Differential spatial repositioning of activated genes in Biomphalaria glabrata snails infected with Schistosoma mansoni

Abstract

Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown in vitro that Biomphalaria glabrata embryonic cells co-cultured with schistosome miracidia display genes changing their nuclear location and becoming up-regulated. This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.

Figure 1. Representative images of the erosion script analysis.

Image A displays a composite cartoon of the nucleus where the computer script has outlined the DAPI signal staining DNA (blue) and created five shells of equal area. The script measures the intensity of the fluorescent signals from both the genes (green) and the DAPI and records these. In order to normalise the data, the percentage of gene signal in each shell is divided by the DAPI signal for the corresponding shell. The data can then be plotted as a bar chart. Images B, C, and D are displaying genes having peripheral, intermediate, and internal positions respectively. Scale bar = 10 µm.

Figure 2. Charts displaying the in vivo radial positioning and expression profile of B. glabrata actin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni to normal miracidia (top row of charts, in red) or irradiated attenuated miracidia (bottom row of charts, in grey) over 30 minutes, 2 hours, 5 hours and 24 hours.

B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and q-RT-PCR was performed (middle chart). In the NMRI snail strain actin is repositioned within interphase nuclei after infection and this is correlated with changes in gene expression (A). No repositioning is observed in the BS-90 snails, however the gene is expressed 30 minutes after infection (B). No repositioning of gene loci or change in expression was observed for actin in snails infected with attenuated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P<0.05). Student's t-test between normalized gene signal in each shell of NMRI snail nuclei compared with BS90 snail nuclei are indicated by a red asterisk (P<0.05). Error bars = S.E.M.

Figure 3. Charts displaying the in vivo radial positioning and expression profile of B. glabrata Hsp 70 gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey).

B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH and RNA was isolated and q-RT-PCR performed (middle chart). In the NMRI strain snail's Hsp 70 gene is repositioned after infection from an intermediate position to a more internal position within the nuclei. This repositioning is directly correlated with changes in gene expression 2 hours after infection (A). No repositioning or change in expression is observed in the BS-90 strain snails (B). No evidence for the relocation of the Hsp 70 gene loci and no induction of Hsp 70 expression were detected by qRT-PCR when the two snail lines were infected with irradiated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P<0.05). Student's t-test between normalized gene signal in each shell of NMRI snail nuclei compared with BS90 snail nuclei are indicated by a red asterisk (P<0.05). Error bars = S.E.M.

Figure 4. Charts displaying the in vivo radial positioning and expression profile of B. glabrata ferritin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey).

B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and a q-RT-PCR was performed (middle chart). In the NMRI strain ferritin is repositioned in snails infected with both normal and irradiated miracidia. However the gene is up-regulated only in snails infected with irradiated miracidia (A). Repositioning of ferritin gene loci is observed in the BS-90 strain snails infected with normal and irradiated miracidia, and this is correlated with up-regulation of its expression (B). Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P<0.05). Student's t-test between normalized gene signal in each shell of NMRI snail nuclei compered with BS90 snail nuclei are indicated by a red asterisk (P<0.05). Error bars = S.E.M.