Larval excretory-secretory products from the parasite Schistosoma mansoni modulate HSP70 protein expression in defence cells of its snail host, Biomphalaria glabrata

Abstract

Synthesis of heat shock proteins (HSPs) following cellular stress is a response shared by many organisms. Amongst the HSP family, the approximately 70 kDa HSPs are the most evolutionarily conserved with intracellular chaperone and extracellular immunoregulatory functions. This study focused on the effects of larval excretory-secretory products (ESPs) from the parasite Schistosoma mansoni on HSP70 protein expression levels in haemocytes (defence cells) from its snail intermediate host Biomphalaria glabrata. S. mansoni larval stage ESPs are known to interfere with haemocyte physiology and behaviour. Haemocytes from two different B. glabrata strains, one which is susceptible to S. mansoni infection and one which is resistant, both showed reduced HSP70 protein levels following 1 h challenge with S. mansoni ESPs when compared to unchallenged controls; however, the reduction observed in the resistant strain was less marked. The decline in intracellular HSP70 protein persisted for at least 5 h in resistant snail haemocytes only. Furthermore, in schistosome-susceptible snails infected by S. mansoni for 35 days, haemocytes possessed approximately 70% less HSP70. The proteasome inhibitor, MG132, partially restored HSP70 protein levels in ESP-challenged haemocytes, demonstrating that the decrease in HSP70 was in part due to intracellular degradation. The extracellular signal-regulated kinase (ERK) signalling pathway appears to regulate HSP70 protein expression in these cells, as the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor, U0126, significantly reduced HSP70 protein levels. Disruption of intracellular HSP70 protein expression in B. glabrata haemocytes by S. mansoni ESPs may be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host.

Fig. 1

Basal levels of HSP70 protein in schistosome-resistant and schistosome-susceptible snail haemocytes are similar. a An equal number of haemocytes (approximately 1 × 10⁵ cells) from each snail strain were lysed and the proteins processed for western blotting with anti-HSP70 antibodies (upper panel). Blots were subsequently stripped of antibodies with a stripping buffer and re-probed with anti-actin antibodies (lower panel), to confirm equal loading of protein between samples. b Quantitative band analysis was used to calculate mean (±SEM) HSP70 protein levels in each snail strain relative to actin levels; n = 4 for each snail strain, performed in two independent experiments

Fig. 2

S. mansoni ESPs affect HSP70 protein expression in haemocytes from schistosome-susceptible (a) and schistosome-resistant (b) B. glabrata. Haemocytes were left to adhere to individual wells of culture plates before being washed with CBBS and challenged with 20 μg/ml ESPs in CBSS, or left in CBSS alone (controls), for 1 h. Western blots were probed with anti-HSP70 antibodies (upper panels), then stripped of antibodies before being re-probed with anti-actin antibodies to confirm equal protein loading (lower panels). Intensities of immuno-reactive bands were measured and mean change in HSP70 expression calculated (a and b, graphs), relative to control values having a relative value of 1 (and represented by the dotted line). *P ≤ 0.05, **P ≤ 0.01 when compared to control values (n = 4, ±SEM)

Fig. 3

S. mansoni ESPs affect HSP70 expression in schistosome-susceptible and schistosome-resistant B. glabrata haemocytes in a dose-dependent manner. Susceptible (a) and resistant (b) snail haemocytes were challenged with a range of concentrations of S. mansoni ESPs in CBSS (0-20 μg/ml) for 1 h, prior to protein extraction and HSP70 protein detection (upper panels) using western blotting with anti-HSP70 antibodies. Blots were stripped and re-probed with anti-actin antibodies (lower panels) to confirm equal loading of protein. The blots are representative of two independent experiments

Fig. 4

Heating of S. mansoni ESPs removes their ability to suppress HSP70 expression in B. glabrata haemocytes. Schistosome-susceptible snail haemocytes were challenged for 1 h with either S. mansoni ESPs (20 μg/ml) in CBSS, ESPs (in CBSS) heated at 90°C for 5 min, or CBSS alone (control). Protein extracts were processed for western blotting with anti-HSP70 antibodies (upper panel) and anti-actin antibodies (lower panel). The blots are representative of two independent experiments

Fig. 5

Cellular distribution and levels of HSP70 in B. glabrata haemocytes exposed or not exposed to S. mansoni ESPs. a Schistosome-susceptible and schistosome-resistant snail haemocytes were challenged with (+) and without (−) 20 μg/ml ESPs in CBSS for 1 h on glass coverslips before being fixed and subsequently stained with anti-HSP70 primary antibodies and FITC-conjugated secondary antibodies (represented by green); haemocytes were also incubated in rhodamine phalloidin (red) to visualise filamentous actin. Panel b shows a typical haemocyte not exposed to ESPs, additionally stained with DAPI (blue) to visualise the nucleus. Haemocytes were observed with a Leica laser scanning confocal microscope; images show z-axis projections in average pixel brightness mode that were captured using Leica software. The results shown are characteristic of those obtained in at least three independent experiments (bar 10 μm)

Fig. 6

HSP70 expression levels recover after 5 h in ESP-exposed haemocytes from schistosome-susceptible B. glabrata only. Susceptible (a) and resistant (b) snail haemocytes were challenged with 20 μg/ml ESPs in CBSS, or CBSS alone (control), for 1 h followed by 5 h recovery period before haemocyte proteins were extracted and processed for western blotting. Blots were probed with anti-HSP70 antibodies (upper panels) before being stripped and re-probed with anti-actin antibodies (lower panels) to confirm equal protein loading. Intensities of immunoreactive bands were measured and mean change in HSP70 expression calculated (c), relative to control values having a relative value of 1 (and represented by the dotted line). *P ≤ 0.05, **P ≤ 0.01 (n = 6 from three independent experiments, ±SEM). S susceptible, SE susceptible snail haemocytes exposed to ESPs, R resistant, RE resistant snail haemocytes exposed ESPs

Fig. 7

Infection of schistosome-susceptible B. glabrata with S. mansoni reduces HSP70 protein expression in haemocytes. Snails were exposed to miracidia and haemolymph containing haemocytes was extracted and pooled from three infected snails 35 days post-exposure; an equal volume of pooled haemolymph was also obtained from three unexposed snails (control). Haemocytes were then recovered, lysed and proteins processed for western blotting with anti-HSP70 and anti-actin antibodies

Fig. 8

MG132 reduces the suppressive effects of S. mansoni ESPs on B. glabrata haemocyte HSP70 expression. Schistosome-susceptible (a) and schistosome-resistant (b) snail haemocytes were treated for 1 h with or without 20 μg/ml ESPs in CBSS containing MG132 (50 μM), or with either ESPs alone or DMSO (vehicle control, c). Blots were probed with anti-HSP70 antibodies (upper panel) and re-probed with anti-actin antibodies (lower panel). Intensities of immuno-reactive bands were measured and mean change in HSP70 expression calculated (graphs), relative to DMSO control values having a relative value of 1 (and represented by the dotted line). Blots are characteristic of those from at least three independent experiments. *P ≤ 0.05, **P ≤ 0.01 when compared to unchallenged DMSO controls (n = 4, ±SEM)

Fig. 9

Inhibition of ERK signalling suppresses HSP70 expression in B. glabrata haemocytes. Haemocytes from schistosome-susceptible snails were exposed to the MEK inhibitor, U0126 (1 μM or 10 μM) or DMSO vehicle (control) for 1 h prior to protein extraction. Western blots (a) were probed with anti-HSP70 antibodies (upper panel) and anti-actin antibodies (lower panel). Intensities of immuno-reactive bands on blots from three independent experiments were measured and mean HSP70 expression levels calculated (b), with respect to control values represented by 1 and shown as the dotted line. **P ≤ 0.01 when compared to DMSO controls (n = 6, ±SEM)