Junling Hou

Identification and functional characterization of alpha-enolase from Taenia pisiformis metacestode.

Enolase belongs to glycolytic enzymes with moonlighting functions. The role of enolase in Taenia species is still poorly understood. In this study, the full length of cDNA encoding for Taenia pisiformis alpha-enolase (Tpeno) was cloned from larval parasites and soluble recombinant Tpeno protein (rTpeno) was produced. Western blot indicated that both rTpeno and the native protein in excretion-secretion antigens from the larvae were recognized by anti-rTpeno monoclonal antibodies (MAbs). The primary structure of Tpeno showed the presence of a highly conserved catalytic site for substrate binding and an enolase signature motif. rTpeno enzymatic activities of catalyzing the reversible dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) and vice versa were shown to be 30.71 ± 2.15 U/mg (2-PGA to PEP) and 11.29 ± 2.38 U/mg (PEP to 2-PGA), respectively. Far-Western blotting showed that rTpeno could bind to plasminogen, however its binding ability was inhibited by ϵ-aminocaproic acid (ϵACA) in a competitive ELISA test. Plasminogen activation assay showed that plasminogen bound to rTpeno could be converted into active plasmin using host-derived activators. Immunohistochemistry and immunofluorescence indicated that Tpeno was distributed in the bladder wall of the metacestode and the periphery of calcareous corpuscles. In addition, a vaccine trial showed that the enzyme could produce a 36.4% protection rate in vaccinated rabbits against experimental challenges from T. pisiformis eggs. These results suggest that Tpeno with multiple functions may play significant roles in the migration, growth, development and adaptation of T. pisiformis for survival in the host environment.

Protection against Asiatic Taenia solium Induced by a Recombinant 45W-4B Protein

ABSTRACT Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates.

Comparative genomics reveals adaptive evolution of Asian tapeworm in switching to a new intermediate host

Taenia saginata, Taenia solium and Taenia asiatica (beef, pork and Asian tapeworms, respectively) are parasitic flatworms of major public health and food safety importance. Among them, T. asiatica is a newly recognized species that split from T. saginata via an intermediate host switch ∼1.14 Myr ago. Here we report the 169- and 168-Mb draft genomes of T. saginata and T. asiatica. Comparative analysis reveals that high rates of gene duplications and functional diversifications might have partially driven the divergence between T. asiatica and T. saginata. We observe accelerated evolutionary rates, adaptive evolutions in homeostasis regulation, tegument maintenance and lipid uptakes, and differential/specialized gene family expansions in T. asiatica that may favour its hepatotropism in the new intermediate host. We also identify potential targets for developing diagnostic or intervention tools against human tapeworms. These data provide new insights into the evolution of Taenia parasites, particularly the recent speciation of T. asiatica.

The Role of Insulin C-Peptide in the Coevolution Analyses of the Insulin Signaling Pathway: A Hint for Its Functions

As the linker between the A chain and B chain of proinsulin, C-peptide displays high variability in length and amino acid composition, and has been considered as an inert byproduct of insulin synthesis and processing for many years. Recent studies have suggested that C-peptide can act as a bioactive hormone, exerting various biological effects on the pathophysiology and treatment of diabetes. In this study, we analyzed the coevolution of insulin molecules among vertebrates, aiming at exploring the evolutionary characteristics of insulin molecule, especially the C-peptide. We also calculated the correlations of evolutionary rates between the insulin and the insulin receptor (IR) sequences as well as the domain-domain pairs of the ligand and receptor by the mirrortree method. The results revealed distinctive features of C-peptide in insulin intramolecular coevolution and correlated residue substitutions, which partly supported the idea that C-peptide can act as a bioactive hormone, with significant sequence features, as well as a linker assisting the formation of mature insulin during synthesis. Interestingly, the evolution of C-peptide exerted the highest correlation with that of the insulin receptor and its ligand binding domain (LBD), implying a potential relationship with the insulin signaling pathway.

Occurrence of Enterocytozoon bieneusi in Donkeys (Equus asinus) in China: A Public Health Concern

Enterocytozoon bieneusi is an important zoonotic parasite. It can infect virtually all animal species and has a global distribution. However, the prevalence of E. bieneusi in donkeys (Equus asinus) has only been reported in Algeria and Spain, and no information is available concerning genotypes of E. bieneusi in donkeys worldwide. In the present study, a total of 301 donkey fecal samples (48 from Jilin Province, 224 from Shandong Province and 29 from Liaoning Province) were collected and examined by PCR amplification of the internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 5.3% (16/301), with 6.3% (3/48) in Jilin Province, 4.9% (11/224) in Shandong Province, and 6.9% (2/29) in Liaoning Province. Prevalence in different age groups ranged from 4.2 to 5.5%. E. bieneusi prevalence in donkeys sampled in different seasons varied from 4.2 to 6.5%. Altogether, four E. bieneusi genotypes were identified in this study, with two known genotypes (J and D) and two novel genotypes (NCD-1and NCD-2). Phylogenetic analysis revealed that genotypes D, NCD-1 and NCD-2 belonged to group 1, while the remaining genotype J was clustered into group 2. These findings revealed the occurrence of E. bieneusi in donkeys in China for the first time. Moreover, the present study also firstly genotyped the E. bieneusi in donkeys worldwide. These findings extend the distribution of E. bieneusi genotypes and provide baseline data for controlling E. bieneusi infection in donkeys, other animals and humans.

Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium

BackgroundCysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets.ResultsWithin the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases. Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases.ConclusionNumerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.

Cloning and characterization of a cathepsin L-like cysteine protease from Taenia pisiformis.

Rabbit cysticercosis, caused by the larval stage of Taenia pisiformis, is a serious parasitic disease of rabbits. It was reported that some cysteine peptidases have potential roles in the pathogenesis of various parasitic infections. To investigate the biochemical characteristics and roles in the pathogenesis/host-invasion of cysteine peptidases, a cDNA sequence encoding for a cathepsin L-like cysteine protease (TpCP) was cloned and identified from the T. pisiformis metacestodes. This sequence was 1220 bp in its length, which included a 1017 bp open reading frame encoding a 339 amino acid peptide. Multiple sequence alignments revealed a 28.9-88.5% similarity with cathepsin L-like cysteine proteases from other helminth parasites and mammals. The recombinant TpCP expressed in Escherichia coli did not show the proteolytic activity by zymography gel assay. However, the TpCP expressed in Pichia pastoris had typical biochemical activities that could hydrolyze rabbit immunoglobulin G, bovine serum albumin and fibronectin. Substrate studies indicated pronounced cleavage of Z-Phe-Arg-AMC. This activity was sensitive to cysteine protease inhibitor E-64 and immunohistochemistry results also indicated that TpCP was distributed as an intense positive reaction in the bladder wall. Our results gave us insights into future studies of TpCPs roles in the infection.

Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18.

A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 10(12) CFU of S. typhimurium χ4558, while all mice infected with 10(7) CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4(+) and CD8(+) T lymphocytes and the percentage of helper CD4(+)/CD8(+) T lymphocytes was significantly increased (p<0.01). After oral administration, S. typhimurium χ4558 was first colonized mainly in the Peyers patches and then predominantly in the mesenteric lymph nodes and spleens in the vaccinated mice. In addition, the high levels of specific anti-TSOL18 antibodies were also observed in pigs administrated with S. typhimurium χ4558. Collectively, these results demonstrate the possibility of use of an attenuated S. typhimurium strain as a vector to deliver protective antigens of T. solium.

Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica

Fasciola gigantica infection in water buffaloes causes significant economic losses especially in developing countries. Although modulation of the host immune response by cytokine neutralization or vaccination is a promising approach to control infection with this parasite, our understanding of cytokines dynamic during F. gigantica infection is limited. To address this, we quantified the levels of serum cytokines produced in water buffaloes following experimental infection with F. gigantica. Five buffaloes were infected via oral gavage with 500 viable F. gigantica metacercariae and blood samples were collected from buffaloes one week before infection and for 13 consecutive weeks thereafter. The levels of 10 cytokines in serum samples were simultaneously determined using ELISA. F. gigantica failed to elicit the production of various pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-2, IL-6, IL-12, and IFN-γ. On the other hand, evidence of a Th2 type response was detected, but only early in the course of parasite colonization and included modest increase in the levels of IL-10 and IL-13. The results also revealed suppression of the immune responses as a feature of chronic F. gigantica infection in buffaloes. Taken together, F. gigantica seems to elicit a modest Th2 response at early stage of infection in order to downregulate harmful Th1- and Th17-type inflammatory responses in experimentally infected buffaloes. The full extent of anti-F. gigantica immune response and its relation to pathogenesis requires further study.

The presence of Giardia intestinalis in donkeys, Equus asinus , in China

BackgroundGiardia intestinalis is one of the most important zoonotic enteric parasites. As no information regarding prevalence and genotype of G. intestinalis in donkeys (Equus asinus) in China is available, 181 faecal samples from 48 donkeys from Jilin Province, from 104 from Shandong Province and from 29 from Liaoning Province were examined between May and December 2015.FindingsTwenty-eight (15.47%) out of 181 donkey samples were tested G. intestinalis-positive by nested amplification of the triosephosphate isomerase (tpi) gene. The prevalence in different regional groups varied from 10.42 to 18.27%. The prevalence in adult and young donkeys was 14.29 and 22.92%, respectively. Otherwise, the prevalence was 11.69% in summer and 18.27% in winter. However, no statistically significant differences were found in relation to region or age group. Sequence analysis of the tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci identified 4, 1 and 3 subtypes of assemblage B, respectively. Moreover, four novel multilocus genotypes (MLGs novel-1 to novel-4) were identified in assemblage B.ConclusionsThis first report of G. intestinalis in donkeys in China indicates that further studies of nation-wide molecular epidemiology and geographical distribution of Giardia in donkeys are warranted. Effective strategies should be implemented to control G. intestinalis infection in donkeys, other animals and humans.