Karl F. Hoffmann

Identification of Schistosoma mansoni gender-associated gene transcripts by cDNA microarray profiling

BackgroundParasitic helminths of the genus Schistosoma mate, achieve sexual maturity and produce eggs in the bloodstream of their definitive hosts, and the most important pathological consequences of the infection are associated with this process. We have used cDNA microarray technology to initiate genome-wide gene-expression studies of sex and sexual development in mature Schistosoma mansoni parasites.ResultsAn S. mansoni-specific cDNA microarray was fabricated using 576 expressed sequence tags selected from three cDNA libraries and originating from two different parasite developmental stages. Five independent cDNA microarray hybridizations were analyzed using stringent filtering criteria and careful quality control, leading to the identification of 12 new female-associated and 4 new male-associated gene transcripts in the mature adult schistosome. Statistical analysis of variation demonstrated high levels of agreement within a cDNA microarray (correlation coefficient 0.91; median coefficient of variation 11.1%) and between cDNA microarrays (correlation coefficient 0.90; median coefficient of variation 14.4%). RT-PCR analysis confirmed the cDNA microarray results, thereby supporting the reliability of the system.ConclusionsOur study expands the list of S. mansoni gender-associated gene transcripts from all previous studies by a factor of two. Among the new associations identified, a tyrosinase ortholog was preferentially expressed in the adult female, and a dynein light-chain ortholog was highly induced in the adult male. cDNA microarrays offer the potential for exponential leaps in the understanding of parasite biology and this study shows how molecules involved in sexual biology can be rapidly identified.

Cytokine-mediated host responses during schistosome infections; walking the fine line between immunological control and immunopathology.

Most helminth infections of humans and animals induce similar immune responses, which are characterised by the production of Th2-associated cytokines (interleukin (IL)-4, IL-5, IL-9, IL-10, IL-13) and antibodies (IgG1--mouse, IgG4--man, IgE--both). This type-2-biased immune phenotype generally persists for the duration of the infection. Although similar types of immune responses are also triggered during allergy, atopy and anaphylaxis, chronic helminth-induced type-2-associated responses are usually held in check by appropriately regulated control mechanisms that limit the destructive potential of prolonged cytokine bias. Among numerous reported activities, helminth-induced type-2-associated immune responses have been linked to the expulsion of gastrointestinal nematodes and the formation of circumoval granulomas during schistosomiasis. However, what happens when this highly regulated, and often beneficial, type-2 immune response becomes chronic, improperly controlled, or exaggerated during helminth infections? Using schistosomiasis as a model disease, we describe the lethal consequences of inappropriate immune response induction by reviewing the literature generated from experimental animal studies and human epidemiological investigations. Development of severe and non-overlapping immunopathological phenotypes will be discussed in the context of immune deviation and in the setting of chronic and/or hyper-polarised cytokine environments.

Anti-schistosomal Intervention Targets Identified by Lifecycle Transcriptomic Analyses

Background Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis. Methodology/Principal Findings Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons. Conclusions/Significance Collectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex.

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type-1 and type-2 cytokine-mediated inflammatory reactions

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type‐1 and type‐2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type‐1 and type‐2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type‐2 response and two linked with an extreme type‐1 cytokine phenotype. Predictions about liver fibrosis, apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining coordinated gene expression profiles but are also highly effective for molecularly “fingerprinting” diseased tissues. Moreover, they illustrate the potential of genome‐wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious disease pathogenesis.

Factors Affecting Human IgE and IgG Responses to Allergen-Like Schistosoma mansoni Antigens: Molecular Structure and Patterns of in vivo Exposure

Background: The human IgE response is associated with allergy and with host defence against parasitic worms. A response to Sm22.6, the dominant IgE antigen in adult Schistosoma mansoni worms, correlates with resistance to re-infection after treatment. Sm22.6 is one of a family of EF-hand containing parasite proteins with sequence similarity to dynein light chain (DLC) and with major non-parasite allergens. Here we compare human IgE and IgG responses to other family members, Sm20.8 and Sm21.7, as well as to SmDLC1, relating these to antigen structure and expression in parasite life stages. Methods: Recombinant antigens were used in ELISA to measure antibody isotype responses in 177 cases from an endemic area, before and 7 weeks after treatment. Parasite antigen expression was assessed by RT-PCR and Western blotting. Results: Levels of antibodies to Sm22.6 and Sm20.8 (but not to Sm21.7 or SmDLC1) showed posttreatment increases in all but young children. Many produced IgE to Sm22.6 and Sm20.8 (2 EF-hands), few to Sm21.7 (1 EF-hand) or SmDLC1 (no EF-hands). Sm21.7 was expressed in cercariae, adults and eggs, Sm22.6 and Sm20.8 were concentrated in the adult. Conclusions: These studies suggest that IgE antigens Sm22.6 and Sm20.8 are only released to boost antibodies when adult worms die, whilst Sm21.7 and SmDLC1 are released constantly from eggs dying in host tissue. IgE responses to these allergen-like molecules may be influenced by patterns of exposure and the number of EF-hand motifs.

Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect Schistosoma viability.

Background Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genome to drug’ lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. Methodology/Principal Findings We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative. Conclusions/Significance The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance.

Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases

Egg production is responsible for life cycle progression and host immunopathology during schistosomiasis, with the associated parasite molecules being investigated as potential novel chemotherapeutic targets. Here, we characterize two Schwtosoma mansoni products, tyrosinase 1 and tyrosinase 2 (SmTYRl / SmTYR2) and show that their diphenol oxidase enzyme activities are critical for eggshell formation and produc‐tion. The genes encoding these bifunctional enzymes (monophenol and diphenol oxidases) result from a duplication event that likely occurred before speciation and exist in the parasites genome as multiple copies, which are linked and localized to chromosomes 4 and W. SmTYRl/SmTYR2 transcription and diphenol oxidase action are developmentally regulated with most enzyme activity localized to the eggshell‐producing cells contained within the vitellaria of adult female worms. Importantly, kojic‐acid mediated inhibition (IC50=0.5μM) of SmTYRl/SmTYR2s diphenol oxidase activity during in vitro culture of sexually mature adult worms resulted in a significant decrease in the production of phenotypically normal eggs. Therefore our data suggest that SmTYRl /2 inhibition represents a novel and potentially effective strategy for combating schistosomiasis and furthermore, it may point to new methods for combinatorial control of immunopathology and egg transmission during platyhelminfh infection.—Fitzpatrick J. M., Hirai Y., Hirai H., Hoffmann K. F. Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases. FASEB J. 21, 823–835(2007)

Cytosine methylation regulates oviposition in the pathogenic blood fluke Schistosoma mansoni

Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain proteins mirrors the detection of cytosine methylation abundance and implicates the presence of a functional DNA methylation machinery. Genome losses in cytosine methylation upon SmDnmt2 silencing and the identification of a hypermethylated, repetitive intron within a predicted forkhead gene confirm this assertion. Importantly, disruption of egg production and egg maturation by 5-azacytidine establishes an essential role for 5-methylcytosine in this parasite. These findings provide the first functional confirmation for this epigenetic modification in any worm species and link the cytosine methylation machinery to platyhelminth oviposition processes.

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

BackgroundBiomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences.ResultsWe have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1α and EF-2.ConclusionProduction of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Human IgE response to the Schistosoma haematobium 22.6 kDa antigen.

In Schistosoma mansoni and S. japonicum infection, the 22·6 kDa tegumental antigens Sm22·6 and Sj22·6 are principal targets for the human IgE response, and levels of IgE to Sm22·6 have been correlated with resistance to re‐infection after chemotherapy. S. haematobium is arguably a more important species in terms of human infection, and in this report we describe for the first time the molecular characterization of a cDNA from S. haematobium (Sh22·6) that is closely homologous to Sm22·6 and Sj22·6. As a member of the tegument‐associated antigen family, Sh22·6 possesses EF‐hand domains and regions homologous to the dynein light chain domains. We have expressed recombinant Sh22·6 and studied the IgE responses to the antigen in a group of 99 infected individuals (68 children and 31 adults) from an endemic area of Gabon who donated blood before and 5 weeks after praziquantel treatment. IgE to Sh22·6 was detected by ELISA in 18 subjects (18%), and in the majority of responders levels rose between pre‐ and post‐treatment. Interestingly, the proportion of adults expressing IgE to Sh22·6 was 35·5%, significantly higher than the 10·3% seen in children. IgE from at least 10 of the 18 ELISA responders recognized Sh22·6 on Western blots of adult worm extract and recombinant antigen. These results demonstrate that like related molecules in other species, Sh22·6 is a target for the human IgE response. The data also indicate that changes in the IgE response occur with age or with progressive exposure to key antigens.