Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

Abstract

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

Keywords: Schistosoma; expressed sequence tags; gene function; gene regulation; protein domains; sequence analysis; serial analysis of gene expression; universal stress proteins.

Figure 1.

Clustal guide tree of the 8 Schistosoma mansoni Universal Stress Proteins. Guide tree of the similarity of the protein sequences to each other as constructed from a multiple sequence alignment of the S. mansoni universal stress protein sequences and visualized using FigTree (http://tree.bio.ed.ac.uk/software/figree).

Figure 2.

Multiple Sequence Alignment of the Schistosoma mansoni and Methanocaldococcus jannaschii universal stress protein sequences. Six USP of S. mansoni and USP (MJ_0577) of Methanocaldococcus jannaschii were aligned using ClustalW. Alignment shows the motif G2xG9xG(S/T) (indicated by series of #) that contain ATP binding residues was present in the six S. mansoni USPs (Smp_001000, Smp_001010, Smp_031300, Smp_043120, Smp_076400) as well as in MJ_0577, a known ATP-binding universal stress protein. Residues making contacts with ATP are indicated with “=” according annotation to Zarembinski et al. Abbreviations: A, Adenine; R, Ribosyl; P, Phosphoyl groups.

Figure 3.

Ligand binding residues in Schistosoma mansoni universal stress protein sequences. Using the 12 binding site residues in 1MJH_B as a reference set, the residues that align to the binding sites in the 8 Schistosoma mansoni universal stress proteins (USPs) were identified. The following 12 binding amino acid residues (Ala, Arg, Asp, Gly, Ile, Leu, Met, Pro, Ser, Thr, Tyr and Val) from the S. mansoni USP sequences were aligned to the ligand binding sites of 1MJH_B. A website that allows for interaction with the dataset presented in Table 4 can be accessed at http://public.tableausoftware.com/views/schisto_mansoni/ligand_residues.

Figure 4.

Homology model of Schistosoma mansoni universal stress protein Smp_031300. On the 3-dimenensional structure, the ligand binding residues are indicated in different colors. Magenta represents location for the ATP binding residues (Gly124, Arg126, Gly127, Gly137, Ser138) and the green represent other binding residues (Pro12, Ile13, Asp14, Val42, Ile123 Val139, Ser140). Ser138 (black) indicates the phosphorylation site.

Figure 5.

Reverse Transcriptase-PCR assessment of mRNA expression for genes encoding universal stress proteins in strain LE of Schistosoma mansoni. (A) and (B): Primers were constructed to assess mRNA expression for genes encoding the 8 S. mansoni USP genes (four shown, A and B) in strain LE of S. mansoni. Sizes of the amplified bands are shown relative to a 1kb DNA Ladder Promega. Life cycle developmental stages examined were Egg (E), Miracidium (Mi), Schistosomula (Sc), Male (M), Female (F). The lane labeled C^– is negative control. (C) The alpha-tubulin (α-tubulin) mRNA was used as a constitutively expressed positive control for the RT-PCR. In the case of the USP genes, only Smp_001000 and Smp_043120 were amplified in at least one stage.

Figure 6.

Integration of evidence of gene expression from Expressed Sequence Tags (EST) and Serial Analysis of Gene Expression (SAGE) of the 8 Schistosoma mansoni USP genes in developmental stages. Red represents expression being detected and green represents no evidence of expression. The SchistoDB IDs are indicated on the right side. Abbreviations: Mir, Miracidia; Spo, Sporocyst; Cer, Cercariae; Sch, Schistosomula; Mal, Male; Fem, Female; Adu, Adult.

Figure 7.

Heat map showing expression of Smp_031300, Smp_043120, and Smp_076400. Heat map visualization of differentially and constitutively expressed transcripts for Smp_031300 (Contig2193), Smp_043120 (Contig1699), and Smp_076400 (CD189581) in the life cycle stages of Schistosoma mansoni.