Hicham El Alaoui

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 ∼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.

Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae.

Background The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. Methodology/Finding Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity. Conclusions/Significance After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite

BackgroundBlastocystis is a highly prevalent anaerobic eukaryotic parasite of humans and animals that is associated with various gastrointestinal and extraintestinal disorders. Epidemiological studies have identified different subtypes but no one subtype has been definitively correlated with disease.ResultsHere we report the 18.8 Mb genome sequence of a Blastocystis subtype 7 isolate, which is the smallest stramenopile genome sequenced to date. The genome is highly compact and contains intriguing rearrangements. Comparisons with other available stramenopile genomes (plant pathogenic oomycete and diatom genomes) revealed effector proteins potentially involved in the adaptation to the intestinal environment, which were likely acquired via horizontal gene transfer. Moreover, Blastocystis living in anaerobic conditions harbors mitochondria-like organelles. An incomplete oxidative phosphorylation chain, a partial Krebs cycle, amino acid and fatty acid metabolisms and an iron-sulfur cluster assembly are all predicted to occur in these organelles. Predicted secretory proteins possess putative activities that may alter host physiology, such as proteases, protease-inhibitors, immunophilins and glycosyltransferases. This parasite also possesses the enzymatic machinery to tolerate oxidative bursts resulting from its own metabolism or induced by the host immune system.ConclusionsThis study provides insights into the genome architecture of this unusual stramenopile. It also proposes candidate genes with which to study the physiopathology of this parasite and thus may lead to further investigations into Blastocystis-host interactions.

New Insights into Blastocystis spp.: A Potential Link with Irritable Bowel Syndrome

Blastocystis spp. belong to the phylum Stramenopila, a complex and heterogeneous evolutionary assemblage of heterotrophic and photosynthetic protozoa [1]. Interestingly, this is the only stramenopile living in the lower digestive tract of humans, and it also lives in other mammals, birds, reptiles, amphibians, and insects [1]. Even though isolates were reported to be morphologically indistinguishable, an extensive genetic variation among isolates from both humans and animals has been observed. Thirteen subtypes (ST1-ST13), with the first nine being found in humans, have been identified based on genes coding for the small-subunit ribosomal RNA [2]. Preferential repartition of STs exists among animals that appear to constitute the main reservoir for environmental dissemination and human contamination.

Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies

ABSTRACT Blastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts—a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders.

Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt

Blastocystis sp. has been described as the most common intestinal parasite in humans and has an increased impact in public health. To improve our understanding of the molecular epidemiology of this human-emerging parasite, we determined the Blastocystis subtypes (STs) and their relative frequency in Egyptian patients living in or in the vicinity of Cairo and presenting gastrointestinal symptoms. We obtained a total of 20 stool samples identified as positive for Blastocystis by microscopic examination of smears. Genotyping using partial small subunit ribosomal RNA gene analysis identified a total of 21 Blastocystis isolates corresponding to 19 single infections and one mixed infection (ST1 and ST3). Three STs were identified: ST3 was the most common ST in the present Egyptian population (61.90%) followed by ST1 (19.05%) and ST2 (19.05%). Together with previous studies carried out in different areas in Egypt, a total of five STs (ST1, ST2, ST3, ST4, and ST6) have been found in symptomatic patients. These data were compared to those available in the literature, and we underlined variations observed in the number and relative proportions of STs between and within countries. On the whole, it seemed that Blastocystis infection is likely not associated with specific STs even if some STs are predominant in the epidemiologic studies, but rather with a conjunction of factors in the course of infection including environmental risk and parasite and host factors.

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Microsporidia: a model for minimal parasite–host interactions

Microsporidia are emerging fungi-like intracellular parasites of economic, veterinary and medical importance. The strategy they use to invade their host is related to the rapid extrusion of a unique and highly specialized organelle, the polar tube, which allows the injection of the infectious spore content within a target cell. This original process seems to be dependent on initial interactions between parasite and host cell components. The extreme reduction and compaction of most microsporidian genomes resulted in the loss of many metabolic pathways, which makes these parasites highly dependent on their host. Recent significant advances have been made in the understanding of mammal and insect immune responses against microsporidian infections with the involvement of both adaptive and innate immunity.

Molecular subtyping of Blastocystis sp. isolates from symptomatic patients in Italy

Blastocystis sp. is the most common eukaryotic parasite in the intestinal tract of humans. Due to its potential impact in public health, we determined the Blastocystis sp. subtypes (STs) and their relative frequency in symptomatic patients living in or in the vicinity of two Italian cities (Rome and Sassari). A total of 34 Blastocystis sp. isolates corresponding to 26 single and 4 mixed infections were subtyped using partial small subunit ribosomal RNA gene sequencing. From this molecular approach, the ST distribution in the present Italian population was as follows: ST3 (47.1%), ST2 (20.6%), ST4 (17.7%), ST1 (8.8%), and ST7, and ST8 (2.9%). As in almost all countries worldwide, ST3 was the most common ST reinforcing the hypothesis of its human origin. Together with a previous preliminary report, a total of seven STs (with the addition of ST5) have been found in Italian symptomatic patients. The wide range of STs identified in the Italian population suggest that Blastocystis sp. infection is not associated with specific STs even if some STs (ST1–ST4) are predominant as reported in all other countries. Since most of the STs identified in Italian patients are zoonotic, our data raise crucial questions concerning the identification of animal reservoirs for Blastocystis sp. and the potential risks of transmission to humans.

Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis

Blastocystis hominis is an anaerobic parasite of the human intestinal tract belonging to the Stramenopile group. Using genome sequencing project data, we describe here the complete sequence of a 29,270-bp circular DNA molecule that presents mitochondrial features (such as oxidative phosphorylation complex I subunits) but lacks complexes III, IV and V. Transmission electron microscopy analyses reveal that this molecule, as well as mitochondrial (NADH:ubiquinone oxidoreductase subunit 7 (NAD7), beta-succinyl-CoA synthetase (beta-SCS)) and hydrogenosomal (pyruvate ferredoxin oxido-reductase (PFOR), iron-hydrogenase) proteins, are located within double-membrane surrounded-compartments known as mitochondria-like organelles (MLOs). As there is no evidence for hydrogen production by this organism, we suggest that MLOs are more likely anaerobic mitochondria.