Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections

Abstract

Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.

Figure 1. BgLBP/BPI1 is expressed in the albumen gland and is the major protein of B. glabrata egg masses.

(A) Relative expression levels of BgLBP/BPI1 transcripts in the albumen gland (AG), hepatopancreas (Hp), Headfoot (HF), gonads (G), digestive tract (DT) and circulating hemocytes (HC) determined using real-time quantitative PCR (data from 3 independent experiments). Expression has been normalized to expression of S19 and EEF1-α reference genes. (B) Representative western blot showing the content in BgLBP/BPI1 protein in 15 µg total protein from the corresponding snail tissues (legend as in (A). (C) Typical live 2-days old B. glabrata egg mass with eggs (e) containing embryos (em) observed under a phase-contrast reversed microscope. (D) Total egg mass protein content as revealed by silver staining (ss) and egg mass BgLBP/BPI1 revealed by western blot (wb) using a custom-made antibody raised against two BgLBP/BPI1 peptides.

Figure 2. BgLBP/BPI1 bind to LPS and lipid A.

(A) Representative plasmon resonance sensorgrams showing the interactions of BSA (negative control), hBPI (positive control), nBgLBP/BPI1 and rBgLBP/BPI1 with LPS. Purified proteins were immobilized on a CM5 sensor chip and serial LPS dilutions (5, 10, 25, 50, 100 and 200 µg/ml) were injected at a flow rate of 50 ul/min. The responses in RU were recorded as a function of time. Data were fitted to a 1∶1 Langmuir binding model. Similar sensorgrams were obtained with lipid A. (B) Dissociation constants (kD) calculated for each protein.

Figure 3. BgLBP/BPI1s permeabilize membranes and induce death of short LPS E. coli.

(A) Representative flow cytometry profiles of E. coli SBS363 cells exposed to BSA (negative control), hBPI (positive control), native (n) BgLBP/BPI1 and recombinant (r) BgLBP/BPI1 at a concentration of 100 µg/ml, for 1 h. Cells have been stained with the Baclight kit (SYTO 9 and PI) and a total of 60,000 events were recorded for each sample. (B) Quantification of the effect of the four proteins on E. coli SBS363 cell death (as measured in (A)). Cell death has been measured by flow cytometry after 1 hour exposure to 10 or 100 µg/ml proteins. Results are the mean percentages of permeabilized cells ± SE of three independent experiments. Asterisks indicate significant differences with negative control (*p<0.05, ***p<0.001).

Figure 4. BgLBP/BPI1 and hBPI display anti-oomycete activity against Saprolegnia parasitica and S. diclina.

(A–C) Pictures of live (green) or dead (red) Saprolegnia zoospores after exposure to BSA (A), or BgLBP/BPI proteins at 10 µg/ml (B) or 100 µg/ml (C). Zoospores have been stained by the live/dead cell assay (Abcam). Scale bar is 20 µm. (D) Phase contrast observation of zoospores exposed to 10 µg/ml BgLBP/BPI. Arrows show examples of dead cells or cell debris. (E, F) Survival rate of Saprolegnia parasitica (E) and S. diclina (F) after 30 min exposure to buffer alone (Control) or increasing concentrations of rBgLBP/BPI1, nBgLBP/BPI1, hBPI and BSA. Results are mean percentages (± SE) of three independent experiments.

Figure 5. BgLBP/BPI1s and hBPI display anti-oomycete activity against P. parasitica zoospores.

Survival rate of P. parasitica zoospores after exposure to BSA, hBPI, rBgLBP/BPI1 and nBgLBP/BPI1. Zoospores were incubated with proteins at 5 (dark blue), 10 (red), 30 (green) and 100 µg/ml (purple). Negative controls (light blue) are zoospores without treatment. Results are mean percentages (± SE) of three independent experiments.

Figure 6. Injection of BgLBP/BPI1 dsRNA results in a substantial decrease in BgLBP/BPI1 protein in the albumen gland and in egg masses.

(A) Western blots of albumen glands from snails at 9 days and 12 days post injection (Days PI) of dsRNA of luciferase (non-relevant dsRNA) (dsLuc) or BgLBP/BPI1 (dsBgLBP/BPI1). Actin was used as an endogenous control for protein loads. (B) Western blots of egg masses laid by snails at 18 and 21 days post injection of dsRNAs. Note that control for actin or total protein contents were not appropriate for egg masses and that protein loads have been standardized towards egg mass dry weights. Western Blots were performed using a custom-made antibody raised against two BgLBP/BPI1 peptides.

Figure 7. BgLBP/BPI1 is essential to protect eggs from oomycete infection.

Typical egg mass from Luc- (A) or BgLBP/BPI1 (B) dsRNA-injected parents, after exposure to S. diclina zoospores. Note the well-developed mycelium (my). Egg masses from (A) and (B) have been laid 19–21 days after dsRNA injection to parents and have been exposed to zoospores at day 22. Observation was made after 8 days exposure to S. diclina zoospores.