Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes

Background

Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.

Methods

Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.

Conclusions

This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.     

Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.     

Genetic diversity and population structuring of Schistosoma mansoni in a Brazilian village

The digenean trematode Schistosoma mansoni is responsible for chronic schistosomiasis worldwide, and in Brazil alone an estimated 35 million people are at risk. To evaluate epidemiological patterns among human definitive hosts, we assessed genetic diversity and population subdivision of S. mansoni infrapopulations in human hosts from the highly endemic village of Virgem das Graças in the state of Minas Gerais, Brazil. We believe this is the largest such survey to date. Genetic diversity of parasites, measured over eight polymorphic microsatellite loci, was relatively high and standard measures of inbreeding indicated that the population was panmictic. Furthermore, there was no significant isolation-by-distance of parasite infrapopulations, and measures of population subdivision indicated significant but low to moderate levels of population differentiation. We conclude that patients within this village sample from a broad range of schistosome genetic diversity and effectively act as “genetic mixing bowls” for the parasites. These results contrast with those previously observed in the Brazilian village of Melqu?´ades and thus provide the opportunity for comparisons of environmental and epidemiological differences that are likely to influence host–parasite coevolution and parasite transmission.     

Evolutionary epidemiology of schistosomiasis: linking parasite genetics with disease phenotype in humans

Here we assess the role of parasite genetic variation in host disease phenotype in human schistosomiasis by implementing concepts and techniques from environmental association analysis in evolutionary epidemiology. Schistosomiasis is a tropical disease that affects more than 200 million people worldwide and is caused by parasitic flatworms belonging to the genus Schistosoma. While the role of host genetics has been extensively studied and demonstrated, nothing is yet known on the contribution of parasite genetic variation to host disease phenotype in human schistosomiasis. In this study microsatellite genotypes of 1561 Schistosoma mansoni larvae collected from 44 human hosts in Senegal were linked to host characteristics such as age, gender, infection intensity, liver and bladder morbidity by means of multivariate regression methods (on each parasite locus separately). This revealed a highly significant association between allelic variation at the parasite locus L46951 and host infection intensity and bladder morbidity. Locus L46951 is located in the 3′ untranslated region of the cGMP-dependent protein kinase gene that is expressed in reproductive organs of adult schistosome worms and appears to be linked to egg production. This putative link between parasite genetic variation and schistosomiasis disease phenotype sets the stage for further functional research.     

A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions

The schistosome egg is the key agent responsible both for transmission of the parasite from human to molluscan host, and is the primary cause of pathogenesis in schistosomiasis. Characterisation of its proteome is a crucial step in understanding the egg’s interactions with the mammalian host. We devised a scheme to isolate undeveloped eggs from mature schistosome eggs by Percoll gradient and then fractionate the mature egg into miracidial, hatch fluid and secreted protein preparations. The soluble proteins contained within the five preparations were separated by two-dimensional electrophoresis and their spot patterns compared by image analysis. Large numbers of representative spots were then excised and subjected to tandem mass spectrometry to obtain identities. In this way, the principal components of each sub-proteome were established. Chaperones were the most abundant category, with heat shock protein 70 (HSP70) dominant in the undeveloped egg and Schistosoma mansoni protein 40 (Smp-40) in the miracidium. Cytoskeletal proteins were expressed at similar levels in the undeveloped egg and miracidium, with tubulins the most abundant. The proteins of energy metabolism reflected the change from anaerobic to aerobic metabolism as the miracidium developed. None of the above categories was abundant in the hatch fluid but this peri-miracidial compartment was highly enriched for defence proteins such as thioredoxin. Hatch fluid also contained several host proteins and schistosome proteins of unknown function, highlighting its distinct nature and potentially its role. The egg secretions could not be compared with the other preparations due to their unique composition featuring the previously characterised IL-4-inducing principal of S. mansoni eggs (IPSE), Omega-1, egg secreted protein 15 (ESP15), a micro-exon gene 2 (MEG-2) protein and two members of the recently described MEG-3 family. This last preparation contains the subset of egg proteins that probably enables eggs to escape from host tissues and may also initiate granuloma formation, emphasising the need to establish fully the roles of its components in schistosome biology.     

Female biased sex-ratio in Schistosoma mansoni after exposure to an allopatric intermediate host strain of Biomphalaria glabrata

For parasites that require multiple hosts to complete their development, the interaction with the intermediate host may have an impact on parasite transmission and development in the definitive host. The human parasite Schistosoma mansoni needs two different hosts to complete its life cycle: the freshwater snail Biomphalaria glabrata (in South America) as intermediate host and a human or rodents as final host. To investigate the influence of the host environment on life history traits in the absence of selection, we performed experimental infections of two B. glabrata strains of different geographic origin with the same clonal population of S. mansoni. One B. glabrata strain is the sympatric host and the other one the allopatric host. We measured prevalence in the snail, the cercarial infectivity, sex-ratio, immunopathology in the final host and microsatellite frequencies of individual larvae in three successive generations.     

Schistosoma spp.: Isolation of microtubule associated proteins in the tegument and the definition of dynein light chains components

Schistosomes are parasitic blood flukes that reside in human mesenteric veins or urinary bladder veins, depending on species of the parasite. The syncytial tegument of these parasites represents a dynamic interface that regulates nutritional and immunological interactions between the parasite and the host. It is known that the components for biogenesis and maintenance of the tegument are supplied via vesicles from the nucleated cell bodies beneath the syncytium and muscle layer. To investigate the common motor components of vesicular transport in the tegument of schistomes, we extracted Schistosoma mansoni tegumental microtubule associated proteins utilizing detergent/high-salt procedure and raised antiserum against these proteins. The antiserum was applied to screen Schistosoma haematobium λgt11 expression library and some of the isolated clones were sequenced. Blast search for the sequences against NCBI database identified clones that are dynein light chains and myosin genes. Further analysis of schistosome dynein genes in the databases identified three families of dynein light chains (Dlcs). The Tctex family protein sequences are significantly different from the mammalian homologs and, therefore, offer a potential vaccine/drug target against schistosomes.     

Conservation of protein kinase a catalytic subunit sequences in the schistosome pathogens of humans

cAMP-dependent protein kinases (PKAs) are central mediators of cAMP signaling in eukaryotic cells. Previously we identified a cDNA which encodes for a PKA catalytic subunit (PKA-C) in Schistosoma mansoni (SmPKA-C) that is required for adult schistosome viability in vitro. As such, SmPKA-C could potentially represent a novel schistosome chemotherapeutic target. Here we sought to identify PKA-C subunit orthologues in the other medically important schistosome species, Schistosoma haematobium and Schistosoma japonicum, to determine the degree to which this potential target is conserved and could therefore be exploited for the treatment of all forms of schistosomiasis. We report the identification of PKA-C subunit orthologues in S. haematobium and S. japonicum (ShPKA-C and SjPKA-C, respectively) and show that PKA-C orthologues are highly conserved in the Schistosoma, with over 99% amino acid sequence identity shared among the three human pathogens we examined. Furthermore, we show that the recently published Schistosoma mansoni and S. japonicum genomes contain sequences encoding for several putative PKA substrates with homology to those found in Homo sapiens, Caenorhabditis elegans, and Saccharomyces cerevisiae.     

Plasmodium yoelii: Adverse outcome of non-lethal P. yoelii malaria during co-infection with Schistosoma mansoni in BALB/c mouse model

Plasmodium yoelii and Schistosoma mansoni co-infections were studied in female BALB/c mice aged 4-6 weeks to determine the effect of time and stage of concomitant infections on malaria disease outcome. Patent S. mansoni infection in BALB/c mice increased malaria peak parasitemia and caused death from an otherwise non-lethal, self-resolving P. yoelii malaria infection. Exacerbation of malaria parasitemia occurred during both pre-patent and patent S. mansoni infection resulting in a delay of 4-8 days in malaria parasite resolution in co-infected mice. Praziquantel administered to mice with patent schistosome infection protected from fatal outcome during co-infection. However, this treatment did not completely clear the worm infestation, nor did it reduce the peak malaria parasitemia reached, which was nonetheless resolved completely. Hepatosplenomegaly was more marked in schistosome and malaria co-infected mice compared to either infection separately. The results suggest a complex relationship between schistosome co-infection and malaria disease outcome in which the timing of malaria infection in relation to schistosome acquisition is critical to disease outcome and pathology.     

Inbreeding within human Schistosoma mansoni: do host-specific factors shape the genetic composition of parasite populations?

The size, structure and distribution of host populations are key determinants of the genetic composition of parasite populations. Despite the evolutionary and epidemiological merits, there has been little consideration of how host heterogeneities affect the evolutionary trajectories of parasite populations. We assessed the genetic composition of natural populations of the parasite Schistosoma mansoni in northern Senegal. A total of 1346 parasites were collected from 14 snail and 57 human hosts within three villages and individually genotyped using nine microsatellite markers. Human host demographic parameters (age, gender and village of residence) and co-infection with Schistosoma haematobium were documented, and S. mansoni infection intensities were quantified. F-statistics and clustering analyses revealed a random distribution (panmixia) of parasite genetic variation among villages and hosts, confirming the concept of human hosts as ‘genetic mixing bowls'' for schistosomes. Host gender and village of residence did not show any association with parasite genetics. Host age, however, was significantly correlated with parasite inbreeding and heterozygosity, with children being more infected by related parasites than adults. The patterns may be explained by (1) genotype-dependent ‘concomitant immunity'' that leads to selective recruitment of genetically unrelated worms with host age, and/or (2) the ‘genetic mixing bowl'' hypothesis, where older hosts have been exposed to a wider variety of parasite strains than children. The present study suggests that host-specific factors may shape the genetic composition of schistosome populations, revealing important insights into host–parasite interactions within a natural system.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

Tetraspanin-2 localisation in high pressure frozen and freeze-substituted Schistosoma mansoni adult males reveals its distribution in membranes of tegumentary vesicles

The tegument, or body wall, of schistosomes is the primary tissue for host interaction and site targeted schistosome vaccination. However, many aspects of the cell biology, particularly differentiation and maintenance, remain uncharacterised. A leading vaccine candidate, Schistosoma mansoni tetraspanin 2 has proven efficacy in experimental models, but its function, precise subcellular location in the tegument and role in tegument biology is not well understood. A primary question is whether this molecule is a true surface molecule, that is, whether it appears within the apical membrane of the tegument. Hitherto, the target sequence for antibody localisation studies had not been available for advanced subcellular localisation studies, such as immuno-electron microscopy, due to aldehyde sensitivity. To circumvent this problem, we adapted the methods of high pressure freezing and cryosubstitution with uranyl acetate for immuno-electron microscopy. The tri-dimensional structure of tegument membranes was resolved using electron tomography. Immunolocalisation of Schistosoma mansoni tetraspanin 2 demonstrates that the molecule is localised to tegument membrane compartments, but predominantly within internal structures associated with surface invaginations and internal vesicles. Surprisingly, no label was found at the virtual surface of the parasite. The significance of this localisation pattern is discussed.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

A Large Repertoire of Parasite Epitopes Matched by a Large Repertoire of Host Immune Receptors in an Invertebrate Host/Parasite Model

For many decades, invertebrate immunity was believed to be non-adaptive, poorly specific, relying exclusively on sometimes multiple but germ-line encoded innate receptors and effectors. But recent studies performed in different invertebrate species have shaken this paradigm by providing evidence for various types of somatic adaptations at the level of putative immune receptors leading to an enlarged repertoire of recognition molecules. Fibrinogen Related Proteins (FREPs) from the mollusc Biomphalaria glabrata are an example of these putative immune receptors. They are known to be involved in reactions against trematode parasites. Following not yet well understood somatic mechanisms, the FREP repertoire varies considerably from one snail to another, showing a trend towards an individualization of the putative immune repertoire almost comparable to that described from vertebrate adaptive immune system. Nevertheless, their antigenic targets remain unknown. In this study, we show that a specific set of these highly variable FREPs from B. glabrata forms complexes with similarly highly polymorphic and individually variable mucin molecules from its specific trematode parasite S. mansoni (Schistosoma mansoni Polymorphic Mucins: SmPoMucs). This is the first evidence of the interaction between diversified immune receptors and antigenic variant in an invertebrate host/pathogen model. The same order of magnitude in the diversity of the parasite epitopes and the one of the FREP suggests co-evolutionary dynamics between host and parasite regarding this set of determinants that could explain population features like the compatibility polymorphism observed in B. glabrata/S. mansoni interaction. In addition, we identified a third partner associated with the FREPs/SmPoMucs in the immune complex: a Thioester containing Protein (TEP) belonging to a molecular category that plays a role in phagocytosis or encapsulation following recognition. The presence of this last partner in this immune complex argues in favor of the involvement of the formed complex in parasite recognition and elimination from the host.     

Schistosoma mansoni and Biomphalaria glabrata share epitopes: antibodies to sporocysts bind host snail hemocytes

Using an independent protocol, we have confirmed that sporocysts of the human blood fluke, Schistosoma mansoni, synthesize antigens which stimulate rabbit antibody activity to epitopes on infermediate snail host hemocytes. This molecular mimicry may aid S. mansoni to escape the innate immune system of this host, Biomphalaria glabrata.     

Host exposure history and priority effects impact the development and reproduction of a dominant parasite

Within a single organism, numerous parasites often compete for space and resources. This competition, together with a parasite’s ability to locate and successfully establish in a host, can contribute to the distribution and prevalence of parasites. Coinfection with trematodes in snail intermediate hosts is rarely observed in nature, partly due to varying competitive abilities among parasite taxa. Using a freshwater snail host (Biomphalaria glabrata), we studied the ability of a competitively dominant trematode, Echinostoma caproni, to establish and reproduce in a host previously infected with a less competitive trematode species, Schistosoma mansoni. Snails were exposed to S. mansoni and co-exposed to E. caproni either simultaneously or 1 week, 4 weeks, or 6 weeks post S. mansoni exposure. Over the course of infection, we monitored the competitive success of the dominant trematode through infection prevalence, parasite development time, and parasite reproductive output. Infection prevalence of E. caproni did not differ among co-exposed groups or between co-exposed and single exposed groups. However, E. caproni infections in co-exposed hosts took longer to reach maturity when the timing between co-exposures increased. All co-exposed groups had higher E. caproni reproductive output than single exposures. We show that although timing of co-exposure affects the development time of parasite transmission stages, it is not important for successful establishment. Additionally, co-exposure, but not priority effects, increases the reproductive output of the dominant parasite.