Schistosoma mansoni: reactivity with infected human sera and monoclonal antibody characterization of a glycoprotein in different developmental stages

Cercarial glycoproteins of Schistosoma mansoni were purified by concanavalin A affinity chromatography. The purified fraction consisted of at least 15 polypeptides when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of infected humans specifically immunoprecipitated all of these polypeptides. These purified glycoproteins were used as antigen for preparing monoclonal antibodies. One of these monoclonal antibodies immunoprecipitated cercarial polypeptides that were identical to polypeptides immunoprecipitated with sera of infected humans as analyzed by two-dimensional gel electrophoresis. Direct binding assays with ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present not only in cercariae (the source of the immunogen) but also in adult male and female worms and in eggs. The protein molecules expressing these antigenic determinants were glycosylated in each of the developmental stages of the larvae, but differed with respect to molecular weight. These findings indicate a role for this monoclonal antibody in serodiagnosis and immunoprophylaxis.     

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.KEYWORDS: amyloid, Hsp104, prion, molecular chaperone, phase separation, protein aggregate, protein misfolding, ubiquitin/proteasome system     

Detection of a sheath on <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.     

Chemical analysis of stalk components of Dictyostelium discoideum

Structural components of the stalks of mature fruiting bodies of Dictyostelium discoideum have been isolated and characterized after solubilizing non-structural components with urea and sodium dodecyl sulfate. The urea/sodium dodecyl sulfate-insoluble stalks are composed of about 52% cellulose, 15% proteins and 3% of a non-cellulosic heteropolymer in a covalently bound matrix. Non-covalently bound fatty acid containing material was also found. The composition and structural interrelationships of these components are essentially identical to that of the urea/sodium dodecyl sulfate-insoluble surface sheath which is produced earlier in development before culmination. These results suggest that the same components are involved in making structural elements which differ substantially in their functional role in the developmental sequence as well as in their spatial and temporal localization and morphological appearance.     

The role of the fibrillar component of the surface sheath in the morphogenesis of Dictyostelium discoideum

We have isolated a fibrillar component of the surface sheath of Dictyostelium discoideum by virtue of its insolubility in 9 M urea-2% sodium dodecyl sulfate (US). The US-insoluble material is primarily composed of cellulose, but also contains other carbohydrate components, protein, and lipid. Evidence is presented that the US-insoluble material is a component of the sheath. Sheath isolated from mutant strains lacking the developmentally regulated N-acetylglucosaminidase (NAG), α-mannosidase, or β-glucosidase activities is similar in composition to sheath isolated from wildtype strain. Strains lacking NAG are unable to migrate normally. This may result from the markedly lower crystallinity of the cellulose in the US-insoluble sheath isolated from these mutants. Strains lacking α-mannosidase or β-glucosidase migrate normally and the crystallinity of the sheath cellulose is not significantly below that of the wildtype. The correlation between the lower crystallinity of cellulose and the inability of strains lacking NAG to migrate suggests that crystallinity is physiologically important and that the degree of crystallinity is controlled by an enzymatic, mutable process. Strains U1 and UN1, which have <1% of the wildtype activity of uridine diphosphate glucose pyrophosphorylase activity, develop in a morphologically normal fashion to the slug stage. However, they cannot form stalk or spore cells, nor can they produce cellulose. These strains do not produce any detectable US-insoluble sheath and are fragile and unable to migrate. A continuous, nonfibrillar sheath surrounds the aggregates and is sufficient for normal morphogenesis up to the slug stage. The fibrillar component gives the aggregate the added rigidity required during migration.     

UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase mediates the initial step in the formation of the methylphosphomannosyl residues on the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins

The Dictyostelium discoideum gene gpt1 encodes a protein XP_638036 with sequence similarity to the α/β subunits of mammalian UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase. We now demonstrate that extracts of D. discoideum clones with mutations in this gene transfer GlcNAc-P from UDP-GlcNAc to mannose residues at less than 5% the wild type value. Further, the lysosomal hydrolases of these mutant clones fail to bind to a cation-independent mannose 6-phosphate receptor affinity column, indicating a lack of methylphosphomannosyl residues on the high mannose oligosaccharides of these proteins. We conclude that the gpt1 gene product catalyzes the initial step in the formation of methylphosphomannosyl residues on D. discoideum lysosomal hydrolases.     

Trypanosoma lewisi: production of exoantigens during infection in the rat

Exoantigens are produced by Trypanosoma lewisi during infections in the rat. They were detected in rat serum and plasma by gel-diffusion techniques with hyperimmune rat sera and with rabbit antiserum to washed, living trypanosomes. Their parasite origin is indicated by their presence in trypanosome homogenates, which also contain bound antigens, the continued reactivity of rabbit antisera after absorption with normal rat serum, and the reactions of identity obtained with rat and rabbit antisera. Moreover, by immunoelectrophoresis, the exontigens are revealed as new components in infected rat serum with a mobility slightly anodal to the origin. The results also show that the exoantigens are continuously released in vivo and that the trypanosomes avidly bind non-antibody rat serum proteins to their surface. Unlike the complete qualitative changes in exoantigens that accompany antigenic variation of pathogenic species of trypanosomes, at least one exoantigen remains unchanged when antigenic variation occurs with T. lewisi although additional exoantigens may appear and disappear. The relation of the exoantigens to the known ablastic and trypanocidal antibodies is difficult to determine since these antibodies and the exoantigens occur simultaneously in the blood during and after the infection. Although it cannot yet be ruled out that the exoantigens elicit the formation of these antibodies, a review of all the available evidence suggests that the exoantigens of T. lewisi may not be immunogenic during a natural course of infection. Possibly they are hemolysins with a nutritive function.     

Modifications of lysosomal enzymes in Dictyostelium discoideum

This paper has two purposes. The first is to review the past studies on the structure, biosynthesis, and immunological properties of a class of glycoproteins, the lysosomal enzymes, in Dictyostelium discoideum. The second purpose is to present new data on the analysis of mutant strains altered in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides, and on the characterization of new carbohydrate antigenic determinants found on multiple proteins in Dictyostelium. We will also show how a combination of genetic, biochemical and immunochemical approaches have been used to unravel a portion of the glycosylation pathway in Dictyostelium.The long-term goal of these studies is to use Dictyostelium discoideum as a model system to understand the functions of a variety of glycoconjugates in a multicellular organism. The existence of a large number of mutant strains which are altered in a variety of cellular functions, development and the posttranslational modification of multiple proteins, offers a great opportunity to explore this area.     

Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum

Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms.     

Giardia lamblia: Immunogenicity and intracellular distribution of GHSP-115, a member of the Giardia head-stalk family of proteins

Giardia lamblia, a protozoan causing diarrheal outbreaks, is one of the main pathogens monitored in developed countries. Immunoscreening of G. lamblia expression library using the monoclonal antibodies (mAb) against G. lamblia, identified a subset of antigenic proteins in this protozoan, which are proteins belonging to GHSP (Giardia head-stalk protein), GHSP115, GHSP138, and GHSP180. In order to map the epitope region of GHSP115, the corresponding open reading frame was dissected into three parts and expressed as recombinant proteins with histidine tags. Western blot analysis of these recombinant proteins with mAbs reacting with GHSP115 indicated that one-third of the C-terminus of GHSP115 showed immunoreactivity with the mAb. Intracellular location of GHSP115 was examined both in trophozoites and encysting cells of G. lamblia by an immunofluorescence assay, indicating that location of GHSP115 varies during encystation. These results suggest that GHSP115 is an abundant and antigenic protein, which is differentially localized during life cycle of G. lamblia.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase

N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling.     

Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Localization of the Tween 20-soluble membrane proteins of Acholeplasma laidlawii by crossed immunoelectrophoresis

The Tween 20-soluble membrane proteins from Acholeplasma laidlawii have previously been fractionated by preparative agarose-suspension electrophoresis. The fractions obtained have now been characterized by crossed immuno-electrophoresis in the presence of Tween 20 and with antiserum containing antibodies directed against the membrane proteins. This antiserum was also utilized in order to get some information about the location of proteins, i.e. whether they are located on the inside or the outside of the membrane. The method used is based upon crossed immunoelectrophoresis of the Tween 20-soluble membrane proteins as antigens and uses an antiserum that has been depleted of the antibodies that are directed against proteins with antigenic determinants exposed either on the outside of the membrane or on both sides. These two types of antisera (called I and II) can be produced by adding intact cells or washed, lysed cells, respectively, to the original antiserum and then removing the cells with the adsorbed antibodies by centrifugation. If there exists in the intact membrane a protein which has antigenic determinants, e.g. only on the inside of the membrane, a precipitation line corresponding to this protein will appear in crossed immunoelectrophoresis experiments with the original antiserum and antiserum type I, but not with antiserum type II. Using this method we found that probably only one of the Tween 20-soluble proteins is exposed on the outside and three on the inside of the A. laidlawii membrane. These findings, combined with results obtained by digesting and labelling erythrocytes and by immunological investigations of protoplasts of Micrococcus lysodeikticus, may reflect a possible, general feature of the structure of the plasma membrane, namely that most of its proteins are associated with the inner surface of the membrane. There is also some evidence that no protein is buried within the lipid layer, which also has been found for erythrocyte ghosts by a labelling technique, and therefore may be another characteristic architectural feature of plasma membranes.     

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein,peroxisomal functions,and autophagosome formation

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)–binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2–like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.     

Variation,Sex, and Social Cooperation: Molecular Population Genetics of the Social Amoeba Dictyostelium discoideum

Dictyostelium discoideum is a eukaryotic microbial model system for multicellular development, cell–cell signaling, and social behavior. Key models of social evolution require an understanding of genetic relationships between individuals across the genome or possibly at specific genes, but the nature of variation within D. discoideum is largely unknown. We re-sequenced 137 gene fragments in wild North American strains of D. discoideum and examined the levels and patterns of nucleotide variation in this social microbial species. We observe surprisingly low levels of nucleotide variation in D. discoideum across these strains, with a mean nucleotide diversity (π) of 0.08%, and no strong population stratification among North American strains. We also do not find any clear relationship between nucleotide divergence between strains and levels of social dominance and kin discrimination. Kin discrimination experiments, however, show that strains collected from the same location show greater ability to distinguish self from non-self than do strains from different geographic areas. This suggests that a greater ability to recognize self versus non-self may arise among strains that are more likely to encounter each other in nature, which would lead to preferential formation of fruiting bodies with clonemates and may prevent the evolution of cheating behaviors within D. discoideum populations. Finally, despite the fact that sex has rarely been observed in this species, we document a rapid decay of linkage disequilibrium between SNPs, the presence of recombinant genotypes among natural strains, and high estimates of the population recombination parameter ρ. The SNP data indicate that recombination is widespread within D. discoideum and that sex as a form of social interaction is likely to be an important aspect of the life cycle.     

Role of Bacterial Surface Structures on the Interaction of Klebsiella pneumoniae with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.