<Emphasis Type="Italic">In silico</Emphasis> identification of essential proteins in <Emphasis Type="Italic">Corynebacterium pseudotuberculosis</Emphasis> based on protein-protein interaction networks

Background

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis.

Results

Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous.

Conclusions

The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

     

Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.     

Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation

BackgroundLeishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4⁺ and CD8⁺ T cell Line 1Lineepitopes. Identification of CD8⁺ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.ConclusionOur results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8⁺ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.     

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.     

A Role for the M9 Transport Signal of hnRNP A1 in mRNA Nuclear Export

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.The transport of macromolecules between the nucleus and cytoplasm is a bi-directional process. The best understood aspect is the import of nuclear proteins that carry a basic nuclear localization signal (NLS)¹ like the simple NLS found in SV-40 T antigen or the bipartite NLS found in nucleoplasmin (Dingwall and Laskey, 1991). Proteins of this class are recognized by the heterodimeric importin receptor, composed of importin α and importin β (for review see Powers and Forbes, 1994; Melchior and Gerace, 1995; Görlich and Mattaj, 1996). The NLS binds directly to the importin α subunit. The importin NLS protein complex docks at the cytoplasmic face of the nuclear pore complex in an energy-independent manner (Newmeyer and Forbes, 1988; Richardson et al., 1988). Subsequently, the small GTPase Ran/TC4 (Melchior et al., 1993; Moore and Blobel, 1993) and a protein of unknown function named variously pp15, p10, or NTF2 (Moore and Blobel, 1994; Paschal and Gerace, 1995) are required for translocation of the NLS-containing complex through the nuclear pore complex.A second major class of imported macromolecules are the uracil rich small nuclear (U sn) RNPs. They do not have a basic NLS but instead have a bipartite nuclear targeting signal. This is composed of an essential signal formed when the Sm core proteins bind to the U snRNA and an additional signal, the trimethyl-guanosine (m₃G) cap, which depending on the cell type or the U snRNA is either essential or required for optimal U snRNP import efficiency (Fischer and Lührmann, 1990; Hamm et al., 1990; Fischer et al., 1993). Kinetic competition experiments have supported the conclusion that U snRNPs require different limiting factors than do NLS-containing proteins for their import and that U snRNPs do not bind to importin α (Fischer et al., 1991, 1993; Michaud and Goldfarb, 1991; van Zee et al., 1993). There is also preliminary evidence that additional different receptors may be required for the nuclear uptake of other RNA species (Michaud and Goldfarb, 1992).Similarly, RNA export from the nucleus relies on recognition of the RNA or RNP export substrates by saturable factors (Zasloff, 1983; Bataillé et al., 1990; Jarmolowski et al., 1994). As for import, evidence for the existence of RNA class-specific export receptors has been obtained from kinetic competition experiments (Jarmolowski et al., 1994). Two RNA-binding proteins have been directly shown to function in RNA export, a nuclear cap binding protein complex in the case of U snRNAs (Izaurralde et al., 1995a ) and the HIV-1 Rev protein in the case of RNAs containing a rev response element (Fischer et al., 1994, 1995). In the case of mRNAs, the best candidates for export mediators are the heterogeneous nuclear (hn) RNP proteins (for review see Piñol-Roma and Dreyfuss, 1993; Izaurralde and Mattaj, 1995).About 20 different hnRNP proteins have been characterized in vertebrate cells (for review see Dreyfuss et al., 1993). The association of hnRNP proteins with mRNA in the nucleus and the cytoplasm suggests that they may regulate and/or facilitate different aspects of gene expression. The possibility that hnRNP proteins might be directly involved in the nucleocytoplasmic trafficking of mRNA molecules was suggested by the observation that several hnRNP proteins, including A1, A2, D, E, I, and K shuttle continuously and rapidly between the nucleus and the cytoplasm and are associated with mRNA in both compartments (Piñol-Roma and Dreyfuss 1992, 1993; Michael et al., 1995a , b). Of these, the best studied example is hnRNP A1. An A1-like hnRNP protein has been shown by immunoelectron microscopy to be associated with a specific mRNA in transit to the cytoplasm through the nuclear pore complex in the insect Chironomus tentans (Visa et al., 1996a ). In mammalian cells, the amount of A1 which is in constant flux between nucleus and cytoplasm is striking. It has been estimated that at least 120,000 molecules of A1 are exported to the cytoplasm per minute but then rapidly reimported such that the steady state localization of A1 is nuclear (Michael et al., 1995a ). Taken together, these results suggest that A1 and other shuttling hnRNP proteins such as A2, D, E, I, and K could play a significant role in the transport of mRNA from the nucleus to the cytoplasm.One key in understanding how hnRNPs may facilitate mRNA export is to determine the signals that mediate their shuttling, i.e., their import into and exit from the nucleus. The nucleocytoplasmic transport of A1 has been recently studied in detail, and the signals that mediate shuttling have been identified (Michael et al., 1995b ; Siomi and Dreyfuss, 1995; Weighardt et al., 1995). Nuclear import of A1 is determined by a 38-amino acid sequence, termed M9, located near the COOH terminus of the protein between amino acids 268 and 305. Its fusion to cytoplasmic reporter proteins such as pyruvate kinase resulted in rapid import of the fusion protein into the nucleus (Siomi and Dreyfuss, 1995). However, the A1 NLS has no sequence similarity to classical protein NLSs such as that of SV-40 large T antigen or nucleoplasmin (Siomi and Dreyfuss, 1995).Surprisingly, M9 also acts as a nuclear export signal (NES). In heterokaryon shuttling assays this domain is necessary and sufficient to allow the export of heterologous proteins, such as the nucleoplasmin core domain (NPLc), which are normally retained in the nucleus (Michael et al., 1995b ). Thus, M9 alone can account for the shuttling of A1. Other hnRNPs such as A2 and B1 bear sequences with striking similarities to M9 (Siomi and Dreyfuss, 1995). Mutagenesis experiments indicate that the NES and NLS activities of M9 are either identical or overlapping as mutants which block M9 NLS activity also abolish NES activity (Michael et al., 1995b ). It is therefore possible that M9 is recognized in the nucleus and the cytoplasm by the same receptor.The second category of NES described was first found in the HIV-1 Rev protein and the inhibitor of protein kinase A (Fischer et al., 1995; Wen et al., 1995; Bogerd et al., 1996; for review see Gerace, 1995). These short, leucine-rich NES sequences bear no relationship to the primary sequence of M9. Furthermore, saturation of the export factor recognized by the Rev NES has no effect on mRNA export (Fischer et al., 1995). A model for mRNA export has been postulated on the basis of the hnRNP data described above. In this model, NES/NLS containing hnRNPs bind in the nucleus to mRNA molecules and deliver them, via the export pathway they access, to the cytoplasm. In the cytoplasm these hnRNP proteins dissociate from the mRNA and return to the nucleus. To further test this model we have analyzed the transport of hnRNP A1 and mRNA in Xenopus laevis oocytes. The oocyte offers a unique opportunity to manipulate specific import or export pathways, like that accessed by M9, and examine the effect on mRNA nuclear export. By using this approach we show here that M9 is, as in somatic cells, a functional NLS in oocytes. Moreover, competition studies indicate that M9 defines a novel class of NLS, since saturation of the M9- mediated import pathway does not interfere with the two previously identified import pathways used by classical NLS-bearing proteins or m₃G-capped-spliceosomal U snRNPs. Injection of an excess of hnRNP A1 but not of a mutant form of the protein lacking the M9 domain, resulted in a specific inhibition of mRNA export, demonstrating that the M9 domain is recognized by a saturable component of the mRNA export machinery. The export of other cellular RNAs such as U snRNAs and tRNA was, in contrast, not affected. Further analysis of mutant hnRNP A1 proteins provides evidence that M9 recognition during mRNA export differs from its recognition during protein transport.     

Recycling of a glycosylphosphatidylinositol-anchored heparan sulphate proteoglycan (glypican) in skin fibroblasts

We have used suramin and brefeldin A to investigate the natureof a heparan sulphate proteoglycan that appears to recycle fromthe cell surface to intracellular compartments which synthesizenew heparan sulphate chains. Suramin, which would block internalizationand deglycanation of a putative recycling cell surface proteoglycan,markedly increases the yield of a membrane-bound proteoglycanwith a core protein of 60–70 kDa and unusually long heparansulphate side chains. When transport of newly made core proteinsto their Golgi sites for glycosaminoglycan assembly is blocked,by using brefeldin A, [³H]glucosamine and [³⁵S]sulphate incorporationinto cell surface-bound heparan sulphate proteoglycan can stilltake place. After chemical biotinylation of cell surface proteinsin brefeldin A-treated cells, followed by metabolic [³⁵S]sulphationin the presence of the same drug, biotin-tagged [³⁵S]proteoglycancan be demonstrated, indicating the presence of recycling proteoglycanspecies. By prelabelling cells with [³H]leucine or [³H]inositolin the presence of suramin, followed by chase labelling with[³⁵S]sulphate in the presence of brefeldin A, a ³H- and ³⁵S-labelled,hydrophobic heparan sulphate proteoglycan with a core proteinof 60–65 kDa is obtained. The proteoglycan loses its hydrophobicitywhen glucosamineinositol bonds are cleaved, indicating thatit is membrane bound via a glycosylphosphatidylinositol anchor.However, treatment with phosphatidylinositol-specific phospholipaseC has no effect, suggesting that the inositol moiety may beacylated. We propose that a portion of the lipid-anchored proteoglycanglypican is internalized, recycled via the Golgi, where heparansulphate chains are added, and finally re-deposited at the cellsurface. glycosylphosphatidylinositol-anchored glypican heparan sulphate proteoglycan recycling     

Light and Electron Microscopical Characterization of Heterophilic Granulocytes in the Intestinal Wall and Islet Parenchyma of the Hagfish,Myxine glutinosa (Cyclostomata)

Heterophilic granulocytes were studied in the blood, intestinal wall, and islet parenchyma of the Atlantic hagfish (Myxine glutinosa) by light and electron microscopical methods. The granulocytes are pseudoeosinophils and show a PAS-positive cytoplasmic reaction. Ultrastructurally, the cells contain evenly distributed pleomorphic cytoplasmic granules with the granule membrane close to the osmiophilic core. Emigrated blood granulocytes are found extra-vascularly in the submucous connective tissue, and obviously they can pass the basal lamina and migrate into the epithelium of the intestine, bile duct, and islet parenchyma. Though the staining characteristics of hagfish granulocytes are different from those of endocrine cells in the intestinal mucosa and islet parenchyma, intraepithelial granulocytes in some locations may sometimes be difficult to distinguish ultrastructurally from insulin-containing B-cells, since heterophil granules have both a size and a shape close to those of secretion granules in B-cells. However, in contrast to B-cells the granulocytes show the following ultrastructural features: a lobated nucleus with peripherally arranged electron-dense chromatin; cytoplasmic processes and often rod-like granules with no clear space between the granule membrane and core; prominent cytoplasmic vacuoles and microtubules; and sparse mitochondria and endoplasmic reticulum. Furthermore, immigrated granulocytes lack desmosomes and annulate lamellae. Some of the intraepithelial granulocytes in the mucosa show signs of disintegration and cell death. Degenerative cell processes are also described in the islet parenchyma.     

Light- and Electronmicroscopic Observations on the Blood Cells of the Atlantic Hagfish,Myxine glutinosa (L.)

In the blood of Myxine glutinosa three cell lines may be distinguished: erythrocytes, granulocytes and agranulocytes. The erythrocytes are remarkably large, oval and nucleated. They are well defined in all their developmental stages by a characteristic micropinocytosis. They originate from blast cells which proliferate in the circulating blood. The blast cells probably form from agranulate stem cells of the intestinal myeloid tissue. The granulocytes constitute about half of the leucocytes. They are neutrophilic with a lobated nucleus. The granulocytopoiesis takes place in the intestinal myeloid tissue. The agranulocytes mainly include two cell types, termed spindle cells and lymphocyte-like cells. These cell types, however, transform into each other. Macrophages occur essentially in the peritoneal cavity rarely in the blood. Transition forms between macrophages and granulocytes may exist. The blood also contains cells which on morphological grounds have been termed thrombocytes. Whether these cells are identical with those necessary for clotting of the blood remains to be proved. With the exception of erythroblasts, the different lines of blast cells are difficult to identify and distinguish from each other. Possibly all lines of blood cells originate from agranulate lymphocyte-like stem cells, most of which are produced by the intestinal myeloid tissue.