Hc-hrg-2, a glutathione transferase gene, regulates heme homeostasis in the blood-feeding parasitic nematode Haemonchus contortus.

BACKGROUND: Haemonchus contortus, a blood-feeding parasite, is constantly surrounded by large quantities of heme released from the catabolism of host red blood cells. To cope with the toxicity of free heme, H. contortus needs to uptake and detoxify the heme, a process believed to be paramount for parasite survival. METHODS: A heme-responsive gene Hc-hrg-2 was identified which is the homologue of Ce-hrg-2. The transcriptional levels in all developmental stages and heme-responsive ability of Hc-hrg-2 were analyzed by qRT-PCR. Immunofluorescence analysis and cell transfections were performed to analyze the expression pattern of Hc-HGR-2. Statistical analyses were performed with GraghPad Prism 6.0 using Student's t-test. RESULTS: To investigate the heme homeostasis of H. contortus, we first identified a heme-responsive gene Hc-hrg-2, a homolog of Ce-hrg-2 that is involved in heme transport in the hypodermis of Caenorhabditis elegans. Using qRT-PCR, we showed that Hc-hrg-2 mRNA was expressed throughout all life-cycle stages of H. contortus with the highest level in the third-stage larvae (L3s). Notably, transcription of Hc-hrg-2 in the exsheathed L3s was significantly upregulated in the presence of high concentration of heme. We found that Hc-HRG-2 protein was mainly located in the hypodermal tissues of adult H. contortus in vivo and the endoplasmic reticulum in the transfected mammalian cells. Our in vitro assay demonstrated that Hc-HRG-2 is a heme-binding protein with glutathione S-transferase activity and heme had a significant effect on its enzymatic activity when a model substrate 1-chloro-2, 4-dinitrobenzene (CDNB) was used. CONCLUSIONS: Hc-hrg-2 is a heme-responsive gene and engaged in heme homeostasis regulation in hypodermal tissues during the free-living stages of H. contortus.

Staphylococcus aureus Protein A induces osteoclastogenesis via the NF­κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor­activated (NF)­κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose­dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast­specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase­9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA­induced osteoclast differentiation was associated with the degradation of inhibitor of κB­α, phosphorylation of NF­κB p65 and increased expression of nuclear factor of activated T­cells. However, by treatment with JSH­23, an NF­κB inhibitor, the formation of osteoclast­like cells and resorption pits was significantly reduced, and the expression of osteoclast­specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF­κB signaling pathway was involved in this process.

Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary Haemonchus contortus infection.

BACKGROUND: Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep. METHODS: Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes. RESULTS: Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection. CONCLUSIONS: Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.

Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus.

Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (P<0.05) and 24.91% reduction in worm burden, but an upward curve with moderate rate of daily FEC in goats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development.