Parasites, proteomes and systems: has Descartes' clock run out of time?

Systems biology aims to integrate multiple biological data types such as genomics, transcriptomics and proteomics across different levels of structure and scale; it represents an emerging paradigm in the scientific process which challenges the reductionism that has dominated biomedical research for hundreds of years. Systems biology will nevertheless only be successful if the technologies on which it is based are able to deliver the required type and quality of data. In this review we discuss how well positioned is proteomics to deliver the data necessary to support meaningful systems modelling in parasite biology. We summarise the current state of identification proteomics in parasites, but argue that a new generation of quantitative proteomics data is now needed to underpin effective systems modelling. We discuss the challenges faced to acquire more complete knowledge of protein post-translational modifications, protein turnover and protein-protein interactions in parasites. Finally we highlight the central role of proteome-informatics in ensuring that proteomics data is readily accessible to the user-community and can be translated and integrated with other relevant data types.

Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii.

To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle.

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. SIGNIFICANCE: The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.

Cervicovaginal microbiome dysbiosis is associated with proteome changes related to alterations of the cervicovaginal mucosal barrier.

Vaginal microbiome (VMB) dysbiosis is associated with increased acquisition of HIV. Cervicovaginal inflammation and other changes to the mucosal barrier are thought to have important roles but human data are scarce. We compared the human cervicovaginal proteome by mass spectrometry of 50 Rwandan female sex workers who had previously been clustered into four VMB groups using a 16S phylogenetic microarray; in order of increasing bacterial diversity: Lactobacillus crispatus-dominated VMB (group 1), Lactobacillus iners-dominated VMB (group 2), moderate dysbiosis (group 3), and severe dysbiosis (group 4). We compared relative protein abundances among these VMB groups using targeted (abundance of pre-defined mucosal barrier proteins) and untargeted (differentially abundant proteins among all human proteins identified) approaches. With increasing bacterial diversity, we found: mucus alterations (increasing mucin 5B and 5AC), cytoskeleton alterations (increasing actin-organizing proteins; decreasing keratins and cornified envelope proteins), increasing lactate dehydrogenase A/B as markers of cell death, increasing proteolytic activity (increasing proteasome core complex proteins/proteases; decreasing antiproteases), altered antimicrobial peptide balance (increasing psoriasin, calprotectin, and histones; decreasing lysozyme and ubiquitin), increasing pro-inflammatory cytokines, and decreasing immunoglobulins immunoglobulin G1/2. Although temporal relationships cannot be derived, our findings support the hypothesis that dysbiosis causes cervicovaginal inflammation and other detrimental changes to the mucosal barrier.

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.

Towards evaluating the economic impact of bovine neosporosis.

In spite of the global importance of neosporosis as a cause of bovine abortion, there is very little information about its economic consequences. The economic costs are a product of estimations of the quantity of the effects attributable to Neospora infection, and the particular unit costs of those effects. In this brief review, which arose from a workshop on the economics of coccidiosis held at the COST 820 meeting, Toledo 1998, we discuss the possible effects of neosporosis which are of economic significance and summarise the available estimates of their magnitude to provide a basis for further economic analysis. Neospora infection has been associated with abortion, increased culling and reduced milk yield. In addition, it has been diagnosed in cases of stillbirth and neonatal mortality, it is likely to contribute to early foetal death and resorption and it is responsible for a reduction in the value of female breeding cattle. In quantifying the role of Neospora, it is important that epidemiologically based, case-controlled studies are conducted because, given the extreme efficiency with which bovine Neospora infection is vertically transmitted, demonstration of prevalence of infection in affected animals (including foetuses) is not a true indicator of the significance of this disease. Relatively few epidemiological studies have been conducted, but in investigations in the USA, Holland and Britain, infected cows have been shown to be about three times more likely to abort than non-infected cattle. In the UK this approach has been used to estimate the proportion of abortions in the national dairy population which may be attributable to Neospora caninum.

Neosporosis. Aspects of epidemiology and host immune response.

Neospora caninum is a recently recognized protozoan parasite which has been described as causing a neuromuscular paralysis in dogs and is emerging as a major cause of bovine infertility and abortion worldwide. The parasite is known to infect a range of warm blooded animals but the disease predominates in dogs and cattle. It is not yet known if N. caninum can infect and cause disease in people. The dog has recently been identified as the definitive host and the parasite may be transmitted through the ingestion of oocysts or congenitally from mother to fetus. N. caninum is known to infect red foxes (Vulpes vulpes) and coyotes (Canis latrans) and the role of wildlife species as reservoirs of infection requires further investigation. Little is known about the range of parasite genotypes within the environment or the variation in virulence between different strains. RAPD-PCR analysis of geographically distinct bovine and canine isolates has revealed little genetic variation. Epidemiological studies from different areas of the world have investigated the importance of N. caninum as an abortifacient agent and longitudinal studies have shown the high rate (approximately 80%) of congenital transmission within infected herds. Information on the rates of repeat abortion due to neosporosis are less well defined however current estimates put this at 5% suggesting that cattle may develop some form of protective immunity against N. caninum-induced abortion. Diagnosis of the disease is based upon detection of the parasite in the tissues, most commonly using immunohistochemistry with additional information provided by serology. However, although positive fetal serology is a strong indicator of exposure to the parasite, care should be taken in the interpretation of maternal serology. As we understand more about the epidemiology of neosporosis we are also better able to interpret the results of diagnostic tests. The mere presence of the parasite does not necessarily infer that this was the primary cause of abortion. CD4+ T-cells, interferon gamma and macrophages have all been found to significantly inhibit multiplication of N. caninum tachyzoites. The nature of a protective immune response and its modulation in the pregnant animal is discussed.

Comparison of two gene amplification methods for the detection of Toxoplasma gondii in experimentally infected sheep.

Efferent lymph and peripheral blood collected from sheep experimentally infected with Toxoplasma gondii strain S48 were analysed for parasite DNA by amplification of the B1 and P30 T. gondii genes by the polymerase chain reaction (PCR). The relative sensitivity of these two gene amplification methods was assessed and compared with parasite detection by mouse injection (MI). B1 PCR was consistently more sensitive than P30 PCR and the results agreed closely with those from MI. By contrast, P30 PCR gave more than twice as many false negatives results than B1 PCR. The few apparent false positive results given by either PCR method were probably due to the inability of MI to detect non-viable parasites. All specimens collected before infection with T. gondii gave negative results by PCR and MI. Parasite DNA was detected by both B1 and P30 PCR in the lymph node of a sheep 12 days after infection but not in other tissues. The results permit a direct comparison between T. gondii detection by P30 and B1 PCR. Moreover, they further confirm the value of PCR detection of toxoplasma as a sensitive, specific and reliable diagnostic and research tool.

The seroprevalence of antibodies to toxoplasma gondii in domestic goats in Uganda.

Only limited epidemiological information is available on the seroprevalence of Toxoplasma gondii in domestic livestock in sub-Saharan Africa. In Uganda, goats are important to the local economy and are also popular food animals. A high incidence of T. gondii infection in goats would have implications both for animal production and for public health, but no data is available on Toxoplasma infection in these animals. In this study we estimated the seroprevalence of antibodies against T. gondii in goats located in both urban and rural environments and from different geographical regions within Uganda. Goat sera were collected using a random, two-stage clustering method. Of 784 samples analysed by antibody-ELISA from various districts in Uganda, 240 tested positive. The combined (cluster-adjusted) seroprevalence was 0.31 (31%) (95% confidence intervals 0.28, 0.34) indicating a substantial level of infection in these regions. Seroprevalence was significantly higher in goats from urban locations. A strong positive relationship between age and seroprevalence was demonstrated and a mathematical model based on continuous exposure proved generally accurate in predicting seroprevalence. Farm environments were identified as being suitable for oocyst survival and transmission, and the reported incidence of caprine abortion was high. The importance of toxoplasmosis to goat production in Uganda has yet to be determined, but the high seroprevalence detected in this study suggests that it may have a significant impact and that the consumption of goat meat may play a role in zoonotic transmission to humans.

The seroprevalence of toxoplasmosis in pigs in Ghana.

A serological survey of toxoplasmosis in pigs in Ghana was carried out between October 1997 and April 1998 in the three ecological zones of Ghana: the Coastal Savannah, the Forest Belt and the Guinea Savannah. Antibody against Toxoplasma gondii was measured in pig serum using a microplate-ELISA which had a sensitivity and specificity of 90.2 and 92.3%, respectively when compared with IFAT. A national seroprevalence of 39% was obtained in pigs, with the ecological distribution being 43.9, 30.5 and 42.5% for the Coastal Savannah, the Forest Belt and the Guinea Savannah, respectively. The age of the animal, the breed, the environmental conditions and the management practices appeared to be the major determinants of prevalence of antibodies against T. gondii. The prevalence of anti-T. gondii antibodies was found to increase with age (P<0.05). Pigs from the two Savannah zones had a significantly higher (P<0.05) antibody prevalence than those sampled from the Forest belt. Antibody prevalence (46.8%) in crossbreed pigs was significantly higher (P<0.05) than that of the exotic Large White breed (38.8%).

The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats.

The enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Toxoplasma gondii antibodies in 1258 small ruminants (732 sheep and 526 goats) sampled from 28 different locations in the three ecological zones of Ghana. The animals sampled had an overall seroprevalence of 30.5% (384 of the total). Sheep had a higher overall prevalence (33.2%) compared to the goats (26.8%). Animals sampled from the Coastal Savannah and the Forest zones had prevalences of 39.4% and 39.1%, respectively, which were significantly higher (P<0.01) than the prevalence recorded for the drier Guinea Savannah zone (20%). Prevalence of antibodies in female animals (35.8%) was significantly higher (P<0.01) than that for males (21.1%). Significant differences were also observed between breeds and age groups. The ELISA was found to be both highly sensitive (92%) and specific (91%) when compared to the IFAT, which was used as a reference test.

A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity.

Kinetics of the local and systemic antibody response to primary and secondary infection with S48 Toxoplasma gondii in sheep.

Vaccination of sheep with live tachyzoites of Toxoplasma gondii, strain S48, affords protection against subsequent challenge with the parasite, but the mechanisms of immunity have not been fully determined. To understand better the nature of the antibody response the kinetics of both local and systemic antibody production were monitored in vaccinated sheep by means of an enzyme-linked immunosorbent assay and Western blotting. Local specific IgG production was analysed in efferent lymph obtained from the cannulated pre-femoral lymph node draining the site of infection. Antibody in efferent lymph plasma and peripheral blood serum from animals vaccinated with S48 tachyzoites was monitored and compared with IgG production in vaccinated sheep given a secondary tachyzoite challenge. Secondary challenge resulted in a clear immunological memory response, antibody being detected in the lymph 3 to 4 days after infection as compared with 7 to 8 days after a primary infection. IgG production was dominated by antibody recognizing a protein with an apparent molecular weight of 30 kDa, but other antigens (32, 24 and 11 kDa) were also readily detected.

Primary and secondary responses of the ovine lymph node to Toxoplasma gondii: cell output in efferent lymph and parasite detection.

Efferent lymphatic cannulation was used to study the dissemination of strain S48 of Toxoplasma gondii and the cell output from the prefemoral lymph node, after infection of both "naive" and vaccinated sheep. In the former the mean cell output decreased for 3 days before reaching a peak at 11 and 12 days, but in vaccinated ewes a similar drop in cell output and subsequent peak occurred significantly earlier, at 24 h and 5 days, respectively. The cellular response in both types of sheep was largely due to a marked increase in blast cells. The detection of live toxoplasms and parasite DNA by mouse inoculation and the polymerase chain reaction, respectively, gave similar results; the parasite was demonstrated in lymph from days 3 to 12 during a primary infection but with a sharp cut-off after day 9 coinciding with the peak blast cell response. Very little evidence of T. gondii was found in lymph of vaccinated sheep after challenge. Immunity, which is thought to be largely T-cell mediated and is sustained without continuous antigenic stimulation, suppresses dissemination of the parasite in the lymph and therefore to other sites, which might include the gravid uterus.

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax).

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of 'incomplete' strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the 'complete' M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.

Toxoplasma gondii--keeping our guests under control.

You might have a closer relationship than you think with one of nature's most widespread and successful parasites. Many of us harbour this subtle organism in our brains and muscles in an uneasy truce; but recent insights into its biochemistry and genetics could provide us with new ways of coping when this 'guest' gets out of hand.

Occurrence and diversity of Giardia duodenalis assemblages in livestock in the UK.

Giardia duodenalis is a common intestinal parasite in humans and a wide range of livestock species. It is a genetically heterogeneous parasite that has been characterized in seven distinct genetic assemblages or cryptic species, and molecular markers can be used to differentiate both animal-specific and potentially zoonotic genotypes. Little is known about G. duodenalis and the range of assemblages occurring in domestic livestock species in the UK. Here, we present data on the occurrence and molecular diversity of G. duodenalis detected in the faeces or large intestinal contents of cattle, sheep, pigs, goats and camelids from farms in the north-west of England. Both healthy and clinically diseased animals were included in the survey. The presence of Giardia spp. and assemblages was determined by sequencing of the small-subunit ribosomal RNA gene. The potential association of infection with various clinical and epidemiological parameters was studied in cattle using both univariate and multivariate analyses. Giardia spp. were detected in 127 (34.3%) of the 370 animals tested. G. duodenalis assemblage E was found to be predominant in cattle and sheep, followed by assemblage A. Mixed infections with assemblages A and E were also detected. Interestingly, some cattle, sheep and pigs were found to be infected with more unexpected assemblages (C, D, F). Pre-weaned calves were more likely to test positive than adult animals, but no association between the occurrence of overt intestinal disease and G. duodenalis infection was detected. The common occurrence of assemblage A and the finding of unusual assemblages in atypical hosts suggest that in future, a multilocus analysis should be used to confirm the actual diversity of G. duodenalis in livestock and the presence of potentially zoonotic genotypes. These data also suggest that there is a need to re-evaluate the clinical significance of G. duodenalis infection in livestock.

Proteomic analysis of the Theileria annulata schizont.

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.