Bang Shen

Characterization and annotation of Babesia orientalis apicoplast genome.

BackgroundBabesia orientalis is an obligate intraerythrocytic protozoan parasite of the buffalo (Bubalus bubalis, Linnaeus, 1758) transmitted by the tick Rhipicephalus heamaphysaloides. It is the causative agent of water buffalo babesiosis, one of the most important pathogens of water buffalo in central and southern China. As a member of the phylum Apicomplexa, B. orientalis possesses a relatively independent and alga originated organelle the apicoplast. Apicoplasts in other apicomplexa parasites are involved in the biosynthesis of haem, fatty acids, iron-sulphur clusters and isoprenoids. Some of these metabolic pathways were shown to be essential for parasite survival, therefore can serve as potential drug targets.Methods30 pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported apicoplast genomes of Babesia bovis and Theileria parva. Conventional PCRs was performed to obtain overlapped fragments to cover the whole apicoplast genome. Then the apicoplast genome of B.orientalis was sequenced, assembled and aligned with reported apicoplast genomes of B. bovis and T. parva. The obtained apicoplast genome was annotated by using Artemis and comparing with published apicomplexan apicoplast genomes. The SSU and LSU nucleotide sequences generated were used in a phylogenetic analysis using the maximum likelihood implemented in MAGE 6.0.ResultsWe have obtained and analyzed the complete genome sequence of the B. orientalis apicoplast. It consisted of a 33.2 kb circular DNA (78.9 % A + T). The apicoplast genome unidirectionally encodes one large and one small subunit ribosomal RNAs, 24 tRNA genes, 4 DNA-dependent RNA polymerase beta subunits (rpoB, rpoC1, rpoC2a and rpoC2b), 17 ribosomal proteins, one EF-Tu elongation factor, 2 Clp protease chaperones, and 14 hypothetical proteins. In addition, it includes two copies of the clpC gene. The structure and organization of the B. orientalis apicoplast genome are most similar to those of the B. bovis apicoplast.ConclusionsThis is the first report of the complete sequence of the B. orientalis apicoplast genome. This information should be useful in the development of safe and efficient treatment against buffalo babesiosis.

Genetic diversity and drug sensitivity studies on Eimeria tenella field isolates from Hubei Province of China

BackgroundAvian coccidiosis is an intracellular intestinal parasitic disease, caused by intracellular intestinal parasites from the genus Eimeria, among which Eimeria tenella is one of the most pathogenic species and causes great economic losses. Frequent applications of anticoccidial drugs have resulted in the development of drug-resistance in E. tenella. In the present study, we sought to determine the genetic diversity of E. tenella isolates prevalent in chicken farms in Hubei Province of China and examine their sensitivity to three anticoccidial drugs. The results provide useful information for the prevention and control of coccidiosis in this region.MethodsEimeria tenella oocysts were isolated from faecal samples collected from different commercial broiler production farms in Hubei Province, China. After oocyst sporulation and animal inoculation for expansion of the field isolates, DNA and RNA were extracted from excysted sporozoites for molecular characterization. Species identification of field isolates were performed by polymerase chain reaction (PCR) amplification of the internal transcribed spacer 1 (ITS1) region of ribosomal DNA. Random amplified polymorphic DNA (RAPD) was used for population genetic analysis. Subsequently, sequences of the major sporozoite surface antigen (SAG), micronemal protein 2 (MIC-2) and cytochrome b (cytb) genes from genomic DNA, and the Eimeria tenella cation-transport ATPase (EtCat ATPase) gene from cDNA were obtained for genotyping using multi-sequence alignments. Finally, sensitivity of the field isolates to three commonly used anticoccidial drugs (diclazuril, decoquinate and maduramycin) were tested to assess the prevalence of drug resistance in E. tenella in Hubei Province of China.ResultsAnalysis of the ITS1 sequences indicated that all the isolates were E. tenella. RAPD analysis and multi-sequence alignments of the SAG, MIC-2, EtCat ATPase and cytb showed genetic diversity among these isolates. Finally, drug sensitivity tests demonstrated that all field isolates were sensitive to diclazuril but resistant to decoquinate (except for the isolates from eastern Hubei) and maduramicin.ConclusionsPopulation genetic analysis indicated that genetic polymorphisms among field isolates were closely related with their regional distributions. Drug sensitivity testing demonstrated that E. tenella isolates in Hubei Province were sensitive to diclazuril, but resistant to maduramycin and decoquinate. The results presented here provide important information for the control and preventions of coccidiosis in the Hubei Province of China.

Sixty Years (1957–2017) of Research on Toxoplasmosis in China—An Overview

Toxoplasma gondii is a ubiquitous zoonotic pathogen belonging to apicomplexan parasites. Infection in humans and animals may cause abortion and other severe symptoms under certain circumstances, leading to great economical losses and public health problems. T. gondii was first discovered in China in 1955 and the corresponding work was published in 1957. Since then, a lot of work has been done on this parasite and the diseases it causes. This review summarizes the major progress made by Chinese scientists over the last 60 years, and gives our perspectives on what should be done in the near future. A wide variety of diagnostic approaches were designed, including the ones to detect T. gondii specific antibodies in host sera, and T. gondii specific antigens or DNA in tissue and environmental samples. Further work will be needed to translate some of the laboratory assays into reliable products for clinic uses. Epidemiological studies were extensively done in China and the sero-prevalence in humans increased over the years, but is still below the world average, likely due to the unique eating and cooking habits. Infection rates were shown to be fairly high in meat producing animals such as, pigs, sheep, and chickens, as well as in the definitive host cats. Numerous subunit vaccines in the form of recombinant proteins or DNA vaccines were developed, but none of them is satisfactory in the current form. Live attenuated parasites using genetically modified strains may be a better option for vaccine design. The strains isolated from China are dominated by the ToxoDB #9 genotype, but it likely contains multiple subtypes since different ToxoDB #9 strains exhibited phenotypic differences. Further studies are needed to understand the general biology, as well as the unique features of strains prevalent in China.

Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China

Several rhoptry proteins (ROPs) have been confirmed to be critical virulence factors of Toxoplasma gondii strains from North America and Europe. The two active kinases ROP17 and ROP18, and pseudokinase ROP5 were thought to be the key determinants of parasites’ virulence in laboratory mice. Given the genetic diversity of Toxoplasma strains from different geographical regions, the virulence determinants in other strains, particularly the ones that are phylogenetically distant to the North American and European strains, are yet to be elucidated. In this study, we sought to examine the contribution of three known virulence factors to the virulence of a type I strain (T.gHB1) isolated from Central China. We deleted ROP17 and ROP18 individually, as well as in combination with GRA7 by the CRISPR-Cas9 system in this local isolate. Subsequent virulence tests in mice indicated that deletion of GRA7, ROP17, or ROP18 in T.gHB1showed similar attenuation in mice as the type I RH strain lacking the corresponding proteins. However, in contrast to the reported double knockouts in RH, double deletions of GRA7 plus ROP17 or GRA7 plus ROP18 in T.gHB1 did not show significant further virulence attenuation compared to the ROP17 or ROP18 single knockouts. These results indicated that GRA7, ROP18 and ROP17 may play different roles in virulence determination in genetically diverse strains of Toxoplasma.

Activation of chronic toxoplasmosis by transportation stress in a mouse model

Toxoplasma gondiiis an obligate intracellular parasite infecting 25% of the world population and enormous number of animals. It can exist in two forms in intermediate hosts: the fast replicating tachyzoites responsible for acute infection and the slowly replicating bradyzoites responsible for life-long chronic infection. The interconversion between tachyzoites and bradyzoites plays critical roles in the transmission and pathogenesis of T. gondii. However, the molecular mechanisms that govern the interconversion are largely unknown. In this study, we established a chronic infection model in mice and examined the impact of transportation stress on the status of chronic infection. Our results demonstrated that, treating chronically infected mice with conditions mimicking transportation stress reduced the levels of several key cytokines that restrict the infection at chronic stage. Increased expression of the tachyzoite specific gene SAG1 (surface antigen 1) was detected in brain cysts of stress treated mice, indicating activation and conversion of bradyzoites to tachyzoites. Using this model, we identified fifteen toxoplasmic proteins that had significant abundance changes during stress induced cysts reactivation. These proteins serve as a basis for further investigation of the mechanisms governing bradyzoite conversion.

Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3

As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.

Identification and characterization of a novel 34 kDa merozoite protein in Babesia orientalis.

A novel Babesia orientalis 34 kDa protein (designated BoP34) was obtained by immunoscreening of a cDNA expression library using B. orientalis infected water buffalo serum. The complete nucleotide sequence of the BoP34 was 1088 bp, which contained one open reading frame (ORF), two untranslated regions (UTRs) and a poly (A) tail. The length of ORF was 933 bp, encoding a polypeptide of 310 aa with a predicted size of 34 kDa. BLAST analysis showed that the nucleotide sequence of BoP34 had 71% similarity with that of the Babesia bovis gene XM_001611335, which encodes a nuclear movement family protein. This suggested that BoP34 is a homologous of the movement family protein. Structural analysis of the BoP34 protein indicated a CS domain which may interact with the ATPase domain of the heat shock protein 90. A truncated version of BoP34 was cloned into the expression vector pET-32a and subsequently expressed and purified from the Escherichia coli Rosetta™ (DE3) pLysS stain as a Trx-fusion (designated rBoP34-T). Antibodies in the serum of a B. orientalis-infected water buffalo were able to recognize this protein in immune-bloting analysis. Rabbit antibodies raised against rBoP34-T could detecte native BoP34 (34 kDa) in B. orientalis-infected water buffalo erythrocytes. These results suggested that BoP34 might be a good diagnostic antigen for the specific detection of anti-B. orientalis antibody in water buffalo. Further research is required to explore the biological function and diagnostic potential of this molecule.

Identification of erythrocyte membrane proteins interacting with Mycoplasma suis GAPDH and OSGEP

Mycoplasma suis (M. suis) is an uncultivable haemotrophic mycoplasma that parasitizes the red blood cells of a wide range of domestic and wild animals. Adhesion of M. suis to host erythrocytes is crucial for its unique RBC-dependent lifecycle. MSG1 protein (now named as GAPDH) with homology to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the first identified adhesion protein of M. suis. In this study, we found that O-sialoglycoprotein endopeptidase (OSGEP) is another M. suis protein capable of binding porcine erythrocytes. Recombinant OSGEP expressed in E. coli demonstrated surface localization similar to GAPDH. Purified rOSGEP bound to erythrocyte membrane preparations in a dose-dependent manner and this adhesion could be specifically inhibited by anti-rOSGEP antibodies. E. coli transformants expressing OSGEP on their surface were able to adhere to porcine erythrocytes. Furthermore, using far-western and pull-down assays, we determined the host membrane proteins that interacted with OSGEP and GAPDH were Band3 and glycophorin A (GPA). In conclusion, our studies indicated that OSGEP and GAPDH could interact with both Band3 and GPA to mediate adhesion of M. suis to porcine erythrocytes.

Identification of novel dense granule proteins in Toxoplasma gondii by two proximity based biotinylation approaches

Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host-parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona fide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite-host interactions.

A Lactate Fermentation Mutant of Toxoplasma Stimulates Protective Immunity Against Acute and Chronic Toxoplasmosis

Toxoplasma gondii is an important zoonotic pathogen infecting one-third of the world’s population and numerous animals, causing significant healthcare burden and socioeconomic problems. Vaccination is an efficient way to reduce global sero-prevalence, however, ideal vaccines are not yet available. We recently discovered that the Toxoplasma mutant lacking both lactate dehydrogenases LDH1 and LDH2 (Δldh) grew well in vitro but was unable to propagate in mice, making it a good live vaccine candidate. Here, we tested the protection efficacy of ME49 Δldh using a mouse model. Vaccinated mice were efficiently protected from the lethal challenge of a variety of wild-type strains, including type 1 strain RH, type 2 strain ME49, type 3 strain VEG, and a field isolate of Chinese 1. The protection efficacies of a single vaccination were nearly 100% for most cases and it worked well against the challenges of both tachyzoites and tissue cysts. Re-challenging parasites were unable to propagate in vaccinated mice, nor did they make tissue cysts. High levels of Toxoplasma-specific IgG were produced 30 days after immunization and stayed high during the whole tests (at least 125 days). However, passive immunization of naïve mice with sera from vaccinated mice did reduce parasite propagation, but the overall protection against parasite infections was rather limited. On the other hand, Δldh immunization evoked elevated levels of Th1 cytokines like INF-γ and IL-12, at early time points. In addition, splenocytes extracted from immunized mice were able to induce quick and robust INF-γ and other pro-inflammatory cytokine production upon T. gondii antigen stimulation. Together these results suggest that cellular immune responses are the main contributors to the protective immunity elicited by Δldh vaccination, and humoral immunity also contributes partially. We also generated uracil auxotrophic mutants in ME49 and compared their immune protection efficiencies to the Δldh mutants. The results showed that these two types of mutants have similar properties as live vaccine candidates. Taken together, these results suggest that mutants lacking LDH were severely attenuated in virulence but were able to induce strong anti-toxoplasma immune responses, therefore are good candidates for live vaccines.