Characterization of immune genes from the schistosome host snail Biomphalaria glabrata that encode peptidoglycan recognition proteins and gram-negative bacteria binding protein

Abstract

Peptidoglycan (PGN) recognition proteins (PGRPs) and gram-negative bacteria binding proteins (GNBPs) play an essential role in Toll/Imd signaling pathways in arthropods. The existence of homologous pathways involving PGRPs and GNBPs in other major invertebrate phyla such as the Mollusca remains unclear. In this paper, we report four full-length PGRP cDNAs and one full-length GNBP cDNA cloned from the snail Biomphalaria glabrata, the intermediate host of the human blood fluke Schistosoma mansoni, designated as BgPGRPs and BgGNBP, respectively. Three transcripts are generated from a long form PGRP gene (BgPGRP-LA) by alternative splicing and one from a short form PGRP gene (BgPGRP-SA). BgGNBP encodes a putative secreted protein. Northern blots demonstrated that expression of BgPGRP-SA and BgGNBP was down-regulated in B. glabrata at 6 h after exposure to three types of microbes. No significant changes in expression were observed in snails at 2 days post-exposure (dpe) to the trematodes Echinostoma paraensei or S. mansoni. However, up-regulation of BgPGRP-SA in M line snails at later time points of infection with E. paraensei (i.e., 12 and 17 dpe) was observed. Our study revealed that exposure to either microbes or trematodes did not alter the expression levels of BgPGRP-LAs, which were consistently low. This study provides new insights into the potential pathogen recognition capabilities of molluscs, indicates that further studies of the Toll/Imd pathways in this phylum are in order, and provides additional ways to judge the importance of this pathway in the evolution of internal defense across the animal phyla.

Fig. 1

Nucleotide and deduced amino acid sequences of a BgPGRP-LA (GAN: EF452347) and b BgPGRP-SA (GAN: EF452346). As compared to BgPGRP-LA, the light shaded region is spliced out of BgPGRP-LA1 (GAN: EF452348). For BgPGRP-LA2 (GAN: EF452349), the sequences in both shaded boxes are spliced out. The primers used for RT-PCR to detect the three isoforms of BgPGRP-LA are indicated by dotted arrow lines. Three nucleotides (tag) which are bolded at the 5′UTR is a stop codon at the open reading frame. The signal peptide (SP) sequence and the putative PGRP domain are underlined by dotted and solid lines, respectively. Numbers in the left margin designate nucleotides (Roman) and amino acids (italicized)

Fig. 2

a Schematic representation of BgPGRP-LA, -LA1, -LA2, and -SA (not drawn to scale). The SP and the putative PGRP and amidase_2 domains were predicted by the SMART program. b The three alternatively spliced forms of BgPGRP-LA revealed by RT-PCR using cDNA as template. The location of the RT-PCR primers is indicated in Fig. 1a. RNA was extracted from M line snails at 2 dpe to S. mansoni (lane 1), 2 dpe to E. paraensei (lane 2) or from unexposed control snails (lane 3). Lane 4 is DNA size markers in base pairs (bp). Band intensity was found to be variable among replicates so does not simply reflect the magnitude of the response to each parasite

Fig. 3

Southern blot analyses of a BgPGRP-LA, b BgPGRP-SA, and c BgGNBP. Three restriction enzymes, EcoRI (lane 1), HindIII (lane 2) and PstI (lane 3) were used to digest genomic DNA. Size markers in kilobases (kb) are indicated on the left

Fig. 4

a Nucleotide and deduced amino acid sequences of BgGNBP (GAN: EF452345). The SP sequence is indicated by a dotted line, and the glucanase-like domain (Pfam: glyc_hydro_16) is underlined. b Schematic diagram of BgGNBP (not drawn to scale)

Fig. 5

a Structure-based sequence alignment of PGRPs guided by the crystal structures of DmPGRP-LB, -SA, and -LE (Kim et al. 2003; Reiser et al. 2004; Lim et al. 2006). The open circles show the five conserved amino acids, and the filled circle indicates the Arg residue critical for the discrimination of DAP-type PGN. The dark and light shaded boxes show regions of sequence identity and of conserved replacements (BLOSUM62 matrix) of greater than 80%, respectively, in the sequences aligned. The five conserved residues critical for enzymatic activity in T7 isozyme are in bold. The numbers on the right side indicate the position from the start codon in each aa sequence. Dm Drosophila melanogaster. The GenBank accession numbers: DmPGRP-LB (Q9VGN3); DmPGRP-LC (Q9GNK5); DmPGRP-LE (Q9VXN9); DmPGRP-SA (Q9VYX7); DmPGRP-SD (Q9VS97); DmPGRP-SC1B (Q95SQ9); bacteriophage T7 isozyme (P00806). b Alignment of the glucanase-like region of the representative GNBPs. The dark and light shaded boxes show regions of sequence identity and of conserved replacements (BLOSUM62 matrix) of greater than 85% (6/7), respectively. Asterisks indicate the residues required for glucanase activity, as assessed from bacterial studies. The numbers on the both sides indicate the position of amino acids related to the start codon of the aa sequence. Bacillius circulans β-1,3-glucanase A1 (P23903), Thermotoga neapolitana β-glucanase (Z47974), Moth (M. sexta) βGRP (AAF44011), DmGNBP1 (AAF33849), DmGNBP3 (AAF33851)

Fig. 6

Northern blots showing expression of a BgPGRP-LAs, b BgPGRP-SA, and c BgGNBP in M line or BS-90 snails (6–9 mm). RNA was extracted from snails that were injected with PBS (lane 1), S. aureus (lane 2), E. coli (lane 3), or S. cerevisiae (lane 4; for details, see “Materials and methods”). Lower panels show rRNA stained with GelRed used as loading controls. Size markers in kilobases (kb) are indicated on the right (same as Fig. 7)

Fig. 7

Northern blots showing the expression of a BgPGRP-LAs, b BgPGRP-SA, and c BgGNBP in M line and BS-90 snails (6–9 mm) exposed to E. paraensei or S. mansoni. For M line snails, lanes 1 and 2 show mRNA from snails at 12 and 17 dpe to E. paraensei. Lanes 3 to 5 show the expression level of three genes in snails that were not exposed (lane 3) or were exposed for 2 days to E. paraensei (lane 4) or to S. mansoni (lane 5). For BS-90 snails, lanes 1 to 3 show mRNA abundance of the three genes from snails that were not exposed (lane 1) or that were exposed for 2 days to E. paraensei (lane 2) or to S. mansoni (lane 3). Please note that the BgGNBP band is faint because the membrane had been stripped three times. Other Northern blots revealed that if the membrane was first probed by BgGNBP, it showed high expression level in all RNA samples, and differential expression was not observed (data not shown). In the case of BgPGRP-SA, the bands were intense, even though the membrane may have been previously stripped (the one shown here was stripped three times). For BgPGRP-LAs, the bands were always faint even on new membranes. Among the three genes, expression from lowest to highest was BgPGRP-LAs, BgGNBP, and BgPGRP-SA

Fig. 8

A gene tree using minimum evolution (ME) showing relationships among a PGRPs or b GNBPs based on amino acid sequences. For each sequence, a protein name and a common name of species are provided. Bootstrap values less than 50% are not shown. Single- or double-filled circles indicated on the branches indicate bootstrap support of 50–80% and 81–100%, respectively. For a depicting relationships among PGRP sequences, the presence/absence of each of the five conserved residues associated with amidase activity is provided under the protein's name, as is the presence/absence of the Arg residue responsible for discrimination of the DAP-type PGN (see Fig. 5a, b for the positions of the residues). In b showing relationships among GNBPs, the delineation between groups A and B is shown with a dotted line (100% bootstrap value). In group A, the presence/absence of the four conserved residues associated with glucanase activity is provided under each protein's name. In group B, the four residues are the same (W, E, D, E) for all sequences. The GenBank accession numbers for all sequences in a are as follows (starting from Human PGRP3, clockwise direction). Human PGRP3 (AAI28115), human PGRP4 (AAI07158), Drosophila melanogaster PGRP-LF (NP_648299), Drosophila PGRP-LC (NP_996030), tsetse fly Glossina morsitans PGRP-LC (ABC25065), Drosophila PGRP-LE (NP_573078), Indian eri silkmoth Samia cynthia PFRP-LC (BAF03521), mosquito Anopheles gambiae PGRP-L (XP_321943), mosquito Aedes aegypti PGRP-S (ABF18154). Drosophila PGRP-LB (NP_731576), Anopheles PGRP (Q7PP76), honey bee Apis mellifera PGRP-SC2 (XP_395941), squid Euprymna scolopes PGRP4 (AAY27976), squid PGRP2 (AAY27974), squid PGRP1 (AAY27973), squid PGRP3 (AAY27975), starfish Asterias rubens PGRP-S1a (DQ222477), starfish PGRP-S2a (DQ222478), Bay scallop Argopecten irradians PGRP (AY437875), Zhikong scallop Chlamys farreri PGRP-S1 (AAY53765), human PGRP-L (NP_443122), sea urchin Strongylocentrotus purpuratus PGRP-2 (XP_796422), human PGRP1 (NP_005082), Drosophila PGRP-SC1B(AAG23736), Drosophila PGRP-SD (NP_648145), yellow mealworm Tenebrio molitor PGRP-SA (BAE78510 ), Drosophila PGRP-SA (NP_572727), honey bee PGRP-SA (XP_001123180), silkworm Bombyx mori PGRP seq 1 (BAA77209), silkmoth Antheraea mylitta PGRP seq 1 (ABG72708), silkmoth PGRP seq 2 (ABG72709), Indian eri silkmoth PGRP-A (BAF03522), hornworm Manduca sexta PGRP-1A (AAO21509), silkworm PGRP seq 2 (AAL32058), Drosophila PGRP-LA (NP_996029). GenBank accession numbers in b are as follows (starting from mosquito Aedes GNBP seq 1, clockwise direction). Aedes aegypti GNBP seq 1 (EAT44801), Anopheles gambiae GNBP seq 1 (Q7QA32), Aedes GNBP seq 2 (EAT41280), Anopheles GNBP seq 2 (Q7PQA4), Aedes GNBP seq 3 (EAT44802), Aedes GNBP seq 4 (EAT 38985), Anopheles GNBP seq 3 (Q7QCT6), Anopheles GNBP seq 4 (Q7QCT7), Aedes GNBP seq 5 (EAT38986), Termite Nasutitermes fumigatus GNBP2 (AAZ08496), Termite GNBP1 (AAZ08483), Snail B. glabrata βGBP (EF121824), Zhikong scallop βGBP (AAP82240), signal crayfish Pacifastacus leniusculus βGBP (CAB65353), black tiger shrimp Penaeus mondon βGBP (AAM21213), blue shrimp Litopenaeus stylirostris βGBP (AAM73871), Pacific white shrimp Litoppenaeus vannamei βGBP (AAW51361), lobster Homarus gammarus βGBP (CAE47485), sea urchin glucanase (AAC47253), earthworm Eisenia fetida CCF (AAC35887), sponge Suberites domuncula βGBP (CAE54585), honey bee GNBP 1 (XP_001121634), honey bee GNBP (XP_395368), hornworm βGRP seq 1 (AAF44011), silkworm βGBP (BAA92243), Indian meal moth Plodia interpunctella βGRP (AAM95970), hornworm βGBP seq 2 (AN10151), Drosophila GNBP1 (AAF33849), yellow mealworm βGRP (BAC99308), red flour beetle Tribolium castaneum GNBP (XP_972063), Aedes βGBP (EAT40654), Anopheles GNBP (Q7QIB2), Drosophila GNBP3 (AF33851), Bacillus circulans glucanase (Z47974), Thermotoga neapolitana glucanses (P23903)