<i>Trichomonas vaginalis</i> degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism?

Besides possessing many physiological roles, nitric oxide (NO) produced by the immune system in infectious diseases has antimicrobial effects. Trichomoniasis, the most widespread non-viral sexually transmitted disease caused by the microaerophilic protist Trichomonas vaginalis, often evolves into a chronic infection, with the parasite able to survive in the microaerobic, NO-enriched vaginal environment. We relate this property to the finding that T. vaginalis degrades NO under anaerobic conditions, as assessed amperometrically. This activity, which is maximal (133 ± 41 nmol NO/108 cells per minute at 20°C) at low NO concentrations (≤ 1.2 μM), was found to be: (i) NADH dependent, (ii) cyanide insensitive and (iii) inhibited by O2. These features are consistent with those of the Escherichia coli A-type flavoprotein (ATF), recently discovered to be endowed with NO reductase activity. Using antibodies against the ATF from E. coli, a protein band was immunodetected in the parasite grown in a standard medium. If confirmed, the expression of an ATF in eukaryotes suggests that the genes coding for ATFs were transferred during evolution from anaerobic Prokarya to pathogenic protists, to increase their fitness for the microaerobic, parasitic life style. Thus the demonstration of an ATF in T. vaginalis would appear relevant to both pathology and evolutionary biology. Interestingly, genomic analysis has recently demonstrated that Giardia intestinalis and other pathogenic protists have genes coding for ATFs

Molecular epidemiology of human Blastocystis isolates in France.

International audienceBlastocystis sp. is the most common eukaryotic parasite in the intestinal tract of humans. Due to its strong impact in public health, in this study, we determined the frequency of different Blastocystis subtypes in patients in France. We hypothesized on the mode of transmission and tested a possible relationship between the subtype and symptomatic status. We obtained a total of 40 stool samples identified as positive for Blastocystis by microscopic examination of smears. Participants consisted of 25 symptomatic and 15 asymptomatic patients, for whom clinical and parasitological data were collected. For nested-polymerase chain reaction and genotyping, DNA was extracted directly from fecal samples or from fecal cultures. Morphological forms observed in fecal cultures were uncorrelated with symptomatic status. Genotyping using partial small subunit rRNA gene analysis identified a total of 43 Blastocystis isolates corresponding to 37 single infections and three mixed infections by two different subtypes. These 43 isolates belonged to five subtypes (1, 2, 3, 4, and 7) with predominance of subtype 3 (53.5%). Patient symptomatic status was uncorrelated with Blastocystis subtype

Microplanktonic Community Structure in a Coastal System Relative to a Phaeocystis Bloom Inferred from Morphological and Tag Pyrosequencing Methods

Background: Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC), have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom. Methodology and Principal Findings: The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates) was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae. Conclusions: Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa) along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the challenge of modelling and predicting ecological change requires the use of different complementary approaches, to link taxonomic data with the functional roles of microbes in biogeochemical cycles

Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt.

International audienceBlastocystis 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

Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing

International audienceWater samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of tax

Molecular subtyping of <i>Blastocystis</i> 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

Molecular characterization of iron-containing superoxide dismutases in the heterotrophic dinoflagellate Crypthecodinium cohnii.

Superoxide dismutases (SODs) are a family of antioxidant enzymes that catalyse the degradation of toxic superoxide radicals in obligate and facultative aerobic organisms. Here, we report the presence of a multi-copy gene family encoding SODs in the heterotrophic dinoflagellate Crypthecodinium cohnii. All the genes identified (sod1 to sod17) have been cloned and sequenced, and shown to encode potentially functional dimeric iron-containing SOD isozymes. Our data revealed a considerable molecular heterogeneity of this enzyme in C. cohnii at both genomic and transcriptional levels. The C. cohnii SOD1, overexpressed in Escherichia coli, was active and its structure obtained by homology modeling using X-ray crystal structures of homologues exhibited the typical fold of dimeric FeSODs. Phylogenetic studies including 110 other dimeric FeSODs and closely related cambialistic dimeric SOD sequences showed that the C. cohnii SODs form a monophyletic group and have all been acquired by the same event of horizontal gene transfer. It also revealed a dichotomy within the C. cohnii SOD sequences that could be explained by an ancestral sod gene duplication followed by subsequent gene duplications within each of the two groups. Enzyme assays of SOD activity indicated the presence of two FeSOD activities in C. cohnii cell lysate whereas MnSOD and Cu/ZnSOD were not detected. These activities contrasted with the SOD repertoire previously characterized in photosynthetic dinoflagellates. To explain these differences, a hypothetical evolutionary scenario is proposed that suggests gains and losses of sod genes in dinoflagellates.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

<i>In situ</i> survey from morphological observations.

<p>Temporal variation of biomasses (µg C L⁻¹) of (a) Phytoplankton and (b) micro- (ciliates and dinoflagellates) and nanoheterotrophic protists (represented mainly by heterotrophic nanoflagellates HF) at the coastal station R1 (50°48′ N, 1°34′ E) in the eastern English Channel (Strait of Dover) from March to April 2009. Results are shown here in terms of biomass – calculated based on abundance and cell size – (see Method section) and to better represent the stocks of the different compartments.</p

Rarefaction curves and richness estimator.

<p>Rarefaction curves representing the numbers of Operational Taxonomic Units (OTU) <i>versus</i> the number of reads plotted from tag pyrosequencing data. The OTUs were determined using the program Uclust <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>, with a cutoff value set to 0.03 (OTUs were grouped when their level of sequence similarity ≥97%) for the analysis. The table indicates the number of reads, the number of OTU, the richness estimator (Schao1 and Margalef indices), and the heterogeneity of the diversity (Shannon and Simpson indices) for each sampling date.</p

Differential infra-specific diversity of Haptophyceae before and during their spring bloom.

<p>Tag pyrosequencing reads assigned to the group of Haptophyceae were independently extracted for both sampling dates. Reads sharing more than 97% sequence identity were grouped under the same OTUs using Uclust <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>. All the reads having a size above 400bp and representative of an OTU were aligned together with reference sequences using Clustalw <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Thompson1" target="_blank">[84]</a>. The representative sequences and reference sequences were aligned using Muscle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>. The resulting alignments, manually curated using the Bioedit software (<a href="http://www.mbio.ncsu.edu/bioedit/bioedit.html" target="_blank">http://www.mbio.ncsu.edu/bioedit/bioedit.html</a>), were used to build phylogenetic trees. For tree construction, the Seaview 4.0 software (<a href="http://pbil.univ-lyon1.fr/software/seaview.html" target="_blank">http://pbil.univ-lyon1.fr/software/seaview.html</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Gouy1" target="_blank">[39]</a> was used with Neighbor-joining (NJ) and K2P substitution method. Bootstrap values were estimated from 1000 replications. The unrooted phylogenetic tree displayed 21 OTUs inferred from 299 reads corresponding to March 31^st sample, and 56 OTUs inferred from 2,759 reads for April 21^st sample. On each branch are indicated the sampling dates with “1-” corresponding to March 31^st and “2-” to April 21^st, the reads I.D. as it was submitted into the SRA database (SRX031036) and between brackets the number of reads composing the OTU. The taxonomical subdivisions are indicated on the left side of the figure. For relevant clades, the sum of reads “n” composing a cluster was given for each sampling dates.</p