Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity

2,7-Dihydroxycadalene and lacinilene C, sesquiterpenoid phytoalexins that accumulate at infection sites during the hypersensitive resistant response of cotton foliage to Xanthomonas campestris pv. malvacearum, have light-dependent toxicity toward host cells, as well as toward the bacterial pathogen. Adaxial epidermal cells surrounding and sometimes covering infection sites turn red. The red cells exhibited 3-4-fold higher absorption at the photoactivating wavelengths of sunlight than nearby colorless epidermal cells. Red epidermal cells protected underlying palisade mesophyll cells from the toxic effects of 2,7-dihydroxycadalene plus sunlight, indicating a role for epidermal pigments in protecting living cells that surround infection sites from toxic effects of the plant’s own phytoalexins. A semi-quantitative survey of UV-absorbing substances extracted from epidermal strips from inoculated and mock-inoculated cotyledons indicated that the principal increase in capacity to absorb the photoactivating wavelengths was due to a red anthocyanin and a yellow flavonol, which were identified as cyanidin-3-O-β-glucoside and quercetin-3-O-β-glucoside, respectively.     

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.     

Development of a broad-spectrum fluorescent heavy metal bacterial biosensor

Bacterial biosensors can measure pollution in terms of their actual toxicity to living organisms. A recombinant bacterial biosensor has been constructed that is known to respond to toxic levels of Zn²⁺, Cd²⁺ and Hg²⁺. The zinc regulatory gene zntR and zntA promoter from znt operon of E. coli have been used to trigger the expression of GFP reporter protein at toxic levels of these ions. The sensor was induced with 3–800?ppm of Zn²⁺, 0.005–4?ppm of Cd²⁺ and 0.001–0.12?ppm of Hg²⁺ ions. Induction studies were also performed in liquid media to quantify GFP fluorescence using fluorimeter. To determine the optimum culture conditions three different incubation periods (16, 20 and 24?h) were followed. Results showed an increased and consistent fluorescence in cells incubated for 16?h. Maximum induction for Zn²⁺, Cd²⁺ and Hg²⁺ was observed at 20, 0.005 and 0.002?ppm, respectively. The pPROBE-zntR-zntA biosensor reported here can be employed as a primary screening technique for aquatic heavy metal pollution.     

Deletion of Thioredoxin Reductase and Effects of Selenite and Selenate Toxicity in Caenorhabditis elegans

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0–2 mM Se as selenite results in inverse, sigmoidal response curves with an LC₅₀ of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and ⁷⁵Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC₅₀ 0.23 mM Se) were identical to selenite, but selenate was 1/4^th as toxic (LC₅₀ 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.     

Genetic regulation of fluxes: iron homeostasis of Escherichia coli

Iron is an essential trace-element for most organisms. However, because high concentration of free intracellular iron is cytotoxic, cells have developed complex regulatory networks that keep free intracellular iron concentration at optimal range, allowing the incorporation of the metal into iron-using enzymes and minimizing damage to the cell. We built a mathematical model of the network that controls iron uptake and usage in the bacterium Escherichia coli to explore the dynamics of iron flow. We simulate the effect of sudden decrease or increase in the extracellular iron level on intracellular iron distribution. Based on the results of simulations we discuss the possible roles of the small RNA RyhB and the Fe–S cluster assembly systems in the optimal redistribution of iron flows. We suggest that Fe–S cluster assembly is crucial to prevent the accumulation of toxic levels of free intracellular iron when the environment suddenly becomes iron rich.     

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.     

Strength of Cu-efflux response in Escherichia coli coordinates metal resistance in Caenorhabditis elegans and contributes to the severity of environmental toxicity

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host–microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host’s response to chronic Cu stress.     

Spatiotemporal Regulation of a Legionella pneumophila T4SS Substrate by the Metaeffector SidJ

Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.     

Schistosoma antigens as activators of inflammasome pathway: from an unexpected stimulus to an intriguing role

Parasites of the genus Schistosoma are organisms capable of living for decades within the definitive host. They interfere with the immune response by interacting with host’s receptors. In this review, we discuss from the first reports to the most recent discoveries regarding the ability of Schistosoma antigens in triggering intracellular receptors and inducing inflammasome activation.     

Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ~300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H₂Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.     

Size-Dependent Toxicity of Silver Nanoparticles to Bacteria,Yeast, Algae,Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.     

Selenium uptake in Zea mays supplied with selenate or selenite under hydroponic conditions

Background and aims

Selenium is an essential micro-nutrient for animals, humans and microorganisms; it mainly enters food chains through plants. This study proposes to explore effect of inorganic Se forms on its uptake and accumulation in Zea mays.

Methods

Zea mays was grown in a controlled-atmosphere chamber for 2 weeks in a hydroponic solution of low-concentration selenium (10 μg/L (i.e.0.12 μM) or 50 μg/L (i.e. 0.63 μM) of Se). For each concentration, four treatments were defined: control (without selenium), selenite alone, selenate alone and selenite and selenate mixed.

Results

At low concentrations, selenium did not affect the biomass production of Zea mays. However, for both concentrations, Se accumulation following a selenite-only treatment was always higher than with selenate-only. Moreover, in the selenate-only treatment, Se mainly accumulated in shoots whereas in the selenite-only treatment, Se was stocked more in the roots. Interactions between selenate and selenite were observed only at the higher concentration (0.63 μM of selenium in the nutrient solution).

Conclusions

Se form and concentration in the nutrient solution strongly influenced the absorption, allocation and metabolism of Se in Zea mays. Selenate seems to inhibit selenite absorption by the roots.     

Combined effect of heavy metals and shungite on the indicator plankton species

The combined effect of salts of heavy metals and shungite on the test organisms of phyto- and zooplankton has been studied. The toxic effect of both cadmium sulfate and potassium dichromate on the culture of Scenedesmus guadricauda was inactivated in the presence of shungite (100 g/L). The efficiency of photosynthesis, the number of cells, the proportion of living cells, and the lifetime of the microalgae cell population increased after adding shungite to the medium (without toxicants). In addition, in acute experiments that lasted up to 96 h, the toxicity of potassium dichromate, copper sulfate, and cadmium sulfate on crustaceans (Daphnia magna and Ceriodaphnia affinis) was studied in the presence of shungite (0.01 g/L) and without it. This study of the effect of shungite’s presence on crustaceans showed that it protected both Daphnia magna and Ceriodaphnia affinis from the action of toxicants at the minimum concentration (0.01 g/L) of the five tested. Daphnia magna died at higher concentrations of shungite. It was shown that the acute toxicity of heavy metals for the two crustacean species decreased in the series Cu–Cd–Cr. The analysis of the obtained data showed that the shungite concentrations necessary for the inactivation of heavy metals were thousands of times higher for algae than for crustaceans. Therefore, when using the shungite as a protector against the toxic effects of various substances, a preliminary laboratory analysis of the survival of different species of hydrobionts in a specific aquatic environment is necessary.     

Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Protective Efficacy of Selenite against Lead-Induced Neurotoxicity in Caenorhabditis elegans

Background

Selenium is an essential micronutrient that has a narrow exposure window between its beneficial and toxic effects. This study investigated the protective potential of selenite (IV) against lead (Pb(II))-induced neurotoxicity in Caenorhabditis elegans.

Principal Findings

The results showed that Se(IV) (0.01 µM) pretreatment ameliorated the decline of locomotion behaviors (frequencies of body bends, head thrashes, and reversal ) of C. elegans that are damaged by Pb(II) (100 µM) exposure. The intracellular ROS level of C. elegans induced by Pb(II) exposure was significantly lowered by Se(IV) supplementation prior to Pb(II) exposure. Finally, Se(IV) protects AFD sensory neurons from Pb(II)-induced toxicity.

Conclusions

Our study suggests that Se(IV) has protective activities against Pb(II)-induced neurotoxicity through its antioxidant property.     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells

Aluminium is a toxic metal whose genotoxicity has been scarcely studied in aquatic species and more generally in mammals. Recently, human and ecological disaster caused by the discharge of red mud in Hungary has revived questions about the toxicity of this metal particularly for the environment. On the contrary, cadmium is a highly toxic metal whose genotoxicity has been well characterized in various mammalian cells. However on non-human cells, little is known about its impact on DNA damage and repair.In this study, the genotoxic potential of both metals on embryonic zebrafish cells ZF4 was analyzed and particularly the impairment of the major DNA double strand breaks (DSB)-repair pathway, i.e. non-homologous end-joining (NHEJ).To this aim, DNA single strand breaks (SSB) and DSB were evaluated using the comet assay and the immunodetection of γ-H2AX proteins, respectively, in AlCl₃ or CdCl₂ exposed ZF4 cells. These exposures result in the production of DSBs a few hours after incubation. The DNA-PK kinase activity, essential for NHEJ, is more affected by the presence of aluminium than cadmium. Altogether our data provide evidence of the high toxicity induced by aluminium in zebrafish and indicates the pertinence of genotoxicity evaluation in organisms living in contaminated water.