Global Gene Expression Profiling through the Complete Life Cycle of Trypanosoma vivax

The parasitic flagellate Trypanosoma vivax is a cause of animal trypanosomiasis across Africa and South America. The parasite has a digenetic life cycle, passing between mammalian hosts and insect vectors, and a series of developmental forms adapted to each life cycle stage. Each point in the life cycle presents radically different challenges to parasite metabolism and physiology and distinct host interactions requiring remodeling of the parasite cell surface. Transcriptomic and proteomic studies of the related parasites T. brucei and T. congolense have shown how gene expression is regulated during their development. New methods for in vitro culture of the T. vivax insect stages have allowed us to describe global gene expression throughout the complete T. vivax life cycle for the first time. We combined transcriptomic and proteomic analysis of each life stage using RNA-seq and mass spectrometry respectively, to identify genes with patterns of preferential transcription or expression. While T. vivax conforms to a pattern of highly conserved gene expression found in other African trypanosomes, (e.g. developmental regulation of energy metabolism, restricted expression of a dominant variant antigen, and expression of 'Fam50' proteins in the insect mouthparts), we identified significant differences in gene expression affecting metabolism in the fly and a suite of T. vivax-specific genes with predicted cell-surface expression that are preferentially expressed in the mammal ('Fam29, 30, 42') or the vector ('Fam34, 35, 43'). T. vivax differs significantly from other African trypanosomes in the developmentally-regulated proteins likely to be expressed on its cell surface and thus, in the structure of the host-parasite interface. These unique features may yet explain the species differences in life cycle and could, in the form of bloodstream-stage proteins that do not undergo antigenic variation, provide targets for therapy

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix.

BACKGROUND Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism. METHODS PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS. RESULTS Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM. CONCLUSION To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

Selection on running capacity has created rat phenotypes of high‐capacity runners (HCRs) that have enhanced cardiac function and low‐capacity runners (LCRs) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCRs and LCRs from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCRs was six‐fold greater than LCRs. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant ( p <0.05; false discovery rate <10%, calculated using q ‐values) differences between HCRs and LCRs. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the β‐oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCRs also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of α B‐crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus, proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86916/1/3369_ftp.pd

The proteome of the infectious bronchitis virus Beau-R Virion

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process

Proposal for a Standard Representation of Two-Dimensional Gel Electrophoresis Data

The global analysis of proteins is now feasible due to improvements in techniques such as two-dimensional gel electrophoresis (2-DE), mass spectrometry, yeast two-hybrid systems and the development of bioinformatics applications. The experiments form the basis of proteomics, and present significant challenges in data analysis, storage and querying. We argue that a standard format for proteome data is required to enable the storage, exchange and subsequent re-analysis of large datasets. We describe the criteria that must be met for the development of a standard for proteomics. We have developed a model to represent data from 2-DE experiments, including difference gel electrophoresis along with image analysis and statistical analysis across multiple gels. This part of proteomics analysis is not represented in current proposals for proteomics standards. We are working with the Proteomics Standards Initiative to develop a model encompassing biological sample origin, experimental protocols, a number of separation techniques and mass spectrometry. The standard format will facilitate the development of central repositories of data, enabling results to be verified or re-analysed, and the correlation of results produced by different research groups using a variety of laboratory techniques

Can policy be risk-based? The cultural theory of risk and the case of livestock disease containment

This article explores the nature of calls for risk-based policy present in expert discourse from a cultural theory perspective. Semi-structured interviews with professionals engaged in the research and management of livestock disease control provide the data for a reading proposing that the real basis of policy relating to socio-technical hazards is deeply political and cannot be purified through ‘escape routes’ to objectivity. Scientists and risk managers are shown calling, on the one hand, for risk-based policy approaches while on the other acknowledging a range of policy drivers outside the scope of conventional quantitative risk analysis including group interests, eventualities such as outbreaks, historical antecedents, emergent scientific advances and other contingencies. Calls for risk-based policy are presented, following cultural theory, as ideals connected to a reductionist epistemology and serving particular professional interests over others rather than as realistic proposals for a paradigm shift

An open-format enteroid culture system for interrogation of interactions between Toxoplasma gondii and the intestinal epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection