Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host

Abstract

Background: The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised.

Methods and principal findings: All gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum.

Conclusions/significance: Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite's energy metabolism or stress responses.

Figure 1. Genes encoding glycosyl transferases.

In this and the subsequent figures the relative levels of transcription are shown, with the stage having the lowest expression set to unity. Each locus is labeled with its systematic identity and gene product. Of the 87 glycosyl transferases in the S. mansoni genome, 21 were differentially expressed in the three life cycle stages studied. The majority of the up-regulated glycosyl transferase genes were in the germ ball (14/21), with fewer in the cercaria and day 3 schistosomulum (five and four, respectively). This correlates with the known high levels of protein glycosylation of the glycocalyx and gland cell secretions of the cercaria.

Figure 2. Genes whose products are involved in aerobic metabolism.

The relative expression levels of the thirteen differentially expressed genes encoding proteins involved in aerobic metabolism are shown. The majority (9/13) were up-regulated in the cercaria, with 7 in the germ ball, and none in the day 3 schistosomulum. This reflects the intensive energy requirements of the rapidly swimming cercaria.

Figure 3. Genes whose products are involved in development.

More than half of the differentially expressed developmental genes were enriched in the germ ball (18/31). In the cercaria, 8/31 were up-regulated, and in the day 3 schistosomulum 11/31. Four genes were up-regulated in both the germ ball and schistosomulum compared to the cercaria, while two were enriched in the germ ball and cercaria compared to the schistosomulum. These data highlight the differences in developmental processes occurring in each of the larval stages.

Figure 4. Genes encoding tegument proteins.

The genes in this custom category encode proteins that have been identified at the adult worm tegument surface by proteomics or localization studies. Of the 30 differentially expressed genes, 20 were up-regulated in the schistosomulum, 13 in the cercaria and three in the germ ball, with seven up-regulated in both the cercaria and schistosomulum. These data reveal the early transcription of genes involved in remodeling the tegument on parasite entry into the mammalian host, a process that continues thereafter with an orthologue of human CD59 enriched 75 times compared to the germ ball.

Figure 5. Genes encoding serine proteases.

The differential expression patterns of 27 of the 78 serine proteases encoded in the S. mansoni genome. Seventeen were up-regulated in the germ ball, of which 10 encoded cercarial elastases, expressed 10–65-fold higher than in the day 3 schistosomulum. Eleven serine proteases were enriched in the cercaria, and five in the schistosomulum. The study reveals a wider range of cercarial elastases potentially involved in skin invasion than hitherto reported.

Figure 6. Genes encoding metalloproteases.

The majority (22/35) of differentially expressed metalloprotease genes were up-regulated in the germ ball. This includes six invadolysins with expression levels ranging from 2 to 27-fold higher than either of the other life cycle stages. By contrast, only seven genes were up-regulated in the schistosomulum, the highest fold change being ×10. These observations strongly implicate a range of metalloproteases in the skin invasion process.

Figure 7. Genes encoding cysteine proteases.

The majority of cysteine protease genes were up-regulated in the schistosomulum, the fold changes for three being >40×, with fewer in the cercaria and germ ball. We infer that the up-regulation seen in the schistosomulum is related to the early differentiation of the parasite gut.

Figure 8. Genes encoding Venom Allergen-like proteins (VALs).

Of the 28 VALs in the genome, 14 were shown to be differentially expressed in the three life cycle stages. Only two were up-regulated in the schistosomulum, six were most highly expressed in the cercaria, three of which were also up-regulated in the germ ball. Six were most highly expressed in the germ ball, two of which were also enriched in the cercaria. These expression patterns imply that the functions of these enigmatic proteins are mostly associated with entry into the mammalian host. Only the gene for VAL 7 appears to be activated after infection.