Search

Nine geographically diverse Moniliophthora perniciosa (witches' broom disease pathogen) isolates were cultured in vitro. They included six C-biotypes differing in virulence on cacao (Theobroma cacao), two S-biotypes (solanaceous hosts), and an L-biotype (liana hosts). Mycelial growth rates and morphologies differed considerably, but no characters were observed to correlate with virulence or biotype. In plant inoculations using basidiospores, one C-biotype caused symptoms on tomato (an S-biotype host), adding to evidence of limited host adaptation in these biotypes. Mycelial proteomes were analysed by two-dimensional gel electrophoresis (2-DE), and 619 gel spots were indexed on all replicate gels of at least one strain. Multivariate analysis of gel spots discriminated the L-biotype, but not the S-biotypes, from the remaining strains. The proteomic similarity of the S- and C-biotypes is consistent with their reported lack of phylogenetic distinction. Sequences from tandem mass spectrometry of tryptic peptides from major 2-DE spots were matched with Moniliophthora genome and transcript sequences on NCBI and WBD Transcriptome Atlas databases. Protein-spot identifications indicated that M. perniciosa saprotrophic mycelial proteomes expressed functions potentially connected with a 'virulence life-style', including peroxiredoxin, heat-shock proteins, nitrilase, formate dehydrogenase, a prominent complement of aldo-keto reductases, mannitol-1-phosphate dehydrogenase, and central metabolism enzymes with proposed pathogenesis functions., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Increasing the rumen-stable protein content of feed would lead to improved nitrogen utilisation in cattle, and less nitrogenous waste. Red clover (Trifolium pratense L.) is a high protein ruminant feed containing high polyphenol oxidase (PPO) activity. PPO mediated protein-quinone binding has been linked to protecting plant proteins from proteolysis. To explore the mechanism underlying the effect of PPO on protein protection in fresh forage feeds, proteomic components of feed down-boli produced from wild-type red clover and a low PPO mutant, at point of ingestion and after 4h in vitro incubation with rumen inoculum were analysed. Significant differences in proteomic profiles between wild-type and mutant red clover were determined after 4h incubation, with over 50% less spots in mutant than wild-type proteomes, indicating decreased proteolysis in the latter. Protein identifications revealed preferentially retained proteins localised within the chloroplast, suggesting that PPO mediated protection in the wild-type operates due to the proximity of target proteins to the enzyme and substrates, either diffusing into this compartment from the vacuole or are present in the chloroplast. This increased understanding of protein targets of PPO indicates that wider exploitation of the trait could contribute to increased protein use efficiency in grazing cattle., Biological Significance: One of the main challenges for sustainable livestock farming is improving capture of dietary nitrogen by ruminants. Typically up to 70% of ingested protein-N is excreted representing a loss of productivity potential and a serious environmental problem in terms of nitrogenous pollution of lands and water. Identification of key characteristics of rumen-protected protein will deliver target traits for selection in forage breeding programmes. The chloroplastic enzyme PPO catalyzes the oxidation of phenols to quinones, which react with protein. Little is currently known about the intracellular protein targets of the products of PPO activity or the mechanism underlying protein complexing, including whether there is any specificity to the reaction. Here we have determined significant differences in the proteomes of freshly ingested down boli corresponding to the presence or absence of active PPO. These results show that in the presence of PPO the forage protein is less amenable to proteolysis and provide the novel information that the protected proteins are putatively chloroplastically located. These data also contribute to a growing evidence base that a chloroplastic PPO substrate exists in red clover in addition to the currently known vacuolar substrates., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for over 50 years. However, the continued development of anthelmintic resistance (AR) threatens this control. Measuring early AR is difficult as there many routes that resistance can arise from within multi-nematode populations operating complex metabolism capabilities coupled to different drug management pressures. There is an urgent need to identify and measure early resistance in the field situation. Proteomic profiling of expressed soluble proteins offers a new approach to reveal a drug resistant phenotype within a complex protein pattern. The hypothesis under test was that established differences in drug response phenotypes between nematode isolates can also be measured in their comparative proteomes. As a case study, proteomic differences were measured between an ivermectin resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females were extracted from the abomasa of six lambs. The nematodes had been maintained in the lambs as monospecific isolates of either ivermectin susceptible or ivermectin resistant worms. Comparative analysis of the soluble proteome was completed along with immuno-proteomic analysis using pooled infection sera from the lambs. Following image analysis, spots of interest were excised and analysed by peptide mass fingerprinting and the proteins putatively identified using BLAST. Overall, a relative increase in the expression of proteins involved in the detoxification metabolic area was observed in the resistant isolate. In addition, Western blotting analysis also revealed differences in immuno-reactivity profiles between resistant and susceptible isolates. It can be concluded from this study that proteomic differences can be detectable between ivermectin susceptible and a resistant isolates of H. contortus, which could be further explored using other isolates to confirm if proteomic based fingerprinting offers molecular phenotyping or a new panel of resistance biomarkers., (Copyright © 2012 Elsevier B.V. All rights reserved.)