The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with salmonid alphavirus subtype 3 (SAV3).

Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, with most outbreaks in Norway caused by SAV subtype 3 (SAV3). This atypical alphavirus is transmitted horizontally causing a significant economic impact on the aquaculture industry. This histopathological and proteomic study, using an established cohabitational experimental model, investigated the correlation between tissue damage during PD and a number of serum proteins associated with these pathologies in Atlantic salmon. The proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting. A number of humoral components of immunity which may act as biomarkers of the disease were also identified. For example, creatine kinase, enolase and malate dehydrogenase serum concentrations were shown to correlate with pathology during PD. In contrast, hemopexin, transferrin, and apolipoprotein, amongst others, altered during later stages of the disease and did not correlate with tissue pathologies. This approach has given new insight into not only PD but also fish disease as a whole, by characterisation of the protein response to infection, through pathological processes to tissue recovery. BIOLOGICAL SIGNIFICANCE: Salmonid alphavirus causes pancreas disease (PD) in Atlantic salmon, Salmo salar, and has a major economic impact on the aquaculture industry. A proteomic investigation of the change to the serum proteome during PD has been made with an established experimental model of the disease. Serum proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting with 72 protein spots being shown to alter significantly over the 12week period of the infection. The concentrations of certain proteins in serum such as creatine kinase, enolase and malate dehydrogenase were shown to correlate with tissue pathology while other proteins such as hemopexin, transferrin, and apolipoprotein, altered in concentration during later stages of the disease and did not correlate with tissue pathologies. The protein response to infection may be used to monitor disease progression and enhance understanding of the pathology of PD.

Changes in the serum proteome of canine lymphoma identified by electrophoresis and mass spectrometry.

The serum proteome of canine lymphoma was characterised by one dimensional (1D) serum protein electrophoresis (SPE) on agarose gels, two dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and tandem mass spectrometry (MS). Results were compared with serum proteome data collected previously from the sera of healthy dogs. Twenty-one dogs with high grade multicentric lymphoma had significantly elevated quantities of α2 globulins on 1D SPE. Further separation of the serum proteins was performed on three dogs using a 2D PAGE system. Thirty-six different proteins were identified in 38 bands submitted for MS. Most of the proteins were the same as those previously identified in the sera of healthy dogs. Haptoglobin was identified in the sera of all three dogs with lymphoma and could account for the increased levels of α2 globulins. α2 Macroglobulin, α-antichymotrypsin and inter-α-trypsin inhibitor were also present in dogs with lymphoma. Clusterin, an anti-apoptotic protein, was identified in the serum of one dog with lymphoma. Kininogen, which is present in the sera of healthy dogs, was absent in all three dogs with lymphoma. The 2D electrophoresis technique identified alterations in the serum proteome of dogs with lymphoma and supported previous findings that canine lymphoma has an inflammatory component.

Characterisation of the normal canine serum proteome using a novel electrophoretic technique combined with mass spectrometry.

One dimensional (1D) serum protein electrophoresis (SPE) on agarose gels is a frequently used diagnostic tool for canine diseases; however, little is known regarding the precise composition of the different protein fractions in normal or diseased animals. In this study, to analyse the canine serum proteome in more detail, conventional 1D SPE was combined with second dimension (2D) polyacrylamide gel electrophoresis (PAGE), followed by tandem mass spectrometry (MS). One dimensional SPE was performed on the sera of 17 healthy dogs to establish normal reference ranges for the albumin and globulin sub-fractions. Two representative serum samples from healthy dogs were further separated using a novel method of 2D PAGE, leading to the generation of 26 distinct bands across the six main sub-fractions, which were subjected to MS analysis. Thirty-two proteins were identified, most of which were found in both dogs. Twenty proteins belonged specifically to the species Canis lupus familiaris, with the remaining 12 proteins belonging to other mammalian species, likely reflecting incomplete sequencing knowledge of canine proteins. Two dimensional electrophoresis and MS allowed identification of canine serum albumin precursor, serpin peptidase inhibitor, kininogen-1, vitamin D binding protein, haemopexin, complement C4 and a variety of immunoglobulin class molecules, along with localisation of these proteins within serum protein subfractions.

Metabolomic profiling using Orbitrap Fourier transform mass spectrometry with hydrophilic interaction chromatography: a method with wide applicability to analysis of biomolecules.

It was shown that coupling hydrophilic interaction chromatography (HILIC) to Orbitrap Fourier transform mass spectrometery (FT-MS) provided an excellent tool for metabolic profiling, principally due to rapid elution of lipids in advance of most metabolites entering the mass spectrometer. We used in vitro cultivated procyclic forms of the protozoan parasite Trypanosoma brucei as a source of metabolites to test the performance of the HILIC column and the mass accuracy of MS. The mass accuracy achieved fell within 2 ppm for all the metabolites identified within samples. It was, for example, possible to identify the signature metabolite of the trypanosome, trypanothione, and also glutathione which were well retained by the HILIC column. By comparing trypanosomes grown in two different media we were able to clearly distinguish the samples in terms of the relative abundance of a number of metabolites using Sieve 1.1 software.

Sleeping sickness and the brain.

Recent progress in understanding the neuropathological mechanisms of sleeping sickness reveals a complex relationship between the trypanosome parasite that causes this disease and the host nervous system. The pathology of late-stage sleeping sickness, in which the central nervous system is involved, is complicated and is associated with disturbances in the circadian rhythm of sleep. The blood-brain barrier, which separates circulating blood from the central nervous system, regulates the flow of materials to and from the brain. During the course of disease, the integrity of the blood-brain barrier is compromised. Dysfunction of the nervous system may be exacerbated by factors of trypanosomal origin or by host responses to parasites. Microscopic examination of cerebrospinal fluid remains the best way to confirm late-stage sleeping sickness, but this necessitates a risky lumbar puncture. Most drugs, including many trypanocides, do not cross the blood-brain barrier efficiently. Improved diagnostic and therapeutic approaches are thus urgently required. The latter might benefit from approaches which manipulate the blood-brain barrier to enhance permeability or to limit drug efflux. This review summarizes our current understanding of the neurological aspects of sleeping sickness, and envisages new research into blood-brain barrier models that are necessary to understand the interactions between trypanosomes and drugs active against them within the host nervous system.

Life in vacuoles--nutrient acquisition by Leishmania amastigotes.

Leishmania have a digenetic life cycle, involving a motile, extracellular stage (promastigote) which parasitises the alimentary tract of a sandfly vector. Bloodfeeding activity by an infected sandfly can result in transmission of infective (metacyclic) promastigotes to mammalian hosts, including humans. Leishmania promastigotes are rapidly phagocytosed but may survive and transform into non-motile amastigote forms which can persist as intracellular parasites. Leishmania amastigotes multiply in an acidic intracellular compartment, the parasitophorous vacuole. pH plays a central role in the developmental switch between promastigote and amastigote stages, and amastigotes are metabolically most active when their environment is acidic, although the cytoplasm of the amastigote is regulated at near-neutral pH by an active process of proton extrusion. A steep proton gradient is thus maintained across the amastigote surface and all membrane processes must be adapted to function under these conditions. Amastigote uptake systems for glucose, amino acids, nucleosides and polyamines are optimally active at acidic pH. Promastigote uptake systems are kinetically distinct and function optimally at more neutral environmental pH, indicating that membrane transport activity is developmentally regulated. The nutrient environment encountered by amastigotes is not well understood but the parasitophorous vacuole can fuse with endosomes, phagosomes and autophagosomes, suggesting that a diverse range of macromolecules will be present. The parasitophorous vacuole is a hydrolytic compartment in which such material will be rapidly degraded to low molecular weight components which are typical substrates for membrane transporters. Amastigote surface transporters must compete for these substrates with equivalent host transporters in the membrane of the parasitophorous vacuole. The elaboration of accumulative transporters with high affinity will be beneficial to amastigotes in this environment. The influence of environmental pH on membrane transporter function is discussed, with emphasis on the potential role of a transmembrane proton gradient in active, high affinity transport.

Hexose transport in asexual stages of Plasmodium falciparum and kinetoplastidae.

The hexose sugar, glucose, is a vital energy source for most organisms and an essential nutrient for asexual stages of Plasmodium falciparum. Kinetoplastid organisms (e.g. Trypanosoma and Leishmania spp) also require glucose at certain critical stages of their life cycles. Although phylogenetically unrelated, these organisms share many common challenges during the mammalian stages of a parasitic life cycle, and possess hexose uptake mechanisms that are amenable to study using similar methods. Defining hexose permeation pathways into parasites might expose an Achilles' heel at which both antidisease and antiparasite measures can be aimed. Understanding the mode of entry of glucose also presents a good general model for substrate acquisition in multicompartment systems. In this review, Sanjeev Krishna and colleagues summarize current understanding of hexose transport processes in P. falciparum and provide a comparison with data obtained from kinetoplastids.

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, K(m) approximately 1.0 mM) also transports fructose (K(m) approximately 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.

Differential regulation of multiple glucose transporter genes in Leishmania mexicana.

We have studied the structure and expression of glucose transporter genes in the parasitic protozoan Leishmania mexicana. Three distinct glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, are encoded by single copy genes that are clustered together at a single locus. Quantitation of Northern blots reveals that LmGT2 mRNA is present at approximately 15-fold higher level in promastigotes, the insect stage of the parasite life cycle, compared with amastigotes, the intracellular stage of the life cycle that lives within the mammalian host. In contrast, LmGT1 and LmGT3 mRNAs are expressed at similar levels in both life cycle stages. Transcription of the LmGT genes in promastigotes and axenically cultured amastigotes occurs at similar levels, as measured by nuclear run-on transcription. Consequently, the approximately 15-fold up-regulation of LmGT2 mRNA levels in promastigotes compared with amastigotes must be controlled at the post-transcriptional level. Measurement of LmGT2 RNA decay in promastigotes and axenic amastigotes treated with actinomycin D suggests that differential mRNA stability may play a role in regulating glucose transporter mRNA levels in the two life cycle stages.

Glucose transport in amastigotes and promastigotes of Leishmania mexicana mexicana.

Promastigotes and amastigotes of Leishmania mexicana mexicana transported 2-deoxy-D-glucose (2-DOG) by a saturable process with a Km of 24 +/- 3 microM and Vmax of 2.21 nmol min-1 (mg protein)-1 for the promastigote and a Km of 29 +/- 8 microM and Vmax of 0.13 nmol min-1 (mg protein)-1 for the amastigote stage. Amastigotes incorporated 2-DOG maximally at pH 5.0, while for promastigotes the optimum was at pH 7.0. Mid-log phase promastigotes were found to accumulate 2-DOG via a stereospecific carrier-mediated process which was competitively inhibited by D-glucose and D-mannose but not L-glucose. Transport was dependent upon temperature, with a Q10 in promastigotes of 1.83 and an optimum rate at 35 degrees C (+/- 4 degrees C) with an activation energy of 50.12 kJ mol-1. Stationary phase promastigotes accumulated 2-DOG at approximately twice the rate of mid-log phase promastigotes. Cytochalasin B, forskolin and phloretin were all found to inhibit human erythrocyte 2-DOG uptake but only cytochalasin B was found significantly to inhibit promastigote 2-DOG uptake. Interestingly, leishmanial 2-DOG uptake was inhibited by a series of membrane potential antagonists including the ionophore monensin, the H+ATPase inhibitor N, N'-dicyclohexylcarbodiimide (DCCD) and uncoupling agent carbonylcyanide-4-(triflouromethoxy) phenylhydrazone (FCCP), as well as, the tricyclic drugs chlomipramine and imipramine, but was insensitive to the Na+/K+ATPase inhibitor ouabain and the antitrypanosomal drugs Pentostam and Suramin. We therefore conclude that there are significant structural and mechanistic differences between the D-glucose uptake systems of Leishmania and the mammalian host to merit the inclusion of glucose transporters as putative targets for rational drug design.

Biochemistry and molecular genetics of Leishmania glucose transporters.

Glucose is utilized as a significant source of metabolic energy by Leishmania parasites. This sugar is accumulated by the parasite via a specific carrier-mediated transport system located in the parasite membrane. Parasites may also contain another transporter that shuttles glucose between the cytoplasm and the glycosome, a membrane-bound organelle where the early steps of glycolysis occur. The transport systems of both the insect stage promastigotes and the intracellular amastigotes have been characterized and shown to have kinetic properties that are consistent with the different physiological environments of the insect gut and the macrophage phagolysosome. Several genes have been cloned from Leishmania species which encode proteins with substantial sequence similarity to glucose transporters from mammals and lower eukaryotes. Two of these genes are expressed preferentially in the promastigote stage of the life cycle, where glucose is more readily available and more rapidly transported and metabolized than in the intracellular amastigotes. One of these two developmentally-regulated genes has been functionally expressed in Xenopus oocytes and shown to encode a glucose transporter. A third gene encodes a protein that is also a member of the glucose transporter family on the basis of sequence similarity and proposed secondary structure. However, the significant differences between this protein and the other two suggest that it is likely to transport a different substrate. Functional expression will be required to define the specific biochemical role of each gene within the parasite.