Cloning and functional characterization of two calmodulin genes during larval development in the parasitic flatworm Schistosoma mansoni

Abstract

Schistosomiasis is endemic in over 70 countries, in which more than 200 million people are infected with the various schistosome species. Understanding the physiological processes underlying key developmental events could be useful in developing novel chemotherapeutic reagents or infection intervention strategies. Calmodulin is a small, calcium-sensing protein found in all eukaryotes and, although the protein has been previously identified in various Schistosoma mansoni stages and implicated in egg hatching and miracidia transformation, few molecular and functional data are available for this essential protein. Herein, we report the molecular cloning, expression, and functional characterization of calmodulin in the miracidia and primary sporocyst stages of S. mansoni. Two transcripts, SmCaM1 and SmCaM2, were cloned and sequenced, and a recombinant SmCaM1 protein was expressed in Escherichia coli and used to generate anti-CaM antibodies. The 2 protein sequences were highly conserved when compared to other model organisms. The alignment of the predicted proteins of both SmCaM1 and SmCaM2 exhibited 99% identity to each other and 97-98% identity with mammalian calmodulins. Analysis of steady-state transcript abundance indicate that the 2 calmodulin transcripts differ in their stage-associated expression patterns, although the CaM protein isotype appears to be constitutively expressed during early larval development. Application of RNAi to larval parasites results in a "stunted growth" phenotype in sporocysts with 30 and 35% reduction in transcript abundance for SmCaM1 and SmCaM2, respectively, and a corresponding 35% reduction in protein level after incubation in double-stranded RNA. Differential expression of CaM transcripts during early larval development and a growth defect-inducing effect associated with partial transcript and protein inhibition as a result of RNAi suggest a potentially important role of calmodulin during early larval development.

Figure 1

(A) Multiple amino acid sequence alignment of the 2 Schistosoma mansoni calmodulin proteins, SmCaM1 and SmCaM2, with other selected calmodulin proteins sharing high homology. Hyphens (-) represent identical amino acid residues and an asterisk (*) represents non-identical amino acid residues. Brackets enclose the EF-hand domains (Ca-binding sites) and the alpha-helix linker domain acting as a flexible tether. Key: FhCaM, Fasciola hepatica (CAL91032); SjCaM, Schistosoma japonicum (AAW27335); HsCaM, Homo sapiens (NP_001734); DmCaMA, Drosophila melanogaster (NP_523710); CeCaM, Caenorhabditis elegans (NP_503386); HcCaM, Haemonchus contortus (CB012646 The alignments were prepared using AlignX from the Vector NTI software package (Invitrogen). (B) Matrix showing percent identity shared between SmCaM1, SmCaM2 and other calmodulins.

Figure 2

Relative transcript abundance of SmCaM1 (A) and SmCaM2 (B) during in vitro larval development of Schistosoma mansoni miracidia, and 1-, 3- and 8-day cultured sporocysts. Transcript levels were measured using realtime quantitative PCR with APDH serving as a non-changing template loading control. Columns represent average transcript abundance in sporocysts relative to miracidia. Error bars represent standard deviation for three separate biological replicates. *P < 0.05 relative to miracidia.

Figure 3

(A) Coomassie blue-stained SDS PAGE gel of purified recombinant (r)SmCaM1 appearing as a single protein band at approximately 18 kDa. This was used for antibody production. (B) Western blot of protein expression levels for native S. mansoni calmodulin during larval development of miracidia and 1-, 3- or 8-day in vitro cultured sporocysts using an anti-rSmCaM1 antibody as probe. Anti-alpha tubulin was used as a control .

Figure 4

Confocal immunofluorescence microscopy of Schistosoma mansoni miracidia (A) and 3-day in vitro developing sporocysts (B). Miracidia and sporocysts were concurrently fixed and permeabilized and probed with anti-SmCaM. SmCaM reactivity was visualized using an Alexa Fluor^®488-conjugated secondary antibody (green). Hoechst was used to visualize DNA/nuclei (blue), while phalloidin (red) was used to visualize actin-rich muscle tissue. In miracidia, arrows indicate non-ciliated sensory papillae, chevrons indicate epidermal plates and pentagons indicate cilia.

Figure 5

A non-denaturing PAGE gel demonstrating the calcium binding properties of SmCaM1. Recombinant SmCaM1 was run out on a nondenaturing PAGE gel containing 1 mM CaCl₂ or 1 mM EDTA in the resolving gel and the gel loading buffer and stained with Coomassie blue. Notice the reduced electrophoretic mobility of SmCaM when run on a PAGE gel containing 1 mM CaCl_2. The gels were aligned with NativeMark protein standards (Invitrogen).

Figure 6

Effects of the CaM antogonist, trifluoperazine, on in vitro miracidium-to-sporocyst transformation in Schistosooma mansoni. Graph depicts a dose-dependent inhibition of miracidial transformation at 18 hr post-treated with 10, 5, 2, and 0.5 µM concentrations of trifluoperazine compared to the DMSO carrier-only control.

Figure 7

RNAi experiments in which larval Schistosoma mansoni were treated with CaM dsRNA or control GFP dsRNA. Relative transcript abundance of SmCaM1 (A) and SmCaM2 (B) after 7 days of incubation in CaM dsRNA or control GFP dsRNA was determined by qPCR using GAPDH as a constitutive template loading control. Transcript abundance levels for SmCaM1 and SmCaM2 dsRNA-treated sporocysts were decreased 30% and 35%, respectively, relative to control treatments. *P < 0.05. (C) Protein levels of SmCaM after treatment with CaM dsRNA compared to GFP dsRNA-treated parasites. Equal concentrations of total proteins were loaded per lane and confirmed using alpha-tubulin as a protein loading control. Protein levels were quantified using scanning densitometry. Notice reduced levels of SmCaM in the dsCaM-treated parasites.

Figure 8

Brightfield images of CaM dsRNA-treated (A) and control GFP dsRNA-treated (B) Schistosoma mansoni sporocysts after 7-days of dsRNA exposure in complete medium. Note the reduced length, indicated with arrows, of more CaM dsRNA-treated sporocysts in comparison to control sporocysts. (C) Sporocyst length measurements are represented by scatter plots with the calculated median values indicated by the horizontal bars within each dsRNA treatment. The two treatments were statistical analyzed using Mann-Whitney U-test within each experiment, P 0.05. This figure is representative of the 3 biological replicates.