Proteomic Analysis of Adult Ascaris suum Fluid Compartments and Secretory Products

Background

Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions.

Methodology/Principal Findings

ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent.

Conclusions/Significance

Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.     

Structural Basis for the Immunomodulatory Function of Cysteine Protease Inhibitor from Human Roundworm Ascaris lumbricoides

Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.     

Role of complex II in anaerobic respiration of the parasite mitochondria from Ascaris suum and Plasmodium falciparum.

Parasites have developed a variety of physiological functions necessary for existence within the specialized environment of the host. Regarding energy metabolism, which is an essential factor for survival, parasites adapt to low oxygen tension in host mammals using metabolic systems that are very different from that of the host. The majority of parasites do not use the oxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during their free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research has shown that the mitochondrial complex II plays an important role in the anaerobic energy metabolism of parasites inhabiting hosts, by acting as quinol-fumarate reductase.     

Molecular and catalytic properties of an arginine kinase from the nematode Ascaris suum

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.     

Nitazoxanide in the treatment of Ascaris lumbricoides in a rural zone of Colima, Mexico

Intestinal parasites in Mexico are an endemic problem. A study was conducted in children, teenagers and adults in a rural community in Colima, Mexico to examine the prevalence and intensity of Ascaris lumbricoides infection and to evaluate the parasitological and clinical efficacy of nitazoxanide (NTZ). Two hundred and eighty children, teenagers and adults participated in this study. Parasitological diagnosis from faeces was confirmed by three consecutive stool samples using the floatation concentration Faust method. Egg counts were performed as described by the Kato-Katz technique before and after treatment. A questionnaire was systematically applied to obtain information about socio-economic status and hygienic habits. One hundred and six participants (38%) were diagnosed as harbouring intestinal parasites, and 86 of them (81%) were infected with A. lumbricoides. All patients with ascariasis infections underwent a complete physical examination before and after NTZ treatment. NTZ resolved 88% of the ascariasis cases, with a 89% clinical efficacy, and there was a 97.5% reduction in the levels of morbidity. The most intense infections for A. lumbricoides were found in housewives, and statistically significant associations were found between ascariasis and the absence of drainage and living in houses with dirt floors.     

Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.     

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

ABSTRACT: BACKGROUND: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. METHODS: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. RESULTS: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signalbackground improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. DISCUSSION: Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events.     

Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins

Leishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes of L. (V.) braziliensis promastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. I. Isolation and characterization of multiple forms

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.     

Acidic ribosomal P proteins are phosphorylated in Trypanosoma cruzi.

Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.     

Helminth assemblages of whitefish (Coregonus lavaretus) in interconnected lakes: similarity as a function of species specific parasites and geographical separation

This article examined the composition of parasite assemblages of whitefish (Coregonus lavaretus) in 8 interconnected lakes in northeastern Finland and evaluated the role of coregonid specific parasites and the geographical distance between populations in determining the similarity of the assemblages. Parasite assemblages were compared using the Jaccard qualitative similarity index and a quantitative similarity index and by incorporating the allogenic-autogenic species concept and the effects of 2 corresponding measures of geographical distance between the lakes. The majority of the parasite species found (10 of 14) were specific to salmonids. Similarity of assemblages of autogenic parasites between the lakes was negatively correlated with geographical distance. The dominance of 2 parasite species, the whitefish specialist Ichthyocotylurus erraticus and the generalist Ergasilus sieboldi, was also demonstrated. We concluded that the high proportion of widespread parasite species specific to coregonids is an important determinant of similarity in these assemblages. However, ecological factors were likely to contribute to qualitative (presence of species) and quantitative (difference in abundances) differences between lakes, in the case of autogenic parasites, their importance increasing with geographical separation.     

Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health

To assess the genetic diversity and evolution of Cryptosporidium parasites, the partial ssrRNA, actin, and 70 kDa heat shock protein (HSP70) genes of 15 new Cryptosporidium parasites were sequenced. Sequence data were analysed together with those previously obtained from other Cryptosporidium parasites (10 Cryptosporidium spp. and eight Cryptosporidium genotypes). Results of this multi-locus genetic characterisation indicate that host adaptation is a general phenomenon in the genus Cryptosporidium, because specific genotypes were usually associated with specific groups of animals. On the other hand, host–parasite co-evolution is also common in Cryptosporidium, as closely related hosts usually had related Cryptosporidium parasites. Results of phylogenetic analyses suggest that the Cryptosporidium parvum bovine genotype and Cryptosporidium meleagridis were originally parasites of rodents and mammals, respectively, but have subsequently expanded their host ranges to include humans. Understanding the evolution of Cryptosporidium species is important not only for clarification of the taxonomy of the parasites but also for assessment of the public health significance of Cryptosporidium parasites from animals.     

Non-physiological expression of UhpT does not lead to uncontrolled leakage of sugar phosphates out of Escherichia coli cells

Abstract A Trichomonas vaginalis cDNA library was constructed and recombinant plaques were screened using rabbit immunoglobulins specific for P65, a protozoan protein involved in pathogenicity that we identified in a previous study. A 1.38 kilobases cDNA fragment coding for the P65 protein was cloned in E. coli and then sequenced. On the basis of of the sequence obtained, six primers were synthesised and used to set up a Polymerase Chain Reaction. The presence of a specific amplicon in all 30 clinical isolates tested shows that P65 is a conserved and stable gene. The reaction is highly sensitive (as few as 5 to 10 parasites can be detected) and specific for Trichomonas vaginalis ; the gene coding P65 adhesin can be therefore considered a very good molecular target for polymerase chain reaction-based diagnostic purposes.     

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.     

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

Immunological identification of a new component of Ascaris pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.