Septins of platyhelminths: identification, phylogeny, expression and localization among developmental stages of Schistosoma mansoni

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 filaments and rings. Here, four septins of Schistosoma mansoni are described, which appear to be conserved within the phylum Platyhelminthes. These orthologues were related to the SEPT5, SEPT10 and SEPT7 septins of humans, and hence we have termed the schistosome septins SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2. Septin transcripts were detected throughout the developmental cycle of the schistosome and a similar expression profile was observed for septins in the stages examined, consistent with concerted production of these proteins to form heterocomplexes. Immunolocalization analyses undertaken with antibodies specific for SmSEPT5 and SmSEPT10 revealed a broad tissue distribution of septins in the schistosomulum and colocalization of septin and actin in the longitudinal and circular muscles of the sporocyst. Ciliated epidermal plates of the miracidium were rich in septins. Expression levels for these septins were elevated in germ cells in the miracidium and sporocyst. Intriguingly, septins colocalize with the protonephridial system of the cercaria, which extends laterally along the length of this larval stage. Together, the findings revealed that schistosomes expressed several septins which likely form filaments within the cells, as in other eukaryotes. Identification and localization demonstrating a broad distribution of septins across organs and tissues of schistosome contributes towards the understanding of septins in schistosomes and other flatworms.NIH Shared Instrumentation (S10RR025565)CNPqInstituto Nacional de Ciência e Tecnologia de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI)CAPES (BEX: 9193/11-1

Interactions of amphipathic α-helical MEG proteins from Schistosoma mansoni with membranes

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole–mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane–active peptides

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins

Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodelling 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, 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 forchlorfenuron. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with forchlorfenuron, 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 forchlorfenuron on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of forchlorfenuron 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 forchlorfenuron 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 forchlorfenuron for anti-schistosomal drugs

Expression profiles of septins among developmental stages of <i>Schistosoma mansoni</i>.

<p>Expression analysis by qPCR of each of four septins in seven developmental stages of the blood fluke. Panels A to D represent expression levels of <i>SmSept5</i>, <i>SmSept10</i>, <i>SmSept7.1</i> and <i>SmSept7.2</i>, respectively. Absolute quantification was used to evaluate the expression levels and is presented as copy number per ng of total RNA. Copy number of each transcript was estimated by interpolation of the sample signal from a standard curve for each gene; bars represent standard deviation of the mean of three technical replicates. Mir, miracidia; Spor, sporocyst; Cerc, cercariae; 2 d S, 2 days old schistosomula; 14 d S, 14 day old schistosomula; Fem, adult female; Mal, adult male. (A biological replicate revealed the same trend among stages; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002602#pntd.0002602.s002" target="_blank">Figure S2</a>.)</p

Septin in germ cells of miracidia and sporocysts of schistosomes.

<p>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-<i>Sm</i>SEPT10 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.</p

Evolutionary relationship among septins of humans and <i>Schistosoma mansoni</i>.

<p>Phylogenetic tree (based on Bayesian inference) generated from a multiple alignment of the conserved GTPase domains of septins from <i>S. mansoni</i> and two other informative protostomes and three deuterostomes. The numbers on the tree nodes are posterior probabilities calculated by MrBayes. Branches with the four discrete groups of septins are enclosed by the dotted lines. Species are identified by the small circles of different shapes and colors as indicated in the lower panel.</p

Superficial structures of miracidia and sporocysts of <i>Schistosoma mansoni</i>.

<p>Panels A and C represent a superficial cross section of miracidia and B and D depict a superficial layer of two-day-old sporocysts. The green panels A and B show F-actin stained with phalloidin conjugated with Alexa Fluor 568. The red panels C and D reveal septin structures labeled with anti-<i>Sm</i>SEPT10 immunoglobulin. The upper left inset in panel C highlights an epidermal plate of a miracidium, rich in septins, and in panel A the same region revealed a muscular structure stained with phalloidin. Scale bar, 20 µm.</p

Septins are ubiquitous in tissues of the schistosomulum.

<p>Cross section at an inner intersection of a schistosomulum cultured for 14 days. Panel A: Nuclei stained with DAPI. B: F-actin structure stained with phalloidin. C: Septin labeled with anti-<i>Sm</i>SEPT5. D: Merged channels. Scale bar, 20 µm.</p