Yan-Zi Wen

Pseudogene-derived small interference RNAs regulate gene expression in African Trypanosoma brucei

Pseudogenes have been shown to acquire unique regulatory roles from more and more organisms. We report the observation of a cluster of siRNAs derived from pseudogenes of African Trypanosoma brucei using high through-put analysis. We show that these pseudogene-derived siRNAs suppress gene expression through RNA interference. The discovery that siRNAs may originate from pseudogenes and regulate gene expression in a unicellular eukaryote provides insights into the functional roles of pseudogenes and into the origin of noncoding small RNAs.

Pseudogenes are not pseudo any more.

Recent significant progress toward understanding the function of pseudogenes in protozoa (Trypanosoma brucei), metazoa (mouse) and plants, make it pertinent to provide a brief overview on what has been learned about this fascinating subject. We discuss the regulatory mechanisms of pseudogenes at the post-transcriptional level and advance new ideas toward understanding the evolution of these, sometimes called “garbage genes” or “junk DNA,” seeking to stimulate the interest of scientists and additional research on the subject. We hope this point-of-view can be helpful to scientists working or seeking to work on these and related issues.

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 μg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 μg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences

Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n=16), Changchun, Jilin province of China (n=1) and from Thailand (n=2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96-97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade.

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

Trypanosoma brucei, a pathogen of human and domestic animals, is an early evolved parasitic protozoan with a complex life cycle. Most genes of this parasite are post-transcriptionally regulated. However, the mechanisms and the molecules involved remain largely unknown. We have deep-sequenced the small RNAs of two life stages of this parasite--the bloodstream form and the procyclic form. Our results show that the small RNAs of T. brucei could derive from multiple sources, including NATs (natural antisense transcripts), tRNAs, and rRNAs. Most of these small RNAs in the two stages were found to share uniform characteristics. However, our results demonstrate that their variety and expression show significant differences between different stages, indicating possible functional differentiation. Dicer-knockdown evidence further proved that some of the small interfering RNAs (siRNAs) could regulate the expression of genes. Based on the genome-wide analysis of the small RNAs in the two stages of T. brucei, our results not only provide evidence to study their differentiation but also shed light on questions regarding the origins and evolution of small RNA-based mechanisms in early eukaryotes.

Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei.

The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei.

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.

Infection by Toxoplasma gondii, a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia

Significance The airway epithelia modulate the inflammatory responses to various pathogens. Pulmonary disease caused by Toxoplasma gondii infection affects human neonates, children, and immunocompromised individuals. However, it is not clear how T. gondii infection impacts airway epithelia. We report the use of a short-circuit current (Isc) technique to determine the Cl− secretion induced by ATP in tracheal epithelia infected by T. gondii. We surprisingly found that the ATP-evoked Cl− secretion in T. gondii-infected mouse tracheal epithelia was significantly suppressed. We also found that the mRNA expression level of the P2Y2 receptor increased significantly in T. gondii-infected mouse trachea, via real-time quantitative PCR. Our study provides previously unidentified insights into the mechanism underlying host impairment caused by T. gondii infection. The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl− secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl− secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl− secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca2+-activated Cl− channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl− secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca2+]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription–PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl− secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

Mitogen-activated protein kinase 5, a novel molecular marker for the identification and detection of Trypanozoon species.

Based on the sequence of mitogen-activated protein kinase 5 (TbMAPK5) gene, we have developed a specific PCR method which can delineate the species within the Trypanozoon subgenus from other parasites or host DNA conveniently. In view of further application in field studies, we performed loop-mediated isothermal amplification (LAMP) employing filter paper flecked with rodent blood. Our data showed that TbMAPK5-specific LAMP was sensitive enough to detect a very low parasitemia during the early stages of the infection and fluctuating parasitemia period, and is below the detection limit by microscope. The detection limit for the infected blood sample was 1000 trypanosomes/ml of blood. Based on these results, we consider that TbMAPK5 locus may be a useful target for LAMP diagnosis providing sensitivity and the potential for the genotyping/identification of Trypanozoon species.