Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib.

In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib's deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6-8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments.

Imatinib treatment causes substantial transcriptional changes in adult Schistosoma mansoni in vitro exhibiting pleiotropic effects.

BACKGROUND: Schistosome parasites cause schistosomiasis, one of the most important infectious diseases worldwide. For decades Praziquantel (PZQ) is the only drug widely used for controlling schistosomiasis. The absence of a vaccine and fear of PZQ resistance have motivated the search for alternatives. Studies on protein kinases (PKs) demonstrated their importance for diverse physiological processes in schistosomes. Among others two Abl tyrosine kinases, SmAbl1 and SmAbl2, were identified in Schistosoma mansoni and shown to be transcribed in the gonads and the gastrodermis. SmAbl1 activity was blocked by Imatinib, a known Abl-TK inhibitor used in human cancer therapy (Gleevec/Glivec). Imatinib exhibited dramatic effects on the morphology and physiology of adult schistosomes in vitro causing the death of the parasites. METHODOLOGY/PRINCIPAL FINDINGS: Here we show modeling data supporting the targeting of SmAbl1/2 by Imatinib. A biochemical assay confirmed that SmAbl2 activity is also inhibited by Imatinib. Microarray analyses and qRT-PCR experiments were done to unravel transcriptional processes influenced by Imatinib in adult schistosomes in vitro demonstrating a wide influence on worm physiology. Surface-, muscle-, gut and gonad-associated processes were affected as evidenced by the differential transcription of e.g. the gynecophoral canal protein gene GCP, paramyosin, titin, hemoglobinase, and cathepsins. Furthermore, transcript levels of VAL-7 and egg formation-associated genes such as tyrosinase 1, p14, and fs800-like were affected as well as those of signaling genes including a ribosomal protein S6 kinase and a glutamate receptor. Finally, a comparative in silico analysis of the obtained microarray data sets and previous data analyzing the effect of a TGFßR1 inhibitor on transcription provided first evidence for an association of TGFß and Abl kinase signaling. Among others GCP and egg formation-associated genes were identified as common targets. CONCLUSIONS/SIGNIFICANCE: The data affirm broad negative effects of Imatinib on worm physiology substantiating the role of PKs as interesting targets.

Protein kinases as potential targets for novel anti-schistosomal strategies.

Schistosome parasites are the causative pathogens of schistosomiasis (bilharzia), a disease of worldwide significance. In terms of patient numbers, schistosomiasis ranks second to malaria as a parasitosis affecting more than 200 million people of the tropics and subtropics. Since the 1970s Praziquantel (PZQ) is the drug of choice and nearly exclusively used for treatment. However, drug resistance is an increasing threat, particularly with respect to large-scale PZQ administration programs. Last decade's research indicated that resistance against PZQ can be induced under laboratory conditions, and field studies provided first indications for the possibility of reduced PZQ efficacy. Furthermore, clear evidence for the molecular armamentarium of schistosomes with multidrug transporters was found, one of which was responding to PZQ challenge. Also the development of a vaccine still represents an elusive goal, although effort and time have been invested in this subject. In light of these facts it is commonly accepted that new drugs are urgently needed. Research on signal transduction processes in Schistosoma mansoni has provided an unexpected and novel perspective towards this end. Molecular, biochemical, and physiological studies elucidating principles of schistosome development have demonstrated the essential role of protein kinases (PKs). In humans, PKs are known to be involved in cancer development. Since a variety of approved anticancer drugs targeting PKs exist, first studies have been performed to investigate whether these drugs are able to also inhibit schistosome PKs. Indeed, promising results have been obtained indicating the potential of PKs as privileged targets for new concepts in fighting schistosomes.

Characterization of the Src/Abl hybrid kinase SmTK6 of Schistosoma mansoni.

Cellular protein-tyrosine kinases play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner, the cellular protein-tyrosine kinase SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in this study confirmed the first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (herbimycin A) and Abl (imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners, we identified among others the known Src kinase SmTK3 and the Venus kinase receptor SmVKR1 of S. mansoni by yeast two-hybrid analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes, it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a receptor tyrosine kinase, Syk and Src kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.

Characterization of the cGMP-dependent protein kinase SmcGK1 of Schistosoma mansoni

Schistosomes are trematode parasites and of worldwide medical importance for humans and animals. Growth and development of these parasites require a specific host environment, but also permanent communication processes between the two genders. Accumulating molecular evidence indicates that the responsible interactions are mediated by signal transduction processes. Conserved signaling molecules were identified, and first approaches made for their characterization. However, no representative of the conserved family of cGMP-dependent protein kinases (cGKs) has been described in this parasite yet. Within the Schistosoma mansoni genome data-set we identified cGK homologs, of which one was investigated in more detail in this study. We present the cloning of SmcGK1, whose sequence shows homology to cGKs of higher eukaryotes. SmcGK1 was found to be gender-independently transcribed in adult schistosomes. The occurrence of SmcGK1 sense and antisense transcripts suggests that the expression of this gene is controlled at the post-transcriptional level. In situ hybridization experiments demonstrated a gonad-preferential expression profile in both genders indicating a role of SmcGK1, at least during sexual development of schistosomes. Using a cGK-specific inhibitor to treat adult schistosomes in vitro finally resulted in a multifaceted phenotype including slow motion, oocyte congestion, and reduced egg production.

Esquistossomos são parasitas trematodos de importância médica em todo o mundo para o homem e os animais. O crescimento e o desenvolvimento destes parasitas requerem um ambiente específico do hospedeiro, mas também um processo de comunicação permanente entre parasitas dos dois sexos. Evidência molecular tem se acumulado e indica que as interações são mediadas por processos de transdução de sinal. Moléculas sinalizadoras conservadas foram identificadas, e as primeiras abordagens têm sido feitas para sua caracterização. Contudo, não foi ainda descrito nenhum representante da família conservada das proteína-quinases dependentes de cGMP (cGKs) neste parasita. Analisando o genoma do Schistosoma mansoni nós identificamos homólogos de cGK, dos quais um foi investigado em mais detalhe no presente estudo. Aqui apresentamos a clonagem do gene SmcGK1, cuja sequência mostra homologia com cGKs de eucariotos superiores. Smc- GK1 foi detectada como sendo transcrita de forma gêneroindependente em esquistossomos adultos. A ocorrência de transcritos de SmcGK1 senso e antisenso sugere que a expressão deste gene é controlada em nível pos-transcricional. Experimentos de hibridização in situ demonstraram uma expressão preferencial nas gônadas em ambos os gêneros, indicando um papel para SmcGK1, pelo menos durante o desenvolvimento de esquistossomos. Usando um inibidor específico de cGK para tratamento de esquistossomos adultos in vitro finalmente resultou em um fenótipo multifacetado, incluindo movimentos lentos, congestão dos oócitos, e redução da produção de ovos.

Schistosoma mansoni Polo-like kinase 1: A mitotic kinase with key functions in parasite reproduction.

Polo-like kinases (Plks) are conserved regulators of mitosis. In mammals, Plk1 is over-expressed in a wide range of tumour cells and constitutes a valuable target for anti-cancer therapy. This work presents the characterisation of the Plk1 homologue (SmPlk1) of Schistosoma mansoni, a trematode responsible for schistosomiasis, one of the most important parasitic diseases, second only to malaria. The intense levels of disease transmission and the severity of pathologies are the consequences of the exceptional reproductive activity of schistosomes, in which Plks may play a decisive role. Structural and functional analyses of SmPlk1 have demonstrated its homology with other Plk1 members and its conserved function in mitotic processes. Activation of SmPlk1 was shown to be dependent on phosphorylation of its conserved threonine residue (T(182)) and the ability of active SmPlk1 to induce mitosis was demonstrated in the Xenopus oocyte model. SmPlk1 transcripts were detected abundantly in parasite stages containing a high amount of germinal cells. A potential role of SmPlk1 in mitosis and/or meiosis in schistosomes was supported by the in situ detection of SmPlk1 transcripts in female vitelline cells and oocytes as well as in male spermatocytes. Several Plk inhibitors were shown to inhibit SmPlk1 activity in Xenopus oocytes, and BI 2536 (the first-in-class prototype Plk1 inhibitor) induced in vitro dramatic alterations in schistosome gonads, which affected oogenesis and spermatogenesis. These results indicate a major role for SmPlk1 in parasite reproduction and suggest its importance as a potential new target against schistosomiasis.

The Syk kinase SmTK4 of Schistosoma mansoni is involved in the regulation of spermatogenesis and oogenesis.

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.

Molecular and functional characterisation of the heat shock protein 10 of Strongyloides ratti.

Strongyloides stercoralis and S. ratti are intestinal parasitic nematodes infecting rats and humans, respectively. Both present extraordinary life cycles comprising a free-living generation in addition to parasitic stages. In search of molecules possibly involved in parasite-host interaction, we performed mass spectrometry to identify excretory/secretory products of S. ratti. Amongst others we detected homologs of the heat shock proteins HSP10 and HSP60 (Sr-HSP10 and Sr-HSP60). HSPs are well known as chaperones involved in stress responses of cells, but recent studies suggest additional roles of small HSPs for parasite biology including immune modulation. To characterise Sr-HSP10, we cloned its full-length cDNA, analysed the genomic organisation, tested its presumptive role as an interaction partner of Sr-HSP60, studied its transcription in the parasite, and expressed the protein to test its immune responses. The cDNA contains an open reading frame of 330bp encoding a polypeptide of 110 amino acids with an approximate molecular weight of 10kDa. The Sr-HSP10 protein is highly homologous to that of the human pathogen S. stercoralis with only eight amino acid substitutions. Analysis of the genomic organisation of the Sr-HSP10 locus revealed that the gene is linked head-to-head to the gene encoding Sr-HSP60, and both share a bidirectional promoter. RT-PCR experiments indicated potential independent expression of the Sr-HSPs genes. In situ hybridisation results demonstrate Sr-HSP10 transcription in the gut area. Mammalian and yeast two-hybrid assays show dimerisation of Sr-HSP10, but no binding to recombinant Sr-HSP60. Immunisation experiments finally revealed a strong immunogenicity of Sr-HSP10 and provided evidence for a role in regulating the host-parasite interaction.

Schistosomiasis and the molecular biology of the male-female interaction of S. mansoni.

Parasitic helminths of the genus Schistosoma are the causative agents of schistosomiasis, an infectious disease affecting humans and animals. For humans, it is one of the most prevalent parasitemias in the world, second behind malaria. Estimates of the World Health Organisation (WHO) indicate that more than 200 million people live in endemic areas (WHO, Fact Sheet No 115). With respect to animals, a number of 530 million cattle is estimated to live in endemic territories of Africa and Asia. In the last two decades ambitious efforts have been made to develop an effective vaccine against schistosomes, but without resounding success. In addition, there is a pressing need to develop new anthelmintics due to the potential emerging resistance against the commonly used drug praziquantel. Therefore, the understanding of essential physiological or developmental processes of schistosome biology and attempts to intervene in these processes may open new ways to control the parasite. Towards this end, one possibility is to study the unusual biology of schistosomes. These digenean parasites differ from other parasitic flukes by living in the blood vessels. Furthermore, schistosomatids are the only bisexual family of the class trematoda. A nearly unique phenomenon in nature is that a continuous pairing-contact is essential for the development of the reproductive organs of the female, an aspect for the possible design of novel control strategies.