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Septin in germ cells of miracidia and sporocysts of schistosomes. Confocal optical sections of a miracidium (panel A) and a two day old sporocyst (B); nuclei stained with DAPI (blue) and actin filaments stained with phalloidin conjugated with Alexa Fluor 568 (green). Probing with anti-SmSEPT10 immunoglobulin (red) revealed the prevalence of septin in germ cells of both miracidia and sporocysts. The insets of A and B highlight germ cell rich regions in these developmental stages. Scale bar, 20 µm.
Source publication
Septins are a family of eukaryotic GTP binding proteins conserved from yeasts to humans. Originally identified in mutants of budding yeast, septins participate in diverse cellular functions including cytokinesis, organization of actin networks, cell polarity, vesicle trafficking and many others. Septins assemble into heteroligomers to form filament...
Citations
... Similar results were also reported for the trematode S. mansoni [102]. In addition, genes encoding these proteins have been found in the genomes of other tapeworms [103] ( Table 1). Tapeworms such as T. solium, E. granulosus, and E. multilocularis have two genes that encode septin 7, one for septin 10 and one for septin 4 [101,103]. ...
... In addition, genes encoding these proteins have been found in the genomes of other tapeworms [103] ( Table 1). Tapeworms such as T. solium, E. granulosus, and E. multilocularis have two genes that encode septin 7, one for septin 10 and one for septin 4 [101,103]. However, ulterior analyses will be useful to understand the role of septins in these parasites. ...
Recent advances have increased our understanding of the molecular machinery in the cytoskeleton of mammalian cells, in contrast to the case of tapeworm parasites, where cytoskeleton remains poorly characterized. The pertinence of a better knowledge of the tapeworm cytoskeleton is linked to the medical importance of these parasitic diseases in humans and animal stock. Moreover, its study could offer new possibilities for the development of more effective anti-parasitic drugs, as well as better strategies for their surveillance, prevention, and control. In the present review, we compile the results of recent experiments on the cytoskeleton of these parasites and analyze how these novel findings might trigger the development of new drugs or the redesign of those currently used in addition to supporting their use as biomarkers in cutting-edge diagnostic tests.
... Based on sequence homology and the number of coiled-coil domains, mammalian septins are classified into four subgroups: septin 2 (septins 3, 9, and 12), septin 6 (septins 6, 8, 10, 11, and 14), and septin 7 (septins 7 and 13) [1,2]. Experimentally, septins have been detected in only two helminths, Schistosoma mansoni [3] and Caenorhabditis elegans [4]. The synthetic cytokinin forchlorfenuron (N-(2chloro-4-pyridyl)-N9-phenylurea or C 12 H 10 ClN 3 O), known 2 Journal of Parasitology Research as FCF, has been shown to alter the stability and function of septin filaments in yeast and mammals [5,6]. ...
... As mentioned earlier, the participation of septins in the cellular biology of parasites is important for the adaptation and survival of these organisms inside their hosts; therefore, it is the key to understand their expression. In the trematode S. mansoni, use of recombinant proteins and confocal microscopy have revealed the existence of four septins [3]. However, no such information has been available for cestodes. ...
... Confocal microscopy observations in the trematode S. mansoni [3] revealed the presence of septins on the surface and in the most adjacent muscle layers of the tegument. There, septins were proposed to interact with muscle fibers expressing actin filaments. ...
Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro , FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.
... Alkaline phosphatase PSp, SSp S. mansoni Ivenchenko et al. 1999 Alkaline phosphatase SSp S. mattheei, S. bovis Kinoti et al. 1971 Calmodulin Peroxiredoxin (Prx1/2) PSp S. mansoni Protein kinase C PSp S. mansoni Ludtmann et al. 2009 Septin/GTP binding protein PSp S. mansoni Zeraik et al. 2013 Serotonin transport receptor PSp S. mansoni Boyle et al. 2000 ...
Features
Explores the biology, pathology, and control of arguably the world's worst parasite
Reviews medication and treatment for Schistosoma
Discusses prevention and diagnosis of Schistosoma
Relates how trapped eggs can cause human morbidity
Summary
Apart from malaria, schistosomiasis is the most prevalent parasitic infection in the world. It affects more than 200 million people in 76 tropical and subtropical countries, causing great suffering and resulting in thousands of deaths. Written by world authorities, this book examines many aspects of the biology, pathology, and control of the schistosoma parasite. Ranging in topic from infection in Pharaonic Egypt, through DNA relationships and biological systems, to advances in development of vaccines against the parasite, this book is a comprehensive text written for researchers and medical professionals alike.
... Here we focus on the septins found in Schistosoma mansoni, the major species responsible for the neglected disease schistosomiasis in America [20]. S. mansoni contains only four septin genes, named with respect to their similarity to human septins-SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2 [21]. As we have previously shown, Schistosome septins can also form heterocomplexes capable of filament assembly, which formed curved and straight bundles as described for other species [6,22]. ...
... The cloning, expression and purification of SmSEPT10, SmSEPT10G and SmSEPT5 was performed as described elsewhere [21]. SmSEPT5G comprises the residues 83e358, SmSEPT10NG and SmSEPT10GC comprises the residues 1e306 and 39e412, respectively. ...
... Based on our findings it is tempting to propose that septins form a mesh on the membrane surface that contributes to the process of membrane deformation and that serves as a scaffold to which other proteins from the exocytotic pathway adhere. Considering the very diverse tissue localization of septins in the Schistosoma life cycle [21], it is expected that septins filaments are very versatile, being involved in several different cellular processes as observed for other models. Therefore, further understating of their properties may help to elucidate mechanism related to several aspects of parasitic lifestyle. ...
Septins are GTP-binding proteins that are highly conserved among eukaryotes and which are usually membrane-associated. They have been linked to several critical cellular functions such as exocytosis and ciliogenesis, but little mechanistic detail is known. Their assembly into filaments and membrane binding properties are incompletely understood and that is specially so for non-human septins where such information would offer therapeutic potential. In this study we use Schistosoma mansoni, exhibiting just four septin genes, as a simpler model for characterizing the septin structure and organization. We show that the biochemical and biophysical proprieties of its SmSEPT5 and SmSEPT10 septins are consistent with their human counterparts of subgroups SEPT2 and SEPT6, respectively. By succeeding to isolate stable constructs comprising distinct domains of SmSEPT5 and SmSEPT10 we were able to infer the influence of terminal interfaces in the oligomerization and membrane binding properties. For example, both proteins tended to form oligomers interacting by the N- and C-terminal interfaces in a nucleotide independent fashion but form heterodimers via the G interface, which are nucleotide dependent. Furthermore, we report for the first time that it is the C-terminus of SmSETP10, rather than the N-terminal polybasic region found in other septins, that mediates its binding to liposomes. Upon binding we observe formation of discrete lipo-protein clusters and higher order septin structures, making our system an exciting model to study interactions of septins with biological membranes.
... This conclusion was based primarily on the ability to obtain crystals/filaments of a fragment of a schistosome septin, SmSEPT10, bound to both GTP and GDP [58]. However, the alpha0 helix was also truncated from the crystallized SmSEPT10 [58], and native SmSEPT10 lacks a polybasic motif [59]. Hence, these results are certainly open to alternative interpretations. ...
Multisubunit protein complexes are essential for cellular function. Genetic analysis of essential processes requires special tools, among which temperature‐sensitive (Ts) mutants have historically been crucial. Many researchers assume that the effect of temperature on such mutants is to drive their proteolytic destruction. In fact, degradation‐mediated elimination of mutant proteins likely explains only a fraction of the phenotypes associated with Ts mutants. Here I discuss insights gained from analysis of Ts mutants in oligomeric proteins, with particular focus on the study of septins, GTP‐binding subunits of cytoskeletal filaments whose structures and functions are the subject of current investigation in my and many other labs. I argue that the kinds of unbiased forward genetic approaches that generate Ts mutants provide information that is largely inaccessible to modern reverse genetic methodologies, and will continue to drive our understanding of higher‐order assembly by septins and other oligomeric proteins.
Also watch the Video Abstract .
... This has allowed for the recent description of four Schistosoma mansoni septins, which have received the names SmSEPT5, SmSEPT7.1, SmSEPT7.2 and SmSEPT10, based on their sequence similarity with human homologues (Zeraik et al., 2013). However, the assembly and specific roles of these proteins in schistosomes has yet to be investigated. ...
... Cercariae were obtained by shedding infected snails under bright light for 2 h at 23 °C. Adult worms were cultured under 5% CO 2 at 37 °C in DMEM, supplemented with 10% FBS and 1×penicillin/streptomycin as described (Dalton et al., 1997;Mann et al., 2009;Zeraik et al., 2013). ...
... The recently described septins from S. mansoni display traits commonly verified in septins, including a conserved GTPase domain containing the G1, G3 and G4 motifs, the septin unique element (SUE), prior to the C-terminal region and the prediction of a coiled-coil structure at the C-terminal domain (Zeraik et al., 2013). Accordingly, it was anticipated that they organize into filamentous heterocomplexes, as in yeast, Drosophila and human cells (Barral and Kinoshita, 2008). ...
Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodeling and scaffolds, and with diverse binding partners. Precise roles for these structural proteins in most processes often remain elusive. Identification of small molecules that inhibit septins could aid in elucidating the functions of septins and has become increasingly important, including the description of roles for septins in pathogenic phenomena such as tumorigenesis. The plant growth regulator forchlorfenuron (FCF), a synthetic cytokinin known to inhibit septin dynamics, likely represents an informative probe for septin function. This report deals with septins of the human blood fluke Schistosoma mansoni and their interactions with FCF. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with FCF, leading to rapid polymerization of filaments. Culturing developmental stages (miracidia, cercariae, adult males) of schistosomes in FCF at 50 - 500 μM rapidly led to paralysis, which was reversible upon removal of the cytokinin. The reversible paralysis was concentration-, time- and developmental stage-dependent. Effects of FCF on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of FCF on fluke motility. The findings implicated a mechanism targeting a molecular system controlling movement in these developmental stages: a direct effect on muscle contraction due to septin stabilization might be responsible for the reversible paralysis, since enrichment of septins has been described within the muscles of schistosomes. This study revealed the reversible effect of FCF on both schistosome motility and its striking impact in hastening polymerization of septins. These novel findings suggested routes to elucidate roles for septins in this pathogen, and exploitation of derivatives of FCF for anti-schistosomal drugs.
... Furthermore, it brings with it also the possibility of identifying new drug and/or vaccine targets (18). Recently, four different septins were identified and described in this organism (19), and these have been classified into three of the four existing subgroups (20 -22). ...
... Expression and Purification of Recombinant SmSEPT10 and SmSEPT10G-SmSEPT10 cDNA was amplified from RNA extracted from adult worms and cloned into the pET28a(ϩ) vector as described previously (19). SmSEPT10G was obtained using the previous construct as template to a new PCR with primers flanking the GTPase domain (residues 39 -306) of SmSEPT10. ...
Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation,
and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins
(1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined.
Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies.
Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage
generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework
for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin
filaments.
Schistosomiasis is a chronic neglected tropical disease, highlighted by the presence of Schistosoma worms, which presents in advanced cases in approximately 80 countries, affecting almost 300 million people. The treatment is based on only one drug, praziquantel, a drug discovered in the 1970s that shows moderate efficacy against the adult parasite, but low efficacy against the larval stages of the parasite. Therefore, the use of only one drug has brought concerns and losses on drug-resistance cases, necessitating the development of new effective chemotherapeutic agents against Schistosoma species. One of the strategies that have been implemented in drug development is the computer-aided drug design (CADD), investigating the structural characteristics of the compounds and targets in order to understand their actions and biological activities through 3D virtual manipulation, as the QSAR applied to ligands and molecular docking applied to a respective biological target. These studies help to extract information and characteristics relevant to the activity, as well as to predict potential applications and activity. Therefore, this chapter will present the main validated biological targets of the genus Schistosoma, as thioredoxin glutathione reductase (TGR), histone deacetylases (HDAC 1, HDAC 8), dihydroorotate dehydrogenase, sirtuin protein and cathepsin L1, as well as reports of CADD in literature applied to the development of drugs against schistosomiasis, providing compounds with high pharmacological potential and high specificity.
Septins are dynamic filament-forming proteins that are recognized as important components of the cytoskeleton and are involved in numerous functions inside the cells, such as cytokinesis, exocytosis, and ciliogenesis and even in defense against pathogenic bacteria. Despite being highly conserved in eukaryotes, there is scarce literature on the role of septins in organisms other than humans and yeast. Therefore, septins from Schistosoma mansoni represent an interesting model to study an unexplored branch of this protein family. Here we described standard protocols for recombinant production and initial characterization of septins from S. mansoni. Septins are notably difficult to purify, mostly due to their tendency to assemble into filaments. Therefore, specific protocols to stabilize these proteins have been developed. In this chapter, we systematically describe protocols to clone, express, and purify schistosome septins. We also describe the use of circular dichroism to assess the folding and stability of septins and use of chromatography to characterize their oligomeric state, bound guanine nucleotide, and GTP hydrolysis. We expect that these protocols may help researchers involved in the study of schistosome septins as well as assist to establish protocols for septins from other organisms.
In the last few years, long non-coding RNAs (lncRNAs) have been widely studied in humans, and their relevance for physiological and pathological conditions has been demonstrated. In parasites, there are only a few works, such as in Plasmodium falciparum, where it was shown that an lncRNA regulates the expression of a gene associated with immune system evasion, also indicating the relevance of understanding the role of this class of RNAs in parasites. In Schistosoma mansoni, in the last 2 years, there were four published articles related to the annotation of lncRNAs in different life cycle stages using RNA-Seq libraries. In order to make this process of lncRNA identification and annotation more accessible to biologists with no bioinformatics training, considering the growing number of S. mansoni RNA-Seq libraries publicly available from different sources, such as ovary tissues from bi-sex and single-sex infections, and the potential of lncRNAs as therapeutic targets, we provide this step-by-step protocol of lncRNA identification and quantification. This guide includes the download of RNA-Seq libraries from a public database and reads processing and mapping against the genome, transcript reconstruction, novel lncRNA identification, transcripts expression level determination, and the identification of differentially expressed lncRNAs.
