Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks.

Triketones, derived chemically from a natural phytotoxin (leptospermone), are a good example of allelochemicals as lead molecules for the development of new herbicides. Targeting a new and key enzyme involved in carotenoid biosynthesis, these latest-generation herbicides (sulcotrione, mesotrione and tembotrione) were designed to be eco-friendly and commercialized fifteen-twenty years ago. The mechanisms controlling their fate in different ecological niches as well as their toxicity and impact on different organisms or ecosystems are still under investigation. This review combines an overview of the results published in the literature on ß-triketones and more specifically, on the commercially-available herbicides and includes new results obtained in our interdisciplinary study aiming to understand all the processes involved (i) in their transfer from the soil to the connected aquatic compartments, (ii) in their transformation by photochemical and biological mechanisms but also to evaluate (iii) the impacts of the parent molecules and their transformation products on various target and non-target organisms (aquatic microorganisms, plants, soil microbial communities). Analysis of all the data on the fate and impact of these molecules, used pure, as formulation or in cocktails, give an overall guide for the assessment of their environmental risks.

On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis.

Nosemosis is one of the most common and widespread diseases of adult honeybees. The causative agents, Nosema apis and Nosema ceranae, belong to microsporidia some obligate intracellular eukaryotic parasites. In this study, 10 sulphated polysaccharides from algae were evaluated for their antimicrosporidian activity. They were first shown to inhibit the in vitro growth of the mammal microsporidian model, Encephalitozoon cuniculi. The most efficient polysaccharides were then tested for their ability to inhibit the growth of Nosema ceranae in experimentally-infected adult honeybees. Two polysaccharides extracted from Porphyridium spp. did not show any toxicity in honeybees and one of them allowed a decrease of both parasite load and mortality rate due to N. ceranae infection. A decrease in parasite abundance but not in mortality rate was also observed with an iota carrageenan. Our results are promising and suggest that algal sulphated polysaccharides could be used to prevent and/or control bee nosemosis.

Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome.

Blastocystis spp. are common anaerobic intestinal protozoa found in both human and animals. They are characterized by a high genetic diversity with at least 17 subtypes (STs) that have been described on the basis of a 600 bp 'barcoding region' from the 18S rDNA gene. However, analysis of the recently sequenced genome of a Blastocystis ST7 isolate (strain B) revealed the presence of multiple variable copies of the 18S rDNA gene, with 17 completely assembled copies. Comparison of the barcoding region from these 17 copies allowed us to classify the 18S rDNA sequences into 6 clusters, each cluster containing identical sequences. Surprisingly, 4 of these clusters had the highest homology with 18S rDNA sequences from 2 other Blastocystis ST7 isolates referred as QQ98-4 and H. These results suggest that the 18S rDNA gene is not the marker of choice to discriminate between strains within STs. In the present study, we identified a single-copy subtyping rDNA marker in the genome of the mitochondria-like organelles (MLOs). Using a partial sequence of the MLO rDNA, we successfully subtyped 66 isolates from both human and animals belonging to Blastocystis ST1 to ST10. Our results also indicate that this mitochondrial marker could be useful to detect co-infections by different isolates of a same ST.

Expression of two cell wall proteins during the intracellular development of Encephalitozoon cuniculi: an immunocytochemical and in situ hybridization study with ultrathin frozen sections.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.

Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

Polymorphism of the gene encoding a major polar tube protein PTP1 in two microsporidia of the genus Encephalitozoon.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78 bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvalI digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

Immunocytochemical identification of spore proteins in two microsporidia, with emphasis on extrusion apparatus.

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera (Glugea atherinae and Encephalitozoon cuniculi) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.

Identification of Encephalitozoon intestinalis in travelers with chronic diarrhea by specific PCR amplification.

With the use of Weber's modified trichrome and Uvitex 2B techniques, spores of microsporidia were detected in the stools of four travelers presenting clinically with chronic diarrhea. The general health of these patients was not impaired, and human immunodeficiency virus screening was negative. Immune evaluation, including the study of lymphocytic subpopulations, assay of serum immunoglobulins, and an intradermal multitest, showed normal results. Molecular identification of microsporidian species was based on the PCR amplification of a small-subunit rRNA sequence followed by HinfI endonuclease restriction. Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of E. intestinalis in immunocompetent individuals remains to be demonstrated.

Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.