Microarray Analysis of Gene Expression Profiles of Schistosoma japonicum Derived from Less-Susceptible Host Water Buffalo and Susceptible Host Goat

Background

Water buffalo and goats are natural hosts for S. japonicum in endemic areas of China. The susceptibility of these two hosts to schistosome infection is different, as water buffalo are less conducive to S. japonicum growth and development. To identify genes that may affect schistosome development and survival, we compared gene expression profiles of schistosomes derived from these two natural hosts using high-throughput microarray technology.

Results

The worm recovery rate was lower and the length and width of worms from water buffalo were smaller compared to those from goats following S. japonicum infection for 7 weeks. Besides obvious morphological difference between the schistosomes derived from the two hosts, differences were also observed by scanning and transmission electron microscopy. Microarray analysis showed differentially expressed gene patterns for parasites from the two hosts, which revealed that genes related to lipid and nucleotide metabolism, as well as protein folding, sorting, and degradation were upregulated, while others associated with signal transduction, endocrine function, development, immune function, endocytosis, and amino acid/carbohydrate/glycan metabolism were downregulated in schistosomes from water buffalo. KEGG pathway analysis deduced that the differentially expressed genes mainly involved lipid metabolism, the MAPK and ErbB signaling pathways, progesterone-mediated oocyte maturation, dorso-ventral axis formation, reproduction, and endocytosis, etc.

Conclusion

The microarray gene analysis in schistosomes derived from water buffalo and goats provide a useful platform to disclose differences determining S. japonicum host compatibility to better understand the interplay between natural hosts and parasites, and identify schistosome target genes associated with susceptibility to screen vaccine candidates.     

Interactions between natural populations of human and rodent schistosomes in the Lake Victoria region of Kenya: a molecular epidemiological approach

Background

Schistosoma mansoni exists in a complex environmental milieu that may select for significant evolutionary changes in this species. In Kenya, the sympatric distribution of S. mansoni with S. rodhaini potentially influences the epidemiology, ecology, and evolutionary biology of both species, because they infect the same species of snail and mammalian hosts and are capable of hybridization.

Methodology/Principal Findings

Over a 2-year period, using a molecular epidemiological approach, we examined spatial and temporal distributions, and the overlap of these schistosomes within snails, in natural settings in Kenya. Both species had spatially and temporally patchy distributions, although S. mansoni was eight times more common than S. rodhaini. Both species were overdispersed within snails, and most snails (85.2% for S. mansoni and 91.7% for S. rodhaini) only harbored one schistosome genotype. Over time, half of snails infected with multiple genotypes showed a replacement pattern in which an initially dominant genotype was less represented in later replicates. The other half showed a consistent pattern over time; however, the ratio of each genotype was skewed. Profiles of circadian emergence of cercariae revealed that S. rodhaini emerges throughout the 24-hour cycle, with peak emergence before sunrise and sometimes immediately after sunset, which differs from previous reports of a single nocturnal peak immediately after sunset. Peak emergence for S. mansoni cercariae occurred as light became most intense and overlapped temporally with S. rodhaini. Comparison of schistosome communities within snails against a null model indicated that the community was structured and that coinfections were more common than expected by chance. In mixed infections, cercarial emergence over 24 hours remained similar to single species infections, again with S. rodhaini and S. mansoni cercarial emergence profiles overlapping substantially.

Conclusions/Significance

The data from this study indicate a lack of obvious spatial or temporal isolating mechanisms to prevent hybridization, raising the intriguing question of how the two species retain their separate identities.     

Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum

Background

Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and Findings

After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions

Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.     

Proteomic analysis of skin invasion by blood fluke larvae

Background

During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.

Methodology and Results

Human skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in “triple play” mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.

Conclusions

This analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.     

Influence of Exposure History on the Immunology and Development of Resistance to Human Schistosomiasis Mansoni

Background

Previous studies suggest that humans can acquire immunity to reinfection with schistosomes, most probably due to immunologic mechanisms acquired after exposure to dying schistosome worms.

Methodology/Principal Findings

We followed longitudinally two cohorts of adult males occupationally exposed to Schistosoma mansoni by washing cars (120 men) or harvesting sand (53 men) in Lake Victoria. Men were treated with praziquantel each time S. mansoni infection was detected. In car washers, a significant increase in resistance to reinfection, as measured by the number of cars washed between cure and reinfection, was observed after the car washers had experienced, on average, seven cures. In the car washers who developed resistance, the level of schistosome-specific IgE increased between baseline and the time at which development of resistance was first evidenced. In the sand harvesters, a significant increase in resistance, as measured by the number of days worked in the lake between cure and reinfection, was observed after only two cures. History of exposure to S. mansoni differed between the two cohorts, with the majority of sand harvesters being lifelong residents of a village endemic for S. mansoni and the majority of car washers having little exposure to the lake before they began washing cars. Immune responses at study entry were indicative of more recent infections in car washers and more chronic infections in sand harvesters.

Conclusions/Significance

Resistance to reinfection with S. mansoni can be acquired or augmented by adults after multiple rounds of reinfection and cure, but the rate at which resistance is acquired by this means depends on immunologic status and history of exposure to S. mansoni infection.     

Comparison of Schistosoma mansoni Soluble Cercarial Antigens and Soluble Egg Antigens for Serodiagnosing Schistosome Infections

A Schistosoma mansoni cercarial antigen preparation (cercarial transformation fluid – SmCTF) was evaluated for detection of anti-schistosome antibodies in human sera in 4 collaborating laboratories. The performance of SmCTF was compared with that of S. mansoni egg antigens (SmSEA) in an indirect enzyme-immunoassay (ELISA) antigen assay, the latter being used routinely in 3 of the 4 participating laboratories to diagnose S. mansoni and S. haematobium infections. In the fourth laboratory the performance of SmCTF was compared with that of S. japonicum egg antigens (SjSEA) in ELISA for detection of anti-S. japonicum antibodies. In all 4 laboratories the results given by SmCTF in ELISA were very similar to those given by the antigen preparation routinely used in the respective laboratory to detect anti-schistosome antibodies in human infection sera. In so far as the ELISA results from SmCTF are thus so little different from those given by schistosome egg antigens and also cheaper to produce, the former is a potentially useful new diagnostic aid for schistosomiasis.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

Mefloquine—An Aminoalcohol with Promising Antischistosomal Properties in Mice

Background

The treatment and control of schistosomiasis, an often neglected tropical disease that exacerbates poverty, depends on a single drug, praziquantel. The large-scale use of praziquantel might select for drug-resistant parasites, hence there is a need to develop new antischistosomal compounds. Here, we report that the antimalarial drug mefloquine possesses promising antischistosomal properties in mice.

Methodology/Principal Findings

A single dose of mefloquine (200 or 400 mg/kg) administered orally to mice infected with adult Schistosoma mansoni or adult S. japonicum resulted in high or complete total and female worm burden reductions (72.3%–100%). Importantly, high worm burden reductions were also observed for young developing stages of S. mansoni and S. japonicum harbored in the mouse. Both mefloquine erythro-enantiomers resulted in high and comparable total and female worm burden reductions when given to mice with either a sub-patent or patent S. mansoni infection.

Conclusions/Significance

Our findings hold promise for the development of a novel antischistosomal drug based on an aminoalcohol functionality. Further in vitro and in vivo studies have been launched to elucidate the possible mechanism of action and to study the effect of mefloquine on S. haematobium and other trematodes. It will be interesting to investigate whether mefloquine, which is widely and effectively used for the treatment of malaria, has an impact on schistosomiasis in areas where both malaria and schistosomiasis co-exist.     

Risk Factors and Spatial Distribution of Schistosoma mansoni Infection among Primary School Children in Mbita District,Western Kenya

Background

An increasing risk of Schistosoma mansoni infection has been observed around Lake Victoria, western Kenya since the 1970s. Understanding local transmission dynamics of schistosomiasis is crucial in curtailing increased risk of infection.

Methodology/Principal Findings

We carried out a cross sectional study on a population of 310 children from eight primary schools. Overall, a total of 238 (76.8%) children were infected with S. mansoni, while seven (2.3%) had S. haematobium. The prevalence of hookworm, Trichuris trichiura and Ascaris lumbricoides were 6.1%, 5.2% and 2.3%, respectively. Plasmodium falciparum was the only malaria parasite detected (12.0%). High local population density within a 1 km radius around houses was identified as a major independent risk factor of S. mansoni infection. A spatial cluster of high infection risk was detected around the Mbita causeway following adjustment for population density and other potential risk factors.

Conclusions/Significance

Population density was shown to be a major factor fuelling schistosome infection while individual socio-economic factors appeared not to affect the infection risk. The high-risk cluster around the Mbita causeway may be explained by the construction of an artificial pathway that may cause increased numbers of S. mansoni host snails through obstruction of the waterway. This construction may have, therefore, a significant negative impact on the health of the local population, especially school-aged children who frequently come in contact with lake water.     

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background

The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.

Methods/Findings

We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D).

Conclusions

Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.     

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10⁶ μm³ sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.     

Effect of Maternal Schistosoma mansoni Infection and Praziquantel Treatment During Pregnancy on Schistosoma mansoni Infection and Immune Responsiveness among Offspring at Age Five Years

Introduction

Offspring of Schistosoma mansoni-infected women in schistosomiasis-endemic areas may be sensitised in-utero. This may influence their immune responsiveness to schistosome infection and schistosomiasis-associated morbidity. Effects of praziquantel treatment of S. mansoni during pregnancy on risk of S. mansoni infection among offspring, and on their immune responsiveness when they become exposed to S. mansoni, are unknown. Here we examined effects of praziquantel treatment of S. mansoni during pregnancy on prevalence of S. mansoni and immune responsiveness among offspring at age five years.

Methods

In a trial in Uganda (ISRCTN32849447, http://www.controlled-trials.com/ISRCTN32849447/elliott), offspring of women treated with praziquantel or placebo during pregnancy were examined for S. mansoni infection and for cytokine and antibody responses to SWA and SEA, as well as for T cell expression of FoxP3, at age five years.

Results

Of the 1343 children examined, 32 (2.4%) had S. mansoni infection at age five years based on a single stool sample. Infection prevalence did not differ between children of treated or untreated mothers. Cytokine (IFNγ, IL-5, IL-10 and IL-13) and antibody (IgG1, Ig4 and IgE) responses to SWA and SEA, and FoxP3 expression, were higher among infected than uninfected children. Praziquantel treatment of S. mansoni during pregnancy had no effect on immune responses, with the exception of IL-10 responses to SWA, which was higher in offspring of women that received praziquantel during pregnancy than those who did not.

Conclusion

We found no evidence that maternal S. mansoni infection and its treatment during pregnancy influence prevalence and intensity of S. mansoni infection or effector immune response to S. mansoni infection among offspring at age five years, but the observed effects on IL-10 responses to SWA suggest that maternal S. mansoni and its treatment during pregnancy may affect immunoregulatory responsiveness in childhood schistosomiasis. This might have implications for pathogenesis of the disease.