Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.     

Analysis on the reactivity of five subunits of antigen B family in serodiagnosis of echinococcosis

In this study, the reactivity and differences of five subunits of echinococcus antigen B (AgB) family, recognizing specific antibodies in echinococcosis patient serum, were analyzed. Eight recombinant subunit antigens from Echinococcus granulosus (EgAgB1-EgAgB4) and Echinococcus multilocularis (EmAgB1-EmAgB3 and EmAgB5) were tested by ELISA using a panel of 243 serum samples collected from cystic echinococcosis (CE), alveolar echinococcosis (AE), cysticercosis (CC) patients and clinically normal individuals (NH). The results showed that the diagnostic sensitivity of the subunits for CE sera were 83.06%, 62.90%, 29.03%, 75.81% and 41.13%, and the specificities were 73.95%, 72.27%, 76.47%, 73.11% and 85.71%, respectively. The reactivity of three paralogous subunits, EgAgB1, EgAgB2 and EgAgB3 from E. granulosus and EmAgB1, EmAgB2 and EmAgB3 from E. multilocularis were compared by serological assay. All of the orthologous subunits showed no statistical difference (P>0.05) in detecting CE and AE sera; it revealed that the reactive epitopes may be similar between the orthologous subunits. In a total of 124 CE sera, the positive recognition rate by EgAgB1 was the highest (103/124), yet cocktail subunit antigens may detect even more positives from 100/124 to 112/124 using different subunit combinations. IgG4 subclass was the predominant antibody in reacting with subunit antigens. To conclude, the epitopes of orthologous AgB subunits from E. granulosus and E. multilocularis that recognize specific antibodies may be similar. The paralogous subunits EgAgB1, EgAgB2 and EgAgB4 were the main reactive subunit in sera detection and may have utility as echinococcosis diagnostics, with EgAgB1 possessing the greatest potential. Cocktail subunits may improve the positive detection rate.     

The Echinococcus granulosus antigen B shows a high degree of genetic variability

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.     

Protective immune mechanisms against the metacestode of Echinococcus multilocularis

Infection with the larval stage of the fox tapeworm Echinococcus multilocularis results in a life-threatening hepatic disease concerning humans and intermediate rodent hosts. Immunoepidemiological surveys provided information that a large proportion of infected individuals may demonstrate either constitutional resistance to early post-oncospheral development of the parasite or late resistance to disease by exhibiting an intrahepatic died-out parasite lesion. Similar events have been found in secondary infections of laboratory rodents. Dissection of humoral and cell-mediated immune responses in susceptible versus resistant individuals provides insight into immunological pathways associated with the different outcome of infection. Survival strategy of the metacestode obviously focuses on the crucial role played by the parasite laminated layer. This layer protects the metacestode from host effector mechanisms which can potentially kill the proliferating germinative compartments in case of resistant hosts. Bruno Gottstein and Richard Felleisen here discuss the need to search for more parameters discriminating between the different immune pathways in order to find out (immunogenetic?) predispositions responsible for the respective phenomena.     

Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.     

In vivo inhibition by ethyloxanilates of alkaline phosphatases of the cestode Echinococcus multilocularis

Alkaline phosphatases (E.C.3.1.3.1), membranous enzymes of the cestode Echinococcus multilocularis have been studied in the parasite and in the experimental host liver. Synthetic inhibitors interaction with metacestode alkaline phosphatases is reported. In regard to the alkaline phosphatases inhibition, the ethyloxanilate 2 is more efficient in the cestode itself than in the host liver.     

ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus

This study investigated sequence heterogeneity in the first internal transcribed spacer (ITS-1) of ribosomal DNA within and among species and strains of Echinococcus. Different ITS-1 sequence variants exist in Echinococcus granulosus and Echinococcus multilocularis, which represent at least four evolutionary lineages: (1) a sheep strain-lineage of E. granulosus, (2) a sister lineage of a cervid and camel E. granulosus ITS-1 variants, (3) a lineage including the ITS-1 variants representing horse, bovine and camel strains of E. granulosus, as well as variants from E. multilocularis, Echinococcus oligarthrus and Echinococcus vogeli and (4) a distinct lineage of ITS-1 variants including E. granulosus strains from sheep and cervid, and E. multilocularis. At least two of the species (E. granulosus and E. multilocularis) were paraphyletic for ITS-1. Divergent ITS-1 variants from these two species shared distinct evolutionary lineages. The sequence data provided evidence that at least two turnover mechanisms, namely slippage and unequal crossing over/transposition, have led to the divergence and maintenance of sequence variants in Echinococcus species and strains.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.     

Effects of cyclosporin A on the course of murine alveolar echinococcosis and on specific cellular and humoral immune responses against Echinococcus multilocularis.

The effects of cyclosporin A (CsA) on Echinococcus multilocularis (E. multilocularis) metacestode growth, and on the specific immune responses of the hosts, were examined in AKR mice. Mice were intra-peritoneally infected with a metacestode homogenate. CsA (40 mg kg(-1) day(-1)) was injected subcutaneously from the 45th day after infection (Group 1), and from the day before infection (Group 2) until the day of autopsy (days 125 and 80, respectively). Results showed that unlike ths situation with some other helminthiases, CsA had no antiparasitic effect, although it lengthened the maturation time of protoscoleces in Group 1. The parasitic burden, unmodified in Group 1, was significantly enhanced in Group 2. This enhancement was associated with a decrease in antibody levels, whereas the delayed-type hypersensitivity was decreased in the two groups. These results confirm the role of cellular immunity in controlling the first stages of the larval development of E. multilocularis and indicate the necessity for a careful follow-up of any recurrence of alveolar echinococcosis in patients treated with CsA after liver transplantation.     

Alveolar Echinococcus species from Vulpes corsac in Hulunbeier, Inner Mongolia, China, and differential development of the metacestodes in experimental rodents

Adults of alveolar Echinococcus species with different uterine structures were collected from Vulpes corsac in the Hulunbeier Pasture of Northeastern China in 2001. They were Echinococcus multilocularis Leuckart, 1863 (type No. 3, similar to E. m. multilocularis), with vaselike uterus; Echinococcus cf. sibiricensis Rausch et Schiller, 1954 (type No. 1), with pyriform uterus; and Echinococcus sp. (type No. 2) with spherical uterus at segment top. The metacestode development in rodents also differed among those 3 parasites. In the case of E. multilocularis (type No. 3), many germinal cells grew on the inner surface of early cysts, most of which metastasized into host tissue to form brood vesicles or from the germinal cell layer on the inner surface of the vesicle wall. Cells also had an appearance of proliferating by means of alveolar buds from alveolar tissue that developed outward to form new alveolar foci. In Echinococcus cf. sibiricensis (type No. 1), the formation of alveolar vesicles was due to the metastasizing of germinal tissue into host tissue; protoscoleces grew in the center of alveolar vesicles. In type No. 2 (Echinococcus sp.), the formation of the alveolar vesicle was by multiplication of germinal cell layers on the inner surface of alveolar cysts; protoscoleces grew from the germinal cell layer and mesh in the vesicles. On the basis of uterine structure and on differences in development of metacestodes in experimental rodents, we propose that the 3 types of Echinococcus represent 3 independent species: E. multilocularis, Echinococcus sibiricensis, and Echinococcus sp. (type No. 2-as yet under study).     

EmTIP,a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

Background

Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.

Principal Findings

We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.

Conclusions

We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.     

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.     

Polymorphism and natural selection of antigen B1 of <Emphasis Type="Italic">Echinococcus granulosus</Emphasis> isolated from different host assemblages in India

Antigen B (AgB) is an excretory–secretory product of larval stage of Echinococcus granulosus. This antigen is abundantly secreted by larval stage of the worm. AgB is encoded by a multigene family and is suggested to be involved in evasion of host immune system. During the present study a total of 110 animal isolates of E. granulosus has been screened by mutation scan screening. A total of 14 conformers were detected on the basis of single strand banding profile of 129 bp fragment of AgB1 subunit. Analysis of sequence information of 14 conformers could cluster the isolates into two different group viz. buffalo–cattle and sheep–goat cluster. Conceptual amino acid sequence information of two clusters could be differentiated in terms of MHCII binding propensity of agretope and B-cell epitope region. This type of analysis of clustering of animal isolates on the basis of binding propensity towards different MHC antigens and B-cell linear epitope is the example of its own kind. Positive Tajima’s D value indicated that, AgB1 is under balancing selection pressure which might be considered as heterogeneous selection pressure exerted by the host.     

Characterization of the laminated layer of in vitro cultivated Echinococcus vogeli metacestodes

The metacestode (larval) stages of the cestode parasites Echinococcus vogeli and E. multilocularis were isolated from the peritoneal cavity of experimentally infected C57BL/6 mice and were cultured in vitro for a period of up to 4 mo under conditions normally applied for the in vitro cultivation of E. multilocularis metacestodes. In contrast to E. multilocularis, E. vogeli did not exhibit extensive exogenous budding and proliferation but increased in size with a final diameter of up to 10 mm. Most metacestodes contained protoscoleces, singly or in groups, either associated with brood capsules or growing directly out of the germinal layer. Each individual metacestode was covered by an acellular translucent laminated layer that was considerably thicker than the laminated layer of E. multilocularis metacestodes. The ultrastructural characteristics, protein content, and carbohydrate composition of the laminated layer of in vitro cultivated E. vogeli and E. multilocularis were assessed using transmission electron microscopy, lectin fluorescence labeling, and lectin blotting assays. The laminated layer of E. vogeli is, as previously described for E. multilocularis metacestodes, largely composed of N-acetyl-beta-D-galactosaminyl residues and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1,3-galactose. However, in contrast to E. multilocularis, N-linked glycopeptides and alpha-D-mannosyl and/or glucosyl residues were also associated with the laminated layer of E. vogeli. The laminated layer from both species was isolated from in vitro cultivated metacestodes, and the purified fractions were comparatively analyzed. The protein:carbohydrate ratio (1:1) was similar in both parasites; however, the protein banding pattern obtained by silver staining following sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested intrinsic differences in protein composition. A polyclonal antiserum raised against the E. multilocularis laminated layer and a monoclonal antibody, G11, directed against the major E. multilocularis laminated layer antigen Em2 did not cross-react with E. vogeli, indicating distinct compositional and antigenic differences between these 2 parasites.     

Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host

Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host‐parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.     

Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-γ mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-β mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.     

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states

Background

Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.

Methodology/Principal Findings

The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.

Conclusions/Significance

For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.