Proteomic characterization of serum proteins from Atlantic salmon (Salmo salar L.) from an outbreak with cardiomyopathy syndrome.

Cardiomyopathy syndrome (CMS), caused by piscine myocarditis virus (PMCV), is a serious challenge to Atlantic salmon (Salmo salar L.) aquaculture. Regrettably, husbandry techniques are the only tool to manage CMS outbreaks, and no prophylactic measures are available at present. Early diagnosis of CMS is therefore desirable, preferably with non-lethal diagnostic methods, such as serum biomarkers. To identify candidate biomarkers for CMS, the protein content of pools of sera (4 fish/pool) from salmon with a CMS outbreak (3 pools) and from clinically healthy salmon (3 pools) was compared using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Overall, seven proteins were uniquely identified in the sera of clinically healthy fish, while 27 proteins were unique to the sera of CMS fish. Of the latter, 24 have been associated with cardiac disease in humans. These were grouped as leakage enzymes (creatine kinase, lactate dehydrogenase, glycogen phosphorylase and carbonic anhydrase); host reaction proteins (acute-phase response proteins-haptoglobin, fibrinogen, α2-macroglobulin and ceruloplasmin; and complement-related proteins); and regeneration/remodelling proteins (fibronectin, lumican and retinol). Clinical evaluation of the suitability of these proteins as biomarkers of CMS, either individually or as part of a panel, is a logical next step for the development of early diagnostic tools for CMS.

Psoroptes ovis-Early Immunoreactive Protein (Pso-EIP-1) a novel diagnostic antigen for sheep scab.

AIMS: Serodiagnosis of sheep scab is an established diagnostic method and has become popular in recent years. However, the current diagnostic antigen, Pso o 2, has shown promise as a component of a recombinant vaccine for scab, making it incompatible with discriminating between infected and vaccinated animals (DIVA). Here, we describe the discovery and characterization of a novel Psoroptes ovis immunodiagnostic antigen, P. ovis-Early Immunoreactive Protein-1 (Pso-EIP-1). METHODS AND RESULTS: Pso-EIP-1 is a highly abundant member of a six-gene family with no known homologs, indicating its potential uniqueness to P. ovis. Expression of recombinant Pso-EIP-1 (rPso-EIP-1) required a C-terminal fusion protein for stability and specific IgG immunoreactivity against rPso-EIP-1 was observed in sheep serum from 1 to 2 weeks post-infestation, indicating its highly immunogenic nature. Two of the three in silico-predicted B-cell epitopes of Pso-EIP-1 were confirmed by in vitro epitope mapping and, in a direct comparison by ELISA, Pso-EIP-1 performed to the same levels as Pso o 2 in terms of sensitivity, specificity and ability to diagnose P. ovis on sheep within 2 weeks of infestation. CONCLUSION: Pso-EIP-1 represents a novel diagnostic antigen for sheep scab with comparable levels of sensitivity and specificity to the existing Pso o 2 antigen.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Carbon Nanoparticles Inhibit the Antimicrobial Activities of the Human Cathelicidin LL-37 through Structural Alteration.

Host defense peptides, also known as antimicrobial peptides, are key elements of innate host defense. One host defense peptide with well-characterized antimicrobial activity is the human cathelicidin, LL-37. LL-37 has been shown to be upregulated at sites of infection and inflammation and is regarded as one of the primary innate defense molecules against bacterial and viral infection. Human exposure to combustion-derived or engineered nanoparticles is of increasing concern, and the implications of nanomaterial exposure on the human immune response is poorly understood. However, it is widely acknowledged that nanoparticles can interact strongly with several immune proteins of biological significance, with these interactions resulting in structural and functional changes of the proteins involved. This study investigated whether the potent antibacterial and antiviral functions of LL-37 were inhibited by exposure to, and interaction with, carbon nanoparticles, together with characterizing the nature of the interaction. LL-37 was exposed to carbon black nanoparticles in vitro, and the antibacterial and antiviral functions of the peptide were subsequently assessed. We demonstrate a substantial loss of antimicrobial function when the peptide was exposed to low concentrations of nanomaterials, and we further show that the nanomaterial-peptide interaction resulted in a significant change in the structure of the peptide. The human health implications of these findings are significant, as, to our knowledge, this is the first evidence that nanoparticles can alter host defense peptide structure and function, indicating a new role for nanoparticle exposure in increased disease susceptibility.

Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen.

Campylobacter jejuni outer membrane vesicles play an important role in bacterial interactions with human intestinal epithelial cells.

Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

Changes in protein expression in the sheep abomasum following trickle infection with Teladorsagia circumcincta.

Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.

Transcriptional changes in Teladorsagia circumcincta upon encountering host tissue of differing immune status.

The aim of this study was to elucidate transcriptional changes in the parasitic nematode Teladorsagia circumcincta upon encountering either naïve or immune ovine hosts. Pools of 100 000 exsheathed 3rd- stage T. circumcincta larvae were exposed in vitro to either an immune or naïve ovine abomasal environment, RNA was extracted from the larvae and sequenced using the Roche 454 platform. Each sample produced approximately 82 000 reads that assembled to give approximately 5500 Isotigs (contigs). The two sequence datasets were clustered together to give a total of 6969 clusters of which 18 were differentially expressed (P<0·001) between the two groups. Clusters with a predominance of reads in larvae exposed to the immune abomasal environment encoded homologues of peptidyl-glycine alpha-amidating monooxygenase, heat shock-protein 16-2 and IDA-1, a tyrosine phosphatase-like receptor protein. Clusters with a predominance of reads in the naïve environment encoded homologues of cytochrome b, EGg Laying defective family member 21 and NADH dehydrogenase subunit 5. Gene ontology analyses indicated that larvae exposed to the immune environment showed an increase in expression of genes involved in 'carbon utilization', 'response to stimulus' and 'developmental process'. These data suggest that T. circumcincta modulates gene expression in response to the immune status of the host.

Characterisation of proteins extracted from the surface of Salmonella Typhimurium grown under SPI-2-inducing conditions by LC-ESI/MS/MS sequencing.

Salmonella enterica has two pathogenicity islands encoding separate type three secretion systems (T3SS). Proteins secreted through these systems facilitate invasion and survival. After entry, Salmonella reside within a membrane bound vacuole, the Salmonella containing vacuole (SCV), where translocation of a second set of effectors by the Salmonella pathogenicity island 2 (SPI-2) T3SS is initiated. SPI-2 secretion in vitro can be induced by conditions that mimic the Salmonella containing vacuole. Utilising high-throughput mass spectrometry, we mapped the surface-attached proteome of S. Typhimurium SL1344 grown in vitro under SPI-2-inducing conditions and identified 108 proteins; using secretion signal prediction software, 43% of proteins identified contained a signal sequence. Of these proteins, 13 were known secreted effector proteins including SPI-2 effector proteins SseB, SseC, SseD, SseL, PipB2 and SteC, although surprisingly five were SPI-1 proteins, SipA, SipB, SipC, SipD and SopD, while 2 proteins SteA and SlrP are secreted by both T3SSs. This is the first in vitro study to demonstrate dual secretion of SPI-1 and SPI-2 proteins by S. Typhimurium and demonstrates the potential of high-throughput LC-ESI/MS/MS sequencing for the identification of novel proteins, providing a platform for subsequent comparative proteomic analysis, which should greatly assist understanding of the pathogenesis and inherent variation between serovars of Salmonella and ultimately help towards development of novel control strategies.

Genomic and surface proteomic analysis of the canine pathogen Staphylococcus pseudintermedius reveals proteins that mediate adherence to the extracellular matrix.

Cell wall-associated (CWA) proteins made by Gram-positive pathogens play a fundamental role in pathogenesis. Staphylococcus pseudintermedius is a major animal pathogen responsible for the canine skin disease bacterial pyoderma. Here, we describe the bioinformatic analysis of the family of 18 predicted CWA proteins encoded in the genome of S. pseudintermedius strain ED99 and determine their distribution among a phylogenetically diverse panel of S. pseudintermedius clinical isolates and closely related species of the Staphylococcus intermedius group. In parallel, we employed a proteomic approach to identify proteins presented on the surface of strain ED99 in vitro, revealing a total of 60 surface-localized proteins in one or more phases of growth, including 6 of the 18 genome-predicted CWA proteins. Based on these analyses, we selected two CWA proteins (SpsD and SpsL) encoded by all strains examined and investigated their capacity to mediate adherence to extracellular matrix proteins. We discovered that SpsD and SpsL mediated binding of a heterologous host, Lactococcus lactis, to fibrinogen and fibronectin and that SpsD mediated binding to cytokeratin 10, a major constituent of mammalian skin. Of note, the interaction with fibrinogen was host-species dependent, suggestive of a role for SpsD and SpsL in the host tropism of S. pseudintermedius. Finally, we identified IgG specific for SpsD and SpsL in sera from dogs with bacterial pyoderma, implying that both proteins are expressed during infection. The combined genomic and proteomic approach employed in the current study has revealed novel host-pathogen interactions which represent candidate therapeutic targets for the control of bacterial pyoderma.

Identification, sequencing and molecular analysis of Chp4, a novel chlamydiaphage of Chlamydophila abortus belonging to the family Microviridae.

Members of the family Microviridae have been identified in a number of chlamydial species infecting humans (phage CPAR39 in Chlamydophila pneumoniae), other mammals (φCPG1 in Chlamydophila caviae, Chp2 in Chlamydophila abortus and Chp3 in Chlamydophila pecorum) and birds (Chp1 in Chlamydophila psittaci). This study describes the identification and genome sequencing of Chp4, an icosahedral, 4530 bp, ssDNA phage in C. abortus. Chp4 is predicted to contain eight ORFs, six of which could be assigned putative functions based on sequence similarity to characterized bacteriophage. Gene order and content were highly conserved amongst chlamydiaphage, with the highest sequence variability occurring in the IN5 and INS variable regions of the VP1 major coat protein, which has been associated with host cell recognition and binding. Phylogenetic analysis of VP1 indicated that Chp4 is a member of the Chlamydiamicrovirus, and is most closely related to phage φCPG1 and CPAR39.

Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches.

Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4(+ve), CD8(+ve), γ/δ TCR(+ve) T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4(+ve) T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4(+ve) T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4(+ve) cells) and the subsequent identification of the stimulating components using tandem mass spectrometry.

Cysteamine Inhibits Glycine Utilisation and Disrupts Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major opportunistic human pathogen which employs a myriad of virulence factors. In people with cystic fibrosis (CF) P. aeruginosa frequently colonises the lungs and becomes a chronic infection that evolves to become less virulent over time, but often adapts to favour persistence in the host with alginate-producing mucoid, slow-growing, and antibiotic resistant phenotypes emerging. Cysteamine is an endogenous aminothiol which has been shown to prevent biofilm formation, reduce phenazine production, and potentiate antibiotic activity against P. aeruginosa, and has been investigated in clinical trials as an adjunct therapy for pulmonary exacerbations of CF. Here we demonstrate (for the first time in a prokaryote) that cysteamine prevents glycine utilisation by P. aeruginosa in common with previously reported activity blocking the glycine cleavage system in human cells. Despite the clear inhibition of glycine metabolism, cysteamine also inhibits hydrogen cyanide (HCN) production by P. aeruginosa, suggesting a direct interference in the regulation of virulence factor synthesis. Cysteamine impaired chemotaxis, lowered pyocyanin, pyoverdine and exopolysaccharide production, and reduced the toxicity of P. aeruginosa secreted factors in a Galleria mellonella infection model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa, further supporting its therapeutic potential in CF and other infections.

Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection.

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE-K mouse small intestinal epithelial cells were protected from histone-induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone-interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone-induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross-linking properties. It was particularly notable that inter-alpha-trypsin inhibitor was prominent among the histone-precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Identification of proteins from the secretory/excretory products (SEPs) of the branchiuran ectoparasite Argulus foliaceus (Linnaeus, 1758) reveals unique secreted proteins amongst haematophagous ecdysozoa.

BACKGROUND: It is hypothesised that being a blood-feeding ectoparasite, Argulus foliaceus (Linnaeus, 1758), uses similar mechanisms for digestion and host immune evasion to those used by other haematophagous ecdysozoa, including caligid copepods (e.g. sea louse). We recently described and characterised glands associated with the feeding appendages of A. foliaceus using histological techniques. The work described in the present study is the first undertaken with the objective of identifying and partially characterising the components secreted from these glands using a proteomic approach. METHODS: Argulus foliaceus parasites were sampled from the skin of rainbow trout (Oncorhynchus mykiss), from Loch Fad on the Isle of Bute, Scotland, UK. The proteins from A. foliaceus secretory/excretory products (SEPs) were collected from the supernatant of artificial freshwater conditioned with active adult parasites (n = 5-9 per ml; n = 560 total). Proteins within the SEPs were identified and characterised using LC-ESI-MS/MS analysis. Data are available via ProteomeXchange with identifier PXD016226. RESULTS: Data mining of a protein database translated from an A. foliaceus dataset using ProteinScape allowed identification of 27 predicted protein sequences from the A. foliaceus SEPs, each protein matching the criteria of 2 peptides with at least 4 contiguous amino acids. Nine proteins had no matching sequence through OmicsBox (Blast2GO) analysis searches suggesting that Argulus spp. may additionally have unique proteins present in their SEPs. SignalP 5.0 software, identified 13 proteins with a signal sequence suggestive of signal peptides and supportive of secreted proteins being identified. Notably, the functional characteristics of identified A. foliaceus proteins/domains have also been described from the salivary glands and saliva of other blood-feeding arthropods such as ticks. Identified proteins included: transporters, peroxidases, metalloproteases, proteases and serine protease inhibitors which are known to play roles in parasite immune evasion/induction (e.g. astacin), immunomodulation (e.g. serpin) and digestion (e.g. trypsin). CONCLUSIONS: To our knowledge, the present study represents the first proteomic analysis undertaken for SEPs from any branchiuran fish louse. Here we reveal possible functional roles of A. foliaceus SEPs in digestion and immunomodulation, with a number of protein families shared with other haematophagous ectoparasites. A number of apparently unique secreted proteins were identified compared to other haematophagous ecdysozoa.

Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning.

The performances of five different ESI sources coupled to a polystyrene-divinylbenzene monolithic column were compared in a series of LC-ESI-MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low-flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest-quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures -- arguably due to an increased number of high intensity precursor ion candidates.

Proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell.

Proteomic characterisation of the Chlamydia abortus outer membrane complex (COMC) using combined rapid monolithic column liquid chromatography and fast MS/MS scanning.

Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)-the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.

Characterisation of a niche-specific excretory-secretory peroxiredoxin from the parasitic nematode Teladorsagia circumcincta.

BACKGROUND: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). RESULTS: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. CONCLUSIONS: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.

The proteome of gastric lymph in normal and nematode infected sheep.

Lymph node cannulation allows the collection of lymph draining from a defined anatomical region. Proteomic analysis of that lymph offers a potentially valuable insight into the immunoinflammatory response of that particular region. In this study, ovine gastric lymph has been used to monitor the proteomic changes occurring in the tissue fluid of the abomasum, in response to infection with the parasitic nematode, Teladorsagia circumcincta. Lymph, collected temporally over an experimental infection period, was analysed by means of 2-DE and subsequent gel analysis using densitometry software. In addition, the composition of the lymphatic proteome was further explored by means of MALDI-TOF and MS/MS analyses. The concentration of gelsolin, alpha-1 beta glycoprotein and haemopexin were altered significantly (p<0.05) with infection.