oryzae pv. oryzae (Xoo) at 8, 16, 24 and 48 h post inoculation. Comprehensive analysis of ECM‐associated proteins was performed by extracting wall‐bound and soluble ECM components using both destructive and non‐destructive procedures. A total of 53 proteins was differentially expressed in citrus–Xanthomonas host and non‐host interaction, out of which 44 were identified by mass spectrometry. The differentially expressed proteins were related to (1) defense‐response (5 pathogenesis‐related proteins, 3 miraculin‐like proteins (MIR, MIR1 and MIR2) and 2 proteases); (2) enzymes of reactive oxygen species (ROS) metabolism [Cu/Zn superoxide dismutase (SOD), Fe‐SOD, ascorbate peroxidase and 2‐cysteine‐peroxiredoxin]; (3) signaling (lectin, curculin‐like lectin and concanavalin A‐like lectin kinase); and (4) cell‐wall modification (α‐xylosidase, glucan 1, 3 β‐glucosidase, xyloglucan endotransglucosylase/hydrolase). The decrease in ascorbate peroxidase and cysteine‐peroxiredoxin could be involved in maintenance of ROS levels. Increase in defense, cell‐wall remodeling and signaling proteins in citrus–Xoo interaction suggests an active involvement of ECM in execution of NHR. Partially compromised NHR in citrus against Xoo, upon Brefeldin A pre‐treatment supported the role of non‐classical secretory proteins in this phenomenon.     

Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells

Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin–Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.     

Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression

The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.     

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation.     

Huanglongbing pathogen Candidatus Liberibacter asiaticus exploits the energy metabolism and host defence responses of its vector Diaphorina citri

Asian citrus psyllid Diaphorina citri is the vector of the citrus Huanglongbing (HLB) associated bacterial agent ‘Candidatus Liberibacter asiaticus’ (CLas). The molecular interactions between CLas and D. citri remain unclear. In the present study, protein profiles of mitochondrial, microsomal and cytosolic fractions from uninfected and CLas‐infected adult D. citri are investigated using two‐dimensional gel electrophoresis. The comparative analysis reveals a total of 18, 24 and 20 protein spots that are unique or differentially expressed in mitochondrial, microsomal and cytosolic proteins fractions respectively. These proteins are successfully identified by mass spectrometry. Among the 62 identified proteins, 30 are up‐regulated, whereas 32 are down‐regulated. These proteins include important components in energy metabolism such as ATP synthase, ATPase, ATP/ADP carrier protein, etc.; host stress responses such as heat shock proteins; host detoxification processes (i.e., cytochrome P450 and glutathione S‐transferase); and the cytoskeleton (such as actin, tubulin, myosin and tropomyosin). These data suggest that, after CLas infection, several proteins of D. citri, especially energy metabolism and protein biosynthesis, are altered, and extensive host defence responses are induced. In conclusion, the present study reports proteomic information that is helpful in understanding the vector–pathogen relationship between CLas and D. citri, and could be used to identify potential targets for limiting the spread of CLas, as well as to provide new insights into HLB management.     

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice

Protein l -isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l -aspartyl and l -asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.     

Quantitative Analysis of the Global Proteome in Peripheral Blood Mononuclear Cells from Patients with New‐Onset Psoriasis

Psoriasis is a common chronic autoimmune skin disease involving the activation of T cells. To explore the proteomic signature of peripheral blood mononuclear cells, a quantitative analysis of their global proteome was conducted in samples from Chinese patients with new‐onset psoriasis (n = 31) and healthy controls (n = 32) using an integrated quantitative approach with tandem mass tag labeling and LC–MS/MS. Protein annotation, unsupervised hierarchical clustering, functional classification, functional enrichment and cluster, and protein–protein interaction analyses were performed. A total of 5178 proteins were identified, of which 4404 proteins were quantified. The fold‐change cutoff was set at 1.2 (patients vs controls); 335 proteins were upregulated, and 107 proteins were downregulated. The bioinformatics analysis indicated that the differentially expressed proteins were involved in processes related to the activation of immune cells including the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway, cellular energy metabolism, and proliferation. Three upregulated proteins and two phosphorylated proteins in the NF‐κB pathway were verified or identified by Western blotting. These results confirm that the NF‐κB pathway is critical to psoriasis. In addition, many differentially expressed proteins identified in this study have never before been associated with psoriasis, and further studies on these proteins are necessary.     

Bacterial glycosyltransferase toxins

Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.     

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Global quantitative proteomic analysis profiles host protein expression in response to Sendai virus infection

Sendai virus (SeV) is an enveloped nonsegmented negative‐strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV‐infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV‐induced innate immune response process, system‐wide evaluations of SeV–host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV–host interaction, a global quantitative proteomic analysis was performed on SeV‐infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in “interferon type I (IFN‐I) signaling pathway” and “defense response to virus,” suggesting that these processes play roles in SeV infection. Further RNAi‐based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV‐induced IFN‐I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV‐induced innate immune response process.     

Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host

Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT‐PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.     

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide‐induced responses in the mosquito.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.