Studies On The Immunomodulatory Properties Of Excretory And Secretory Proteins (ESP) Of Haemonchus Contortus

Haemonchosis is one of the most important and highly pathogenic diseases of small ruminant caused by H. contortus. Heavily infected animal can lose over 250 mL of blood per day, leading to decreased wool production, reduced carcass quality, anemia and death.Excretory and secretory Products (ESPs) are molecules produced and released by parasites during in vitro cultivation and to be released in vivo. ESP contains various proteins and glycoproteins with functions including depression of host immunity and probably also initiating the host immune response and pathology. The sheep acquire resistance to the H.contortus infection associated with breed, age and expose to previous infection, however this immunological phenomenon are not very clear. In the present studies impacts of the H.contortus excretory and secretory proteins on the immune-modulation were screen out and studies were carried out as follows.1- Effects of the Haemonchus contortus excretory and secretory proteins (HcESPs) on the functions of goat peripheral blood mononuclear cells (PBMCs) in vitroHaemonchus contortus excretory and secretory proteins (HcESP) from the adult worms cultured in RPMI 1640 medium (100 worms/ml) at 37 ? under 5% CO2. After incubation,supernatant was collected, centrifuged, filter-sterilized (0.2 m), concentrated, and desalted (10 mM Tris, NaCl pH7.4) in 3-kDa filters. Protein profile of HcESP was checked by SDS-PAGE and the results showed different band pattern ranging from 13 to 180 kDa and these bands were detected through western blotting probed by IgGHcESP. Binding of HcESP to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay. Effects of the HcESP on cytokine production, cell proliferation, cell migration, nitric oxide (NO)production and apoptosis were observed by co-incubation of goat PBMCs with HcESP. ELISA was performed to evaluate the effects of HcESP on the cytokine production and results showed that, the production of IL-4 and IFN? was significantly decreased by HcESP in dose depended manner. However, the production of IL10 and IL-17 was significantly increased. Cell migration was significantly increased by HcESP, whereas, HcESP treatment significantly suppressed the proliferation and nitric oxide production in dose depended manner. Induction of the apoptosis was also observed in PBMCs incubated with the different concentration of HcESP. Findings of our study provided the better knowledge and insights of the host parasite interaction and regulatory effects of the HcESP on the goat PBMCs.2. Proteomic analysis of Haemonchus contortus excretory and secretory proteins(HcESPs) binding to goat peripheral blood mononuclear cells (PBMCs) in vitro.The present study has been conducted to identify the interacting protein of Haemonchus contortus excretory and secretory protein (HcESP)in vitro. In this regard,goat PBMCs were incubated with HcESPs and the binding was confirmed by an immunofluorescence assay.Proteins were extracted from the PBMCs incubated with HcESPs (in vitro) were analyzed through Co- immunoprecipitation (CO-IP) probed by rat anti HcESP IgG (IgGHcESP).Immunoprecipitates were analyzed through Liquid chromatography- tandem mass spectrometry (LC-MS/MS) for the protein identification. Proteins were identified as Actin,Heat shock protein 70 (HSP70), myosin, elongation factor alpha, 14-3-3, ubiquitin, Tubulin alpha, tubulin FtsZ, histone 3 , histone 4 and Lipoma HMGIC fusion partner protein,ISE/inbred ISE genomic scaffold, WORM6 protein kinase and Protein kinase domain containing protein. Identified protein were divided into molecular function and biological process on the basis of their gene ontology (GO). Binding (54%) catalytic activity (31.42 %)two major molecular function categories were found. In case of biological GO terms cellular process, single-organism process, metabolic process, biological regulation, developmental process,multicellular organismal process, growth, response to stimulus, localization,reproduction, cellular component organization or biogenesis, locomotion and signaling GO terms were found most abundantly in in vitro of the annotated sequences at blast level 2. This is the first global report regarding the identification of the binding protein of the HcESP with goat PBMCs in vitro.3. Proteomic analysis of the excretory and secretory proteins of Haemonchus contortus(HcESP) binding to goat PBMCs in vivo at different developmental stages of the wormIn this chapter, we identified the binding proteins of HcESPs at different developmental stages to goat peripheral blood mononuclear cells (PBMCs) in vivo using liquid chromatography-tandem mass spectrometry. Blood samples were collected from experimentally infected goat after 7 (L4 stage), 15 (L5 stage), 40 (early adult stage) and 60 days(late adult stage) post infection. PBMCs were separated by the standard Ficoll-Hypaque gradient centrifugation method. Isolated PBMCs were used to identify HcESP-PBMC-binding proteins by co-immunoprecipitation (Co-IP) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 407 HcESP proteins that bound goat PBMCs at different time points were identified from a H. contortus protein database using SEQUEST searches.The L4and L5 stages ofH. contortus represented a higher proportion of the identified proteins compared with the early and late adult stages. Both stage-specific binding proteins and proteins that were common to multiple stages were identified. Forty-seven binding proteins were shared among all stages. The gene ontology (GO) distributions of the identified goat PBMC-binding proteins were nearly identical among all developmental stages, with high representation of binding and catalytic activity. Cellular, metabolic and single-organism processes were also annotated as major biological processes, but interestingly, more proteins were annotated as localization processes at the L5 stage than at the L4 and adult stages. Based on the clustering of homologous proteins, we improved the functional annotations of un-annotated proteins identified at different developmental stages. Some unnamed H. contortus ATP-binding cassette proteins, including ADP-ribosylation factor and P-glycoprotein-9, were identified by string protein clustering analysis.4. Molecular cloning, expression and regulatory effects of recombinant protein of 14-3-3 isoform 2 (rHcftt-2) on the goat PBMCs functionsA novel Haemonchus contortus 14-3-3 gene (Hcflt-2) identified as binding protein to goat PBMCs at L4- to adult stage in vivo was isolated from the adult Haemonchus contortus by using the specific primers designed based on the CDC of the 14-3-3 protein domain containing protein (GenBank: accession No. CDJ94531.1). The ORF of Hcftt-2 contains 750 nucleotides and encode a peptide of 132 amino acids with molecular weight about 28 kDa. Hcftt-2 protein was predicted for 13 B and 13 T cell epitopes at multiple locations. Sequence analysis showed that it has significant homology with the known 14-3-3 protein isoform 2. Binding activity of rHcftt-2 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and it's modulatory effects on cytokine production,cell proliferation, cell migration, nitric oxide (NO) production and apoptosis were observed by co-incubation of rHcftt-2 with goat PBMCs. ELISA was performed to evaluate the effects of rHcftt-2 on the cytokine production and results showed that, the production of IL10, IL-17 and IFNy was increased in cells incubated with rHCftt-2, however, the production of IL-4 was significantly decreased. Cell migration was significantly increased by rHcftt-2, whereas,rHcftt-2 treatment significantly suppressed the proliferation of the PBMCs in dose depended manner. NO production by PBMCs was also significantly suppressed by rHcftt-2. The apoptosis of PBMCs were also observed, but no significant changes were found between the control group and the rHcftt-2 protein treated groups The present study is the first molecular and functional characterization of Hcftt-2. Our results provide the better knowledge and insights of the host parasite interaction and regulatory effects of the rHcftt-2 on the goat PBMCs.5. Molecular Cloning, Expression and effects on the goat PBMCs functions of small GTPase ADP-ribosylation factor gene (HcARF1)In this chapter, H. contortus small GTPASE ADP-ribosylation gen (HcARF1) identified as binding protein to goat PBMCs at L4- to adult stage in vivo was cloned by using specific primers based on the conserved domain sequences (CDS) of H. contortus ARF gene (GI:533372025), gene bank accession number HF964523.1. The ORF of HcARF1 was comprised of 546 bp,encodes a 181 amino acid. Predicted HcARF1 protein molecular weight was about 20 kDa. HcARF1 was predicted for 6 B and 9 T cell epitopes at multiple locations. Sequence analysis showed that it has significant homology with the known ARF1 protein. The recombinant protein of rHcARFlwas expressed in a histidine-tagged fusion protein soluble form in Escherichia coli. Binding activity of rHcARF1 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effect on cytokine secretion, cell proliferation,cell migration,nitric oxide production and apoptosis were observed by co-incubation of rHcARF1. IF A results revealed that rHcARFl could bind to the PBMCs. The interaction of rHcARF1 modulate the cytokine production, the production of IL4, IL10 and IL17 was increased in dose depended manner, however, it was significantly decreased the IFNy production. Cell migration was significantly increased by rHcARF1, whereas, rHcARF1 treatment significantly suppressed the proliferation of the PBMC in dose depended manner.Apoptosis and Nitric oxide (NO) production by PBMCs was also significantly induced by rHcARF1.6. Molecular Cloning, Expression and effects on the goat PBMCs functions cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (Hc-24)In this chapter, H. contortus cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (Hc-24) was cloned by using specific primers based on based on the H.contortus 24kDa excretory/secretory protein mRNA, gene bank accession number AY821551.1. The ORF of HcCAP-2 was comprised of 609 bp,encodes a 202 amino acid.Predicted HcCAP-2 protein molecular weight was about 24 kDa. HcCAP-2 was predicted for 11 peptides for B and 3 peptides T cell epitopes at multiple locations. Sequence analysis showed that it has significant homology with the H. contortus SCP extracellular domain containing protein (98%),H. contortus 24 kDa excretory/secretory protein (97%),H.contortus cap-2 (94%), H. contortus cap-3 (93%) and H. contortus cap-4 (78%). The recombinant protein of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (rHc-24) was expressed in a histidine-tagged fusion protein soluble form in Escherichia coli.Binding activity of rHc-24 to goat PBMCs was confirmed by immunofluorescence assay (IFA)and its immunomudulatory effect on cytokine secretion, cell proliferation, cell migration, nitric oxide production, and apoptosis were observed by co-incubation of rHc-24. IFA results revealed that rHc-24 could bind to the PBMCs. The interaction of rHc-24 modulate the cytokine production, the production of IL4, IL10 and IL17 was increased in dose depended manner, whereas, rHc-24 treatment significantly suppressed the production of IFNy,proliferation of the PBMC and Nitric oxide (NO) production in dose depended manner.Apoptosis and cell migration by PBMCs was also significantly induced by rHc-24.