A proteasomal ß5 subunit of Haemonchus contortus with a role in the growth, development and life span.

BACKGROUND: The proteasome in eukaryotic cells can degrade a variety of proteins and plays an important role in regulating the cell cycle, cell survival and apoptosis. The proteasome receives much attention as a potential chemotherapeutic target for treatment of a variety of infectious parasitic diseases, but few studies of proteasomes have been done on parasitic nematodes. METHODS: A proteasomal ß5 subunit encoding gene (named Hc-pbs-5) and its inferred product (Hc-PBS-5) in Haemonchus contortus were identified and characterized in this study. Then, the transcriptional profiles and anatomical expression were studied using an integrated molecular approach. Finally, a specific proteasome inhibitor bortezomib (BTZ), together with RNA interference (RNAi), was employed to assess the function of Hc-PBS-5. RESULTS: Bioinformatic analysis revealed that the coding sequence of Hc-pbs-5 was 855 bp long and encoded 284 amino acids (aa). The predicted protein (Hc-PBS-5) had core conservative sequences (65-250 aa) belonging to N-terminal nucleophile (Ntn) family of hydrolases. Real-time PCR results revealed that Hc-pbs-5 was continuously transcribed in eight developmental stages with higher levels at the infective third-stage larvae (L3s) and adult males of H. contortus. Immunohistochemical results revealed that Hc-PBS-5 was expressed in intestine, outer cuticle, muscle cells under the outer cuticle, cervical glands and seminal vesicles of male adults and also in intestine, outer cuticle, cervical glands, uterine wall, eggs and ovaries of female adults of H. contortus. BTZ could reduce proportions of egg hatching, and the fourth-stage larvae (L4s) developed from the exsheathed L3s (xL3s) of H. contortus. In addition, silencing Hc-pbs-5 by soaking the specific double-stranded RNA (dsRNA) could decrease the transcription of Hc-pbs-5 and result in fewer xL3s developing to L4s in vitro. CONCLUSIONS: These results indicate that proteasomal ß5 subunit plays an important role in the growth, development and life span of H. contortus.

Effect of primary and secondary Fasciola gigantica infection on specific IgG responses, hepatic enzyme levels and weight gain in buffaloes.

Buffaloes, as highly susceptible definitive hosts of Fasciola gigantica, suffer from a high infection rate of fasciolosis, which causes enormous economic losses. Repeat infection is responsible for this high rate; thus, elucidating the protective immunity mechanism in repeat infection is decisive in fasciolosis prevention. Herein, a secondary experimental infection model was established to preliminarily reveal the protective immunity that occurs in repeat infection. In brief, animals were assigned to three groups: group A (uninfected control), group B (primary infection) and group C (secondary infection). Buffaloes were autopsied 20 weeks post-infection for measurements of the recovered flukes and hepatic examination. In addition, the detection of specific antibody (IgG) responses to F. gigantica excretory-secretory product (FgESP) throughout the whole period and weight gain throughout the first 4 months as a percentage (%) of the starting weight were also determined. The serum hepatic enzyme gamma glutathione transferase (GGT) levels were monitored to assess hepatic damage throughout the study period. Infection establishment was compared between group B and group C. Similar specific IgG patterns were observed between group B and group C, and hepatic damage was more severe in group C than group B. Significant differences in weight gain as a percentage of the start weight were observed between group A and group B at the 3rd and 4th months postprimary infection, while significant differences were not observed between group A and group C or group B and group C. Our results suggest that challenge infection cannot induce resistance against F. gigantica in buffaloes, which is consistent with the protective immunity against Fasciola hepatica reinfection observed in sheep and goats.

A daf-7-related TGF-ß ligand (Hc-tgh-2) shows important regulations on the development of Haemonchus contortus.

BACKGROUND: In most multicellular organisms, the transforming growth factor-ß (TGF-ß) signalling pathway is involved in regulating the growth and stem cell differentiation. Previous studies have demonstrated the importance of three key molecules in this pathway in the parasitic nematode Haemonchus contortus, including one TGF-ß type I receptor (Hc-tgfbr1), one TGF-ß type II receptor (Hc-tgfbr2), and one co-Smad (Hc-daf-3), which regulated the developmental transition from the free-living to the parasitic stages of this parasite. However, almost nothing is known about the function of the TGF-ß ligand (Hc-tgh-2) of H. contortus. METHODS: Here, the temporal transcription profiles of Hc-tgh-2 at eight different developmental stages and spatial expression patterns of Hc-TGH-2 in adult female and male worms of H. contortus have been examined by real-time PCR and immunohistochemistry, respectively. In addition, RNA interference (RNAi) by soaking was employed to assess the importance of Hc-tgh-2 in the development from exsheathed third-stage larvae (xL3s) to fourth-stage larvae (L4s) in H. contortus. RESULTS: Hc-tgh-2 was continuously transcribed in all eight developmental stages of H. contortus studied with the highest level in the infective third-stage larvae (iL3) and Hc-TGH-2 was located in the muscle of the body wall, intestine, ovary of adult females and testes of adult males. Silencing Hc-tgh-2 by the specific double-stranded RNA (dsRNA), decreased the transcript level of Hc-tgh-2 and resulted in fewer xL3s developing to L4s in vitro. CONCLUSIONS: These results suggested that the TGF-ß ligand, Hc-TGH-2, could play important roles in the developmental transition from the free-living (L3s) to the parasitic stage (L4s). Furthermore, it may also take part in the processes such as digestion, absorption, host immune response and reproductive development in H. contortus adults.

Identification and characterization of an R-Smad homologue (Hco-DAF-8) from Haemonchus contortus.

BACKGROUND: Smad proteins are essential cellular mediators within the transforming growth factor-ß (TGF-ß) superfamily. They directly transmit incoming signals from the cell surface receptors to the nucleus. In spite of their functional importance, almost nothing is known about Smad proteins in parasitic nematodes including Haemonchus contortus, an important blood-sucking nematode of small ruminants. METHODS: Based on genomic and transcriptome data for H. contortus and using bioinformatics methods, a Smad homologue (called Hco-daf-8) was inferred from H. contortus and the structural characteristics of this gene and its encoded protein Hco-DAF-8 established. Using real-time PCR and immunofluorescence assays, temporal transcriptional and spatial expression profiles of Hco-daf-8 were studied. Gene rescue in Caenorhabditis elegans was then applied to assess the function of Hco-daf-8 and a specific inhibitor of human Smad3 (called SIS3) was employed to evaluate the roles of Hco-DAF-8 in H. contortus development. RESULTS: The features of Hco-DAF-8 (502 amino acids), including conserved R-Smad domains and residues of the L3-loop that determine pathway specificity, are consistent with a TGF-ß type I receptor-activated R-Smad. The Hco-daf-8 gene was transcribed in all developmental stages of H. contortus studied, with a higher level of transcription in the fourth-stage larval (L4) females and the highest level in adult males. Hco-DAF-8 was expressed in the platymyarian muscular cells, intestine and reproductive system of adult stages. Gene rescue experiments showed that Hco-daf-8 was able to partially rescue gene function in a daf-8 deficient mutant strain of C. elegans, leading to a resumption of normal development. In H. contortus, SIS3 was shown to affect H. contortus development from the exsheathed third-stage larvae (L3s) to L4s in vitro. CONCLUSIONS: These findings suggest that Hco-DAF-8, encoded by the gene Hco-daf-8, is an important cellular mediator of H. contortus development via the TGF-ß signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important parasitic nematodes.

Molecular characterization of the Haemonchus contortus phosphoinositide-dependent protein kinase-1 gene (Hc-pdk-1).

BACKGROUND: Phosphoinositide-dependent protein kinase-1 (PDK-1), which functions downstream of phosphoinositide 3-kinase (AGE-1) and activates protein kinases of the AGC family, plays critical roles in regulating biology processes, such as metabolism, growth, development and survival. In the free-living nematode Caenorhabditis elegans, PDK-1 is a key component of the insulin-like signalling pathway, regulating the entry into and exit from dauer (arrested development). Although it is proposed that similar molecular mechanisms control the transition from the free-living to the parasitic stages of nematodes, nothing is known about PDK-1 in Haemonchus contortus, a socioeconomically important gastric nematode of ruminants. METHODS: Here, we isolated and characterized the pdk-1 gene (Hc-pdk-1) and its inferred product (Hc-PDK-1) from H. contortus. Using in vitro and in vivo methods, we then studied the transcriptional profiles of Hc-pdk-1 and anatomical gene expression patterns of Hc-PDK-1 in different developmental stages of C. elegans. RESULTS: In silico analysis of Hc-PDK-1 displayed conserved functional domains, such as protein kinase and pleckstrin homology (PH) domains and two predicted phosphorylation sites (Thr226/Tyr229), which are crucial for the phosphorylation of downstream signalling. The Hc-pdk-1 gene is transcribed in all of the main developmental stages of H. contortus, with its highest transcription in the infective third-stage larvae (iL3) compared with other stages. Transgene constructs, in which respective promoters were fused to the coding sequence for green fluorescent protein (GFP), were used to transform C. elegans, and to localize and compare the expression of Hc-pdk-1 and Ce-pdk-1. The expression of GFP under the control of the Hc-pdk-1 promoter was localized to the intestine, and head and tail neurons, contrasting somewhat the profile for the C. elegans ortholog, which is expressed in pharynx, intestine and head and tail neurons. CONCLUSIONS: This is the first characterization of pdk-1/PDK-1 from a trichostrongyloid nematode. Taken together, the findings from this study provide a first glimpse of the involvement of Hc-pdk-1 in the insulin-like signalling pathway in H. contortus.