Balancing selection in Pattern Recognition Receptor signalling pathways is associated with gene function and pleiotropy in a wild rodent.

Pathogen-mediated balancing selection is commonly considered to play an important role in the maintenance of genetic diversity, in particular in immune genes. However, the factors that may influence which immune genes are the targets of such selection are largely unknown. To address this, here we focus on Pattern Recognition Receptor (PRR) signalling pathways, which play a key role in innate immunity. We used whole-genome resequencing data from a population of bank voles (Myodes glareolus) to test for associations between balancing selection, pleiotropy and gene function in a set of 123 PRR signalling pathway genes. To investigate the effect of gene function, we compared genes encoding (a) receptors for microbial ligands versus downstream signalling proteins, and (b) receptors recognizing components of microbial cell walls, flagella and capsids versus receptors recognizing features of microbial nucleic acids. Analyses based on the nucleotide diversity of full coding sequences showed that balancing selection primarily targeted receptor genes with a low degree of pleiotropy. Moreover, genes encoding receptors recognizing components of microbial cell walls etc. were more important targets of balancing selection than receptors recognizing nucleic acids. Tests for localized signatures of balancing selection in coding and noncoding sequences showed that such signatures were mostly located in introns, and more evenly distributed among different functional categories of PRR pathway genes. The finding that signatures of balancing selection in full coding sequences primarily occur in receptor genes, in particular those encoding receptors for components of microbial cell walls etc., is consistent with the idea that coevolution between hosts and pathogens is an important cause of balancing selection on immune genes.

Molecular identification and characterization of prohibitin from Echinococcus granulosus.

Prohibitin (PHB) is a widely distributed protein that functions as a molecular chaperone, is involved in the regulation of cell cycle, and maintains mitochondrial structure and functions of the anti-apoptosis, senescence, and proliferation. The aim of this study was to characterize PHB in Echinococcus granulosus (EgPHB), a harmful cestode parasite of humans, many livestock species, and wild animals. We found that EgPHB is a conserved SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain-containing protein, consisting of 289 amino acids, which shares 42.66-99.31% identity with PHBs from other parasites and mammals. EgPHB was located mainly in the tegument issue of protoscoleces, in the inner body of adult worms, and was expressed widely in the germinal layer. This is the first report on prohibitin from E. granulosus, and EgPHB is considered to be a valuable protein to study more in the future.

Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene.

We evaluated genetic diversity and structure of Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1-G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies.

Sequence analysis of cytb gene in Echinococcus granulosus from Western China.

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.

Explore the shared molecular mechanism between dermatomyositis and nasopharyngeal cancer by bioinformatic analysis.

BACKGROUND: Dermatomyositis (DM) is prone to nasopharyngeal carcinoma (NPC), but the mechanism is unclear. This study aimed to explore the potential pathogenesis of DM and NPC. METHODS: The datasets GSE46239, GSE142807, GSE12452, and GSE53819 were downloaded from the GEO dataset. The disease co-expression module was obtained by R-package WGCNA. We built PPI networks for the key modules. ClueGO was used to analyze functional enrichment for the key modules. DEG analysis was performed with the R-package "limma". R-package "pROC" was applied to assess the diagnostic performance of hub genes. MiRNA-mRNA networks were constructed using MiRTarBase and miRWalk databases. RESULTS: The key modules that positively correlated with NPC and DM were found. Its intersecting genes were enriched in the negative regulation of viral gene replication pathway. Similarly, overlapping down-regulated DEGs in DM and NPC were also enriched in negatively regulated viral gene replication. Finally, we identified 10 hub genes that primarily regulate viral biological processes and type I interferon responses. Four key genes (GBP1, IFIH1, IFIT3, BST2) showed strong diagnostic performance, with AUC>0.8. In both DM and NPC, the expression of key genes was correlated with macrophage infiltration level. Based on hub genes' miRNA-mRNA network, hsa-miR-146a plays a vital role in DM-associated NPC. CONCLUSIONS: Our research discovered pivot genes between DM and NPC. Viral gene replication and response to type I interferon may be the crucial bridge between DM and NPC. By regulating hub genes, MiR-146a will provide new strategies for diagnosis and treatment in DM complicated by NPC patients. For individuals with persistent viral replication in DM, screening for nasopharyngeal cancer is necessary.

Disulfide-stabilized trimeric hemagglutinin ectodomains provide enhanced heterologous influenza protection.

Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund's adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

Divergence in Coding Sequence and Expression of Different Functional Categories of Immune Genes between Two Wild Rodent Species.

Differences in immune function between species could be a result of interspecific divergence in coding sequence and/or expression of immune genes. Here, we investigate how the degree of divergence in coding sequence and expression differs between functional categories of immune genes, and if differences between categories occur independently of other factors (expression level, pleiotropy). To this end, we compared spleen transcriptomes of wild-caught yellow-necked mice and bank voles. Immune genes expressed in the spleen were divided into four categories depending on the function of the encoded protein: pattern recognition receptors (PRR); signal transduction proteins; transcription factors; and cyto- and chemokines and their receptors. Genes encoding PRR and cyto-/chemokines had higher sequence divergence than genes encoding signal transduction proteins and transcription factors, even when controlling for potentially confounding factors. Genes encoding PRR also had higher expression divergence than genes encoding signal transduction proteins and transcription factors. There was a positive correlation between expression divergence and coding sequence divergence, in particular for PRR genes. We propose that this is a result of that divergence in PRR coding sequence leads to divergence in PRR expression through positive feedback of PRR ligand binding on PRR expression. When controlling for sequence divergence, expression divergence of PRR genes did not differ from other categories. Taken together, the results indicate that coding sequence divergence of PRR genes is a major cause of differences in immune function between species.

Comparison of spleen transcriptomes of two wild rodent species reveals differences in the immune response against Borrelia afzelii.

Different host species often differ considerably in susceptibility to a given pathogen, but the causes of such differences are rarely known. The natural hosts of the tick-transmitted bacterium Borrelia afzelii, which is one of causative agents of Lyme borreliosis in humans, include a variety of small mammals like voles and mice. Previous studies have shown that B. afzelii-infected bank voles (Myodes glareolus) have about ten times higher bacterial load than infected yellow-necked mice (Apodemus flavicollis), indicating that these two species differ in resistance. In this study, we compared the immune response to B. afzelii infection in these host species by using RNA sequencing to quantify gene expression in spleen. Gene set enrichment analysis (GSEA) showed that several immune pathways were down-regulated in infected animals in both bank voles and yellow-necked mice. Moreover, IFNα response was up-regulated in B. afzelii-infected yellow-necked mice, while IL6 signaling and the complement pathway were down-regulated in infected bank voles; differences in regulation of these three pathways between bank voles and yellow-necked mice could thus contribute to the difference in resistance to B. afzelii between the species. This study provides knowledge of gene expression induced by a zoonotic pathogen in its natural host, and possible species-specific regulation of immune responses associated with resistance.

Codon optimization with deep learning to enhance protein expression.

Heterologous expression is the main approach for recombinant protein production ingenetic synthesis, for which codon optimization is necessary. The existing optimization methods are based on biological indexes. In this paper, we propose a novel codon optimization method based on deep learning. First, we introduce the concept of codon boxes, via which DNA sequences can be recoded into codon box sequences while ignoring the order of bases. Then, the problem of codon optimization can be converted to sequence annotation of corresponding amino acids with codon boxes. The codon optimization models for Escherichia Coli were trained by the Bidirectional Long-Short-Term Memory Conditional Random Field. Theoretically, deep learning is a good method to obtain the distribution characteristics of DNA. In addition to the comparison of the codon adaptation index, protein expression experiments for plasmodium falciparum candidate vaccine and polymerase acidic protein were implemented for comparison with the original sequences and the optimized sequences from Genewiz and ThermoFisher. The results show that our method for enhancing protein expression is efficient and competitive.

Colonization and pathology of Borrelia afzelii in its natural hosts.

Studies of Borrelia burgdorferi sensu lato in laboratory mice and humans have shown that spirochaetes disseminate from the site of infection (skin) to internal tissues, and cause various pathological effects. However, less is known about colonization and pathology of Lyme borreliosis spirochaetes in their natural hosts. In the present study, we assessed the colonization and manifestations during B. afzelii infection in reservoir hosts (yellow-necked mouse, Apodemus flavicollis; bank vole, Myodes glareolus; common shrew, Sorex araneus) infected in the wild. The infection prevalence and bacterial load was measured in skin (ear), joints and heart by quantitative PCR, and pathology in infected joints was evaluated by histology. The prevalence of B. afzelii was higher in skin than in joints and heart, but most animals that were positive in skin were also positive in internal tissues, and there was no difference between species in tissue-specific prevalence. Thus, spirochaetes disseminated from skin to other tissues in a similar way in all species. The bacterial load varied among host species and among different tissues within the same host species. In the case of skin and joints, bank voles and common shrews had higher bacterial loads than yellow-necked mice. In hearts, voles had higher bacterial loads than shrews and mice. Histological analyses showed no inflammation in joints of infected animals when compared to controls. We conclude that B. afzelii disseminates to internal tissues in natural hosts, but that levels of colonization vary between both species and tissues. There is as yet little evidence for pathological effects in natural hosts.

Molecular and biochemical characterization of calmodulin from Echinococcus granulosus.

BACKGROUND: Echinococcus granulosus is a harmful cestode parasite that causes cystic echinococcosis in humans as well as various livestock species and wild animals. Calmodulin (CaM), a Ca2+ sensor protein, is widely expressed in eukaryotes and mediates a variety of cellular signaling activities. METHODS: In the present study, the cDNA encoding CaM in Echinococcus granulosus (rEgCaM) was successfully cloned and the molecular and biochemical characterizations carried out. The antigenicity and immunoreactivity of rEgCaM was detected and the preliminary enzyme-linked immunosorbent assay (ELISA)-based serodiagnostic potential of EgCaM was assessed. The locations of this protein in the adult worm and larval stage, and the mRNA expression in different states of E. granulosus protoscoleces (PSCs) were defined clearly. Moreover, the Ca2+-binding properties of EgCaM were measured. RESULTS: rEgCaM is a highly conserved calcium-binding protein, consisting of 149 amino acids. Immunoblotting analysis revealed that rEgCaM could be identified using E. granulosus infected sheep serum. The use of rEgCaM as an antigen was evaluated by indirect ELISA which exhibited a high sensitivity (90.3%), but low specificity (47.1%). rEgCaM was ubiquitously expressed in protoscoleces and adults of E. granulosus, as well as in the germinal layer of the cyst wall. The mRNA expression level of rEgCaM was increased from the start of H2O2 exposure and then gradually decreased because of the increased apoptosis of PSCs. In electrophoretic mobility tests and 1-anilinonaphthalene-8-sulfonic acid assays, rEgCaM showed a typical characteristic of a calcium-binding protein. CONCLUSIONS: To our knowledge, this is the first report on CaM from E. granulosus and rEgCaM is likely to be involved in some important biological function of E. granulosus as a calcium-binding protein.

Characterization of a Secretory Annexin in Echinococcus granulosus.

Cystic echinococcosis, caused by Echinococcus granulosus, is a widespread parasitic zoonosis causing economic loss and public health problems. Annexins are important proteins usually present in the plasma membrane, but previous studies have shown that an annexin B33 protein of E. granulosus (Eg-ANX) could be detected in the excretory/secretory products and cyst fluid. In this study, we cloned and characterized Eg-ANX. In silico analysis showed that the amino acid sequence of Eg-ANX was conserved and lacked any signal peptides. The phospholipid-binding activity of recombinant Eg-ANX (rEg-ANX) was tested; liposomes could bind to rEg-ANX only in the presence of Ca(2+). In addition, we performed western blotting and immunohistochemical analyses to further validate the secretory properties of Eg-ANX. The protein could be detected in the cyst fluid of E. granulosus and was also present in the intermediate host tissues, which suggested that Eg-ANX might play an important role in parasite-host interaction.

The complete mitochondrial genome of G3 genotype of Echinococcus granulosus (Cestoda: Taeniidae).

As a cryptic species complex, the taxonomy of Echinococcus granulosus has long been controversial. The complete mitochondrial (mt) genome of the E. granulosus G3 genotype was sequenced and characterized. The G3 mt genome was 13,607 bp in length, and contained 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 12 protein-coding genes, with a gene order identical to those of other reported genotypes. The overall nucleotide composition (coding strand) for the mt genome sequence of G3 was 19.1% A, 47.8% T, 25.0% G, and 8.1% C, with T being the most common base and C the least common.

Genetic variability of Echinococcus granulosus based on the mitochondrial 16S ribosomal RNA gene.

Echinococcus granulosus is the etiological agent of cystic echinococcosis, a major zoonotic disease of both humans and animals. In this study, we assessed genetic variability and genetic structure of E. granulosus in the Tibet plateau, using the complete mitochondrial 16 S ribosomal RNA gene for the first time. We collected and sequenced 62 isolates of E. granulosus from 3 populations in the Tibet plateau. A BLAST analysis indicated that 61 isolates belonged to E. granulosus sensu stricto (genotypes G1-G3), while one isolate belonged to E. canadensis (genotype G6). We detected 16 haplotypes with a haplotype network revealing a star-like expansion, with the most common haplotype occupying the center of the network. Haplotype diversity and nucleotide diversity were low, while negative values were observed for Tajima's D and Fu's Fs. AMOVA results and Fst values revealed that the three geographic populations were not genetically differentiated. Our results suggest that a population bottleneck or population expansion has occurred in the past, and that this explains the low genetic variability of E. granulosus in the Tibet Plateau.

Molecular insights into a tetraspanin in the hydatid tapeworm Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (hydatid disease), caused by the tapeworm Echinococcus granulosus (class Cestoda; family Taeniidae), is a neglected tropical disease that results in morbidity and mortality in millions of humans, as well as in huge economic losses in the livestock industry globally. Proteins from the tetraspanin family in parasites have recently become regarded as crucial molecules in interaction with hosts in parasitism and are therefore suitable for the development of vaccines and diagnostic agents. However, no information is available to date on E. granulosus tetraspanin. METHODS: In this study, a uroplakin-I-like tetraspanin (Eg-TSP1) of E. granulosus was cloned and expressed in E. coli. The immunolocalization of Eg-TSP1 in different life stages of E. granulosus was determined using specific polyclonal antibody. The antibody and cytokine profiles of mice that immunized with recombinant Eg-TSP1 (rEg-TSP1) were measured for the immunogenicity analysis of this protein. Additionally, we use RNA interference method to explore the biological function of Eg-TSP1 in larva of E. granulosus. RESULTS: Immunofluorescence analysis showed that endogenous Eg-TSP1 mainly localized in the tegument of larvae and adults. Significantly elevated levels of antibodies IgG1 and IgG2a and of cytokines IFN-γ and IL-12 were observed in the sera of mice after immunization with rEg-TSP1, suggesting a typical T helper (Th)1-mediated immune response elicited by rEg-TSP1. On further probing the role of Eg-TSP1 in E. granulosus by RNA interference, we found that a thinner tegmental distal cytoplasm was induced in protoscoleces treated with siRNA-132 compared to controls. CONCLUSIONS: This is the first report characterizing a tetraspanin from the tapeworm E. granulosus. Our results suggest that Eg-TSP1 is associated with biogenesis of the tegument and maintenance of structural integrity of E. granulosus and could therefore be a candidate intervention target for control of hydatid disease.

Intraspecific genetic variation and phylogenetic analysis of Dirofilaria immitis samples from Western China using complete ND1 and 16S rDNA gene sequences.

Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1.