The Schistosoma mansoni tegumental-allergen-like (TAL) protein family: influence of developmental expression on human IgE responses

Abstract

Background: A human IgE response to Sm22.6 (a dominant IgE target in Schistosoma mansoni) is associated with the development of partial immunity. Located inside the tegument, the molecule belongs to a family of proteins from parasitic platyhelminths, the Tegument-Allergen-Like proteins (TALs). In addition to containing dynein-light-chain domains, these TALs also contain EF-hand domains similar to those found in numerous EF-hand allergens.

Methodology/principal findings: S. mansoni genome searches revealed 13 members (SmTAL1-13) within the species. Recent microarray data demonstrated they have a wide range of life-cycle transcriptional profiles. We expressed SmTAL1 (Sm22.6), SmTAL2, 3, 4, 5 and 13 as recombinant proteins and measured IgE and IgG4 in 200 infected males (7-60 years) from a schistosomiasis endemic region in Uganda. For SmTAL1 and 3 (transcribed in schistosomula through adult-worms and adult-worms, respectively) and SmTAL5 (transcribed in cercariae through adult-worms), detectable IgE responses were rare in 7-9 year olds, but increased with age. At all ages, IgE to SmTAL2 (expressed constitutively), was rare while anti-SmTAL2 IgG4 was common. Levels of IgE and IgG4 to SmTAL4 and 13 (transcribed predominantly in the cercariae/skin stage) were all low.

Conclusions: We have not measured SmTAL protein abundance or exposure in live parasites, but the antibody data suggests to us that, in endemic areas, there is priming and boosting of IgE to adult-worm SmTALs by occasional death of long-lived worms, desensitization to egg SmTALs through continuous exposure to dying eggs and low immunogenicity of larval SmTALs due to immunosuppression in the skin by the parasite. Of these, it is the gradual increase in IgE to the worm antigens that parallels age-dependent immunity seen in endemic areas.

Figure 1. Alignment of amino acids sequences of the SmTAL family.

Helix-loop-helix EF-hand domains (Pfam00036) are indicated. The canonical aspartic acid residues at the start of the loop are denoted D on black background. Residues in the dynein light chain domain (EMBL/EBI IPR001372) are shaded grey. Alignment was performed using Clustal W.

Figure 2. Transcription profiles of SmTALs.

Profiles from the S. mansoni lifecycle microarray data available via Array express (31) under the experimental accession number E-MEXP-2094. Values are mean normalized fluorescence units ± sem. In primate infections, larvae remain in the skin for 2–5 days (Wilson et al. 1990). In the figure cercariae to 3 d schistosomula are denoted “skin stage”.

Figure 3. Electrophoresis of purified SmTAL proteins.

2 µg of each of the indicated proteins was run under reducing conditions on a 4–12% gradient SDS-PAGE gel and stained with Coomassie blue.

Figure 4. Antibody responses to SmTAL1, SmTAL3 and SmTAL5 in the S. mansoni infected cohort.

Recombinant SmTAL1, 3 and 5 were used in ELISA to measure antigen-specific IgE (A) or IgG4 (B) before and after praziquantel treatment in 200 males infected with S. mansoni. Only individuals whose levels exceeded the seropositive threshold (mean+3xSD uninfected controls) for each response are graphed. For the whole cohort, the prevalence of each response (C) is shown in 5 age groups, 7–9 (n = 36), 10–14 (n = 43), 15–24 (n = 35), 25–34 (n = 43) and 35–60 (n = 43) Shown is % seropositive for each group after treatment +95% confidence intervals.

Figure 5. Antibody responses to SmTAL2, SmTAL4 and SmTAL13 in the S. mansoni infected cohort.

Recombinant antigens were used in ELISA to measure IgE (A) or IgG4 (B) before and after treatment in infected cohort. Only individuals whose levels exceeded the seropositive threshold (mean+3xSD uninfected controls) for each response are graphed. The prevalence of each response (C) is shown (% seropositive for each group after treatment +95% confidence intervals).

Figure 6. Expression of SmTAL4 in cercarial tail only.

For PCR analysis (A), total RNA from isolated from heads (H) and tails (T) after mechanical separation and used to prepare cDNA for use with specific primers to generate amplicons for Sm ß-actin (203 bp), SmTAL4 (152 bp), SmTAL5(228 bp) and SmTAL13(206 bp). Products were separated on a 2% agarose gel and detected with ethidium bromide. For immunostaining (B) 8 µ sections of frozen sections of whole cercariae were fixed and stained with rat anti-SmTAL4 antiserum and TRITC -anti-rat antibody (red). Nuclei were counterstained with DAPI (blue). In the negative control (insert) anti- SmTAL4 antiserum was pre-absorbed with recombinant SmTAL4.