Improved Expression of a Thermostable GH18 Bacterial Chitinase in Two Different Escherichia coli Strains and Its Potential Use in Plant Protection and Biocontrol of Phytopathogenic Fungi.

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability. Escherichia coli strains Origami B and BL21 (DE3) were selected for their distinct cytosolic environment to express BhChitA chitinase of Bacillus halodurans C-125 and to investigate the role of disulfide bond formation and proper folding on its stability and activity. Expression of the recombinant BhChitA in bacterial strain containing oxidative cytosol (Origami B) improved its activity and stability. Although both expression systems have comparable biochemical properties (temperature range 20-80 °C and pH spectrum 3-10), BhChitA expressed in Origami strain seems more stable than expressed in BL21. Furthermore, the optimal expression conditions of the recombinant BhChitA has been carried out at 30 °C during 6 h for the Origami strain, against 20 °C during 2 h for BL21. On the other hand, no significant differences were detected between the two enzymes when the effect of metal ions was tested. These findings correlate with the analysis of the overall structure of BhChitA. The model structure permitted to localize disulfide bond, which form a stable connection between the substrate-binding residues and the hydrophobic core. This link is required for efficient binding of the chitin insertion domain to the substrate. BhChitA exhibited in vitro antifungal effect against phytopathogenic fungi and suppressed necrosis of Botrytis cinerea on detached tomato leaves. In vitro assays showed the influence of BhChitA on growth suppression of Botrytis cinerea (53%) Aspergillus niger (65%), Fusarium graminearum (25%), and Fusarium oxysporum (34%). Our results highlight the importance of the bacterial expression system with oxidative cytosol in producing promising biopesticides that can be applied for post-harvest processing and crop protection.

Biological and Molecular Characterization of the Lytic Bacteriophage SoKa against Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast and Black Pit in Tunisia.

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between -80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol.

Diversity of pathogenic Pseudomonas isolated from citrus in Tunisia.

The damages observed in Tunisian citrus orchards have prompted studies on the Pseudomonas spp. responsible for blast and black pit. Prospective orchards between 2015 and 2017 showed that the diseases rapidly spread geographically and to new cultivars. A screening of Pseudomonas spp. isolated from symptomatic trees revealed their wide diversity according to phylogenetic analysis of their housekeeping rpoD and cts genes. The majority of strains were affiliated to Pseudomonas syringae pv. syringae (Phylogroup PG02b), previously described in Tunisia. However, they exhibited various BOX-PCR fingerprints and were not clonal. This work demonstrated, for the first time in Tunisia, the involvement of Pseudomonas cerasi (PG02a) and Pseudomonas congelans (PG02c). The latter did not show significant pathogenicity on citrus, but was pathogenic on cantaloupe and active for ice nucleation that could play a role in the disease. A comparative phylogenetic study of citrus pathogens from Iran, Montenegro and Tunisia revealed that P. syringae (PG02b) strains are closely related but again not clonal. Interestingly P. cerasi (PG02a) was isolated in two countries and seems to outspread. However, its role in the diseases is not fully understood and it should be monitored in future studies. The diversity of pathogenic Pseudomonas spp. and the extension of the diseases highlight that they have become complex and synergistic. It opens questions about which factors favor diseases and how to fight against them efficiently and with sustainable means.

New species of pathogenic Pseudomonas isolated from citrus in Tunisia: Proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016-2017 from the fruits and leaves of Citrus sinensis cv. 'Valencia Late' and Citrus limon cv. 'Eureka' plants with symptoms of blast and black pit disease. A phylogenetic analysis of the housekeeping gene rpoD was used for strain identification at the species level. The results demonstrated the affiliation of these strains with the genus Pseudomonas and revealed the presence of 11 strains representing two putative new species in two monophyletic branches. These strains were analyzed morphologically and genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences, and their phenotypic characteristics by API 20NE and Biolog GEN III. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv. 'Eureka'. Fatty acids and WC MALDI-TOF MS major protein profiles were determined. The genomes of both representatives were sequenced. The average nucleotide index and genome-to-genome distance from KC12T and E10BT are below the cut-off established for a described species. These results support the conclusion that the strains KC12T, KC17, KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12T (=CECT9766 and CFBP 8662). The strains E10BT, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10BT (=CECT9765 and CFBP 8661).

Production and identification of iturin A lipopeptide from Bacillus methyltrophicus TEB1 for control of Phoma tracheiphila.

A lipopeptide-producing endophytic Bacillus methyltrophicus TEB1 strain exhibited potent antifungal activity against Phoma tracheiphila. Lipopeptide production started at the early growth phase plateaued after 36 h of culture where it reduced the mycelium growth by 80%. The crude lipopeptide extract harvested at the stationary phase efficiently inhibited the growth of P. tracheiphila mycelium and MIC values displaying 50 and 90% inhibition of conidia germination were around 47.5 and 100 µg ml(-1) , respectively. Increasing lipopeptide extract till 3 mg ml(-1) induced 10% swelling and 3% crumbling of P. tracheiphila conidia whereas 5 mg ml(-1) induced 40% swelling and 20% crumbling. Mass spectrometry analysis of the lipopeptide extract indicated that surfactin production took place from 12 to 20 h, iturin A from 16 to 72 h, and fengycin from 12 to 72 h and that the main active compound against P. tracheiphila was identified as C15 iturin A lipopeptide. Iturin A appeared as a potential biological control agent able to substitute the currently used chemical pesticides in agriculture.

Combined effects of alternariols mixture on human colon carcinoma cells.

Mycotoxins are naturally occurring contaminants encountered at high levels in a wide variety of agricultural products intended for human and animal consumptions. Various Alternaria mycotoxins may occur simultaneously in small grain cereals. Considering the concomitant production of alternariol (AOH) and alternariol monomethyl ether (AME), it is expected that humans and animals are exposed to the mixture rather than to individual compounds. Therefore, we studied the interactive effects of binary mixture of alternariols (AOH and AME) on the human intestinal cell line, HCT116 cells. Exposure of HCT116 cells to low cytotoxic alternariols doses, resulted in a moderate cytotoxicity manifested by a loss in the cell viability mediated by an activation of the mitochondrial apoptotic process, associated with the opening of mitochondrial permeability transition pore (PTP) and the loss of the mitochondrial transmembrane potential (ΔΨm). However, when combined, they exert a significant increase in their toxic potential. Altogether, our study showed that AOH and AME combination is obviously additive.

In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone.

It is expected that humans are exposed to combined mycotoxins, which occur simultaneously in the food items, than to individual compounds and that can increase their potential toxicity. Considering this coincident production, deoxynivalenol (DON) and zearalenone (ZEN) as they are produced by several Fusarium species, can interfere at a cellular level. Therefore, these two toxins were chosen to study their interactive effects on human colon carcinoma cells (HCT116), using the endpoints including cell viability, cell cycle analysis, mitochondrial transmembrane potential (ΔΨm) determination and permeability transition pore (PTP) opening. Our results showed that DON and ZEN caused a marked decrease of cell viability in a dose-dependent manner, mediated by an activation of the mitochondrial apoptotic process; characterized by PTP opening and the loss of ΔΨm. Nevertheless, combined DON and ZEN reduced all the toxicities observed with the mycotoxins separately. Therefore, the combination of the two mycotoxins appears as a sub-additive response.

Cell death induced by the Alternaria mycotoxin Alternariol.

Mycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one of the primary targets of these toxins, respond to their toxic effects. In this study, human colon carcinoma cells were used to elucidate the cell death mode and the pathways triggered by Alternariol (AOH), the most important mycotoxin produced by Alternaria species, which are the most common mycoflora infecting small grain cereals worldwide. Treatment of cells with AOH resulted in a loss of cell viability by inducing apoptosis. AOH-induced apoptosis was mediated through a mitochondria-dependent pathway, characterized by a p53 activation, an opening of the mitochondrial permeability transition pore (PTP), a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O(2)(*-) and caspase 9 and 3 activation. Besides, deficiency of the pro-apoptotic protein Bax partially protected cells against AOH-induced mitochondrial alterations. In addition, experiments performed on purified mitochondria indicated that AOH does not directly target this organelle to induce cell death. Our results demonstrate for the first time that AOH-induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells.

Involvement of mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity.

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates cereal crops and harmfully affects the gastrointestinal tract. Since it is well known that mitochondria play a central role in apoptosis triggered by many stimuli, an effort was made to examine whether DON-induced cytotoxicity occurs through mitochondria-mediated apoptotic pathway. The intestinal system being one of the primary targets of mycotoxins, the human colon carcinoma cell line HCT116 was used in this study. Using flow cytometric analyses and immunofluorescence, we showed that DON at 100 µM induced a mitochondria-dependent apoptotic pathway associated with opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm), downstream generation of O2·â» and cytochrome c release. The DON-induced apoptosis was accompanied by an activation of caspase 9 and 3, as demonstrated by Western blot and caspase activity assay. In addition, by taking advantage of HCT116 cells invalidated for Bax, we showed that this pro-apoptotic protein favored mitochondrial alterations induced by the mycotoxin. Besides, incubation of purified mitochondria with DON indicated that this mycotoxin does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Altogether, our results indicate that mitochondria-related caspase-dependent apoptotic pathway is involved in this in vitro model of DON induced-cytotoxicity.

Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells.

Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm) downstream generation of O(2)(-), cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis.

Trichothecene chemotypes of Fusarium culmorum infecting wheat in Tunisia.

Fusarium culmorum is a major pathogen associated with Fusarium head blight (FHB) of wheat in Tunisia. It may cause yield loss or produce mycotoxins in the grain. The objectives of the present study were threefold: to evaluate by PCR assays the type of mycotoxins produced by 100 F. culmorum isolates recovered from different regions in Northern Tunisia, to determine the amount of mycotoxin production by HPLC analysis, and to analyse for correlations between the amount of mycotoxin produced and the aggressiveness of isolates. PCR assays of Tri5, Tri7, Tri13, and Tri3 were used to predict whether these isolates could produce nivalenol, 3-acetyl-deoxynivalenol, or 15-acetyl-deoxynivalenol. Two of the isolates were predicted to produce NIV, whereas the others were predicted to produce 3-AcDON. Trichothecene production was confirmed and quantified by high pressure liquid chromatography (HPLC) in 28 isolates, after growth on wheat grains, and in a liquid Mycotoxin Synthetic medium (MS). All strains produced DON/3-AcDON at detectable levels ranging from 21 microg/g to 11.000 microg/g of dry biomass on MS medium and from 10 microg/g to 610 microg/g on wheat grain. The evaluation of the relationship between 3-AcDON production and aggressiveness of 17 strains revealed a significant difference in aggressiveness among the isolates. Moreover, only a significant correlation was revealed between aggressiveness and the amount of 3-AcDON produced on MS medium (r=0.36). Chemotyping of F. culmorum isolates is reported for the first time for isolates from Tunisia, and highlights the important potential of F. culmorum to contaminate wheat with 3-AcDON trichothecenes.

Mode of action of thuricin S, a new class IId bacteriocin from Bacillus thuringiensis.

Different methods were used to elucidate the mode of action of thuricin S, a new class IId bacteriocin produced by Bacillus thuringiensis subsp. entomocidus HD198. According to cell viability tests, thuricin S was shown to exert a bactericidal effect on the sensitive cells of Bacillus thuringiensis subsp. darmastadiensis 10T. The use of the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide as an indicator proved that thuricin S interacts with the cytoplasmic membrane to dissipate the transmembrane potential. It was also demonstrated that thuricin S acts as a pore-forming bacteriocin, since it allows the nonpermeable stain propidium iodide to enter the cells. The loss of membrane integrity and the morphological changes in sensitive cells were visualized by scanning electron microscopy.

Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells.

The mycotoxin, deoxynivalenol (DON), is generally detected in cereal grains and grain-based food products worldwide. Therefore, DON has numerous toxicological effects on animals and humans. The present investigation was conducted to determine the molecular aspects of DON toxicity on human colon carcinoma cells (HT 29). To this aim, we have monitored the effects of DON on (i) cell viability, (ii) Heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage and (iv) cell death signalling pathway. Our results clearly showed that DON treatment inhibits cell proliferation, did not induce Hsp 70 protein expression and reactive oxygen species generation. We have also demonstrated that this toxin induced a DNA fragmentation followed by p53 and caspase-3 activations. Finally, our findings suggested that oxidative damage is not the major contributor to DON toxicity. This mycotoxin induces direct DNA lesions and could be considered by this fact as a genotoxic agent inducing cell death via an apoptotic process.

Purification and partial amino acid sequence of thuricin S, a new anti-Listeria bacteriocin from Bacillus thuringiensis.

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3-10.5). The monoisotopic mass of thuricin S purified by high performance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.