Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

Influence of sudden salinity variation on the physiology and domoic acid production by two strains of Pseudo‐nitzschia australis

Several coastal countries including France have experienced serious and increasing problems related to Pseudo‐nitzschia toxic blooms. These toxic blooms occur in estuarine and coastal waters potentially subject to fluctuations in salinity. In this study, we document for the first time the viability, growth, photosynthetic efficiency, and toxin production of two strains of Pseudo‐nitzschia australis grown under conditions with sudden salinity changes. Following salinity variation, the two strains survived over a restricted salinity range of 30–35, with favorable physiological responses, as the growth, effective quantum yield and toxin content were high compared to the other conditions. In addition, high cellular quotas of domoic acid (DA) were observed at a salinity of 40 for the strain IFR‐PAU‐16.1 in comparison with the other strain IFR‐PAU‐16.2 where the cell DA content was directly released into the medium. On the other hand, the osmotic stress imposed at lower salinities, 20 and 10, resulted in cell lysis and a sudden DA leakage in the medium. Intra‐specific variability was observed in growth and toxin production, with the strain IFR‐PAU‐16.1 apparently able to withstand higher salinities than the strain IFR‐PAU‐16.2. On the whole, DA does not appear to act as an osmolyte in response to sudden salinity changes. Since most of the shellfish harvesting areas of bivalve molluscs in France are located in areas where the salinity generally varies between 30 and 35, Pseudo‐nitzschia australis blooms might potentially impact public health and commercial shellfish resources in these places.     

Influence of muskmelon cell wall polysaccharides on roridin E production by a pathogenic strain of Myrothecium roridum

Addition of fruit cell wall extracts from two muskmelon cultivars into liquid media affected mycotoxin production by a strain of Myrothecium roridum pathogenic to muskmelon. Cell wall extracts from a susceptible cultivar (Iroquois) significantly increased toxin production while cell wall extracts from a resistant cultivar (Hales Best) significantly inhibited toxin production. Media containing 0.1 or 1.0 mg ml^–1 stimulated toxin production more than media containing 10 or 100 mg ml^–1 of cell wall extracts. Previous studies in our laboratory suggest that roridin E may be involved in virulence or pathogenicity of M. roridum; the present study indicates that cell wall polysaccharides as well as other materials present in cell wall preparations from susceptible host tissue provide a better substrate for toxin production than cell wall preparation from resistant host tissue.     

High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full‐length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his‐tagged recombinant protein from E. coli extracts (CNF1‐H8). CNF1‐H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1‐H8 functionality was confirmed by testing its activity on purified RhoA and on HEp‐2 cultured cells. Finally, a first structural characterization of the full‐length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150–159, 2018     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa

The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. An E. coli-expressed NIT2/-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements. Nuclear extracts prepared from a wild-type N. crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein. The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences. The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein. Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2. Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain. These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified. The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.     

Resistance of sugarcane borer to Bacillus thuringiensis Cry1Ab toxin

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), strain (F52‐3‐R) was developed from F₃ survivors of a single‐pair mating on commercial Cry1Ab Bacillus thuringiensis (Bt) corn plants in the greenhouse. The susceptibility of a Bt‐susceptible and the F52‐3‐R strain of D. saccharalis to trypsin‐activated Cry1Ab toxin was determined in a laboratory bioassay. Neonate‐stage larvae were fed a meridic diet incorporating Cry1Ab toxin at a concentration range of 0.0625 to 32 µg g^?1. Larval mortality, larval weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded on the 7th day after inoculation. The F52‐3‐R strain demonstrated a significant level of resistance to the activated Cry1Ab toxin. Larval mortality of the Bt‐susceptible strain increased in response to higher concentrations of Cry1Ab toxin, exceeding 75% at 32 µg g^?1, whereas mortality of the F52‐3‐R strain was below 8% across all Cry1Ab concentrations. Using a measure of practical mortality (larvae either died or gained no weight), the median lethal concentration (LC₅₀) of the F52‐3‐R strain was 102‐fold greater than that of the Bt‐susceptible insects. Larval growth of both Bt‐susceptible and F52‐3‐R strains was inhibited on Cry1Ab‐treated diet, but the inhibition of the F52‐3‐R strain was significantly less than that of the Bt‐susceptible insects. These results confirm that the survival of the F52‐3‐R strain on commercial Bt corn plants was related to Cry1Ab protein resistance and suggest that this strain may have considerable value in studying resistance management strategies for Bt corn.     

INFLUENCE OF PHOSPHORUS LIMITATION ON TOXICITY AND PHOTOSYNTHESIS OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) MONITORED BY IN‐LINE DETECTION OF VARIABLE CHLOROPHYLL FLUORESCENCE1

The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexandrium minutum were examined in batch culture experiments. Cell division was greatly impaired in P‐limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equivalent to P‐replete cultures. The cellular concentration of paralytic shellfish toxins was consistent over the growth cycle of control cultures from lag phase into logarithmic growth phase, with toxins repeatedly lost to daughter cells during division. The low level of cell division in P‐limited cultures resulted in a 10‐fold increase of cellular toxin compared with controls, but this dropped upon P spiking due to increased rates of cell division. The history of phosphorus supply had an important effect on toxin concentration, with the P‐limited and the P‐spiked cultures showing values 2‐fold higher than the P‐replete cultures. Toxin profiles of the A. minutum strain used in these experiments were dominated by the N₁‐hydroxy toxins, gonyautoxins (GTX) GTX1 and GTX4, which were approximately 40 times more abundant than their analogues, GTX2 and GTX3, in P‐limited cultures. The dominance of the N₁‐hydroxy toxins increased significantly in control cultures as they advanced through logarithmic growth. In‐line measurements of the variable chl fluorescence of light‐adapted cells indicated consistent photochemical efficiency under P‐replete conditions. P limitation induced a drop in fluorescence‐based photochemical efficiency that was reversible by P spiking. There was an inverse linear relationship between in‐line fluorescence and cell toxin quota (r = ?0.88). Monitoring fluorescence in‐line may be valuable in managing efficient biotechnological production of toxins.     

Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP‐31

Light supply is one of the most important factors affecting autotrophic growth of microalgae. This study investigated the effect of the type and light intensity of artificial light sources on the cell growth of an indigenous microalga Chlorella vulgaris ESP‐31 obtained from southern Taiwan. In addition, a dissolved inorganic carbon source (i.e. sodium bicarbonate) was used to improve the biomass production of strain ESP‐31. The results show that a new fluorescent light source (TL5) was effective in indoor cultivation of microalgae. Better overall productivity of 0.029 g dry cell weight/L‐d was obtained when using TL5 lamps as the light source with a light intensity of 9 W/m². A carbon source (sodium bicarbonate) concentration of 1000 mg/L was found to be optimal for the growth of strain ESP‐31 in terms of both biomass production and carbon source utilization. Under the optimal growth conditions, the resulting microalgal biomass consisted of 25–30% protein, 6–10% carbohydrate, and 30–40% lipid.     

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R,and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.

     

FTIR spectra of algal species can be used as physiological fingerprints to assess their actual growth potential

Fourier transform infrared (FTIR) spectra were measured from cells of Microcystis aeruginosa and Protoceratium reticulatum, whose growth rates were manipulated by the availability of nutrients or light. As expected, the macromolecular composition changed in response to the treatments. These changes were species‐specific and depended on the type of perturbation applied to the growth regime. Microcystis aeruginosa showed an increase in the carbohydrate‐to‐protein ratio with decreased growth rates, under nutrient limitation, whereas light limitation induced a decrease of the carbohydrate‐to‐protein ratio with decreasing proliferation rates. The macromolecular pools of P. reticulatum showed a higher degree of compositional homeostasis. Only when the lowest light irradiance and nutrient availability were supplied, an increase of the carbohydrate‐to‐protein FTIR absorbance ratio was observed. A species‐specific partial least squares (PLS) model was developed using the whole FTIR spectra. This model afforded a very high correlation between the predicted and the measured growth rates, regardless of the growth conditions. On the contrary, the prediction based on absorption band ratios generally used in FTIR studies would strongly depend on growth conditions. This new computational method could constitute a substantial improvement in the early warning systems of algal blooms and, in general, for the study of algal growth, e.g. in biotechnology. Furthermore, these results confirm the suitability of FTIR spectroscopy as a tool to map complex biological processes like growth under different environmental conditions.     

Effect of salinity on growth and toxin production of <Emphasis Type="Italic">Alexandrium minutum</Emphasis> isolated from a shrimp culture pond in northern Vietnam

A clonal culture of a Vietnamese strain of Alexandrium minutum, AlexSp17, was subjected to different salinity treatments to determine the growth and toxin production of this strain that produces a novel toxin analogue, deoxy GTX4-12ol. The experiment was carried out in batch cultures without pre-acclimatization at seven salinity treatments from 5 to 35 psu, under constant temperature of 25°C, illumination of 140 μmol photon m⁻² s⁻¹, and 12:12 light/dark photoperiod. The strain grew in all salinity treatments, with optimum growth at 10–15 psu. However, the specific growth rate (0.2 day⁻¹) was lower than those reported in Malaysian strains and other strains from different geographical areas. The optimum range of salinity for the growth of this species agreed with field observations of the locality of origin. No significant change in toxin profiles was observed at different salinities. The cellular toxin quota, Qt, was not affected by the salinity-dependent growth rate. The toxin GTX4-12ol is presumed to be a transformation product of GTX4 from specific cellular reductase enzymes. Further investigation at the molecular level of toxin biosynthesis and subcellular enzyme activities is needed to provide insight in the production of this unique toxin analogue.     

Medium N:P Ratios and Specific Growth Rate ComodulateMicrocystin and Protein Content in Microcystis aeruginosa PCC7806 and M. aeruginosa UV027

Hepatotoxin production in cyanobacteria has been shown to correlate to external stimuli such as light and nutrient concentrations and ratios, although conflicting results have been reported. Specific growth rates and protein and microcystin content of M. aeruginosa PCC7806 and M. aeruginosa UV027 were determined under nonlimiting batch culture conditions for a range of medium nitrogen and phosphorous atomic ratios. Both strains exhibited a similar optimal medium N:P ratio for increased cellular microcystin levels. Additionally, total cellular protein content and intracellular microcystin content were significantly correlated to each other (r² = 0.81, p < 0.001). Microcystin and protein content increased considerably as the maximum specific growth rate for the experimental conditions was reached. The significant correlation of cellular protein and microcystin content and their relative increase with increasing specific growth rate, within defined ranges of medium N:P ratios, suggest a close association between microcystin production and N:P ratio–dependent assimilation of nitrogen, and resulting total cellular protein levels, which may be further modulated by specific growth rate.