Si-Ming Zhang

Invertebrate immune systems – not homogeneous, not simple, not well understood

Summary:  The approximate 30 extant invertebrate phyla have diversified along separate evolutionary trajectories for hundreds of millions of years. Although recent work understandably has emphasized the commonalities of innate defenses, there is also ample evidence, as from completed genome studies, to suggest that even members of the same invertebrate order have taken significantly different approaches to internal defense. These data suggest that novel immune capabilities will be found among the different phyla. Many invertebrates have intimate associations with symbionts that may play more of a role in internal defense than generally appreciated. Some invertebrates that are either long lived or have colonial body plans may diversify components of their defense systems via somatic mutation. Somatic diversification following pathogen exposure, as seen in plants, has been investigated little in invertebrates. Recent molecular studies of sponges, cnidarians, shrimp, mollusks, sea urchins, tunicates, and lancelets have found surprisingly diversified immune molecules, and a model is presented that supports the adaptive value of diversified non‐self recognition molecules in invertebrates. Interactions between invertebrates and viruses also remain poorly understood. As we are in the midst of alarming losses of coral reefs, increased pathogen challenge to invertebrate aquaculture, and rampant invertebrate‐transmitted parasites of humans and domestic animals, we need a better understanding of invertebrate immunology.

Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection

Invertebrates lack adaptive immune systems homologous to those of vertebrates, yet it is becoming increasingly clear that they can produce diversified antigen recognition molecules. We have previously noted that the snail Biomphalaria glabrata produces a secreted lectin, fibrinogen-related protein 3 (FREP3), unusual among invertebrate defense molecules because it is somatically diversified by gene conversion and point mutation. Here we implicate FREP3 in playing a central role in resistance to a major group of snail pathogens, digenetic trematodes. FREP3 is up-regulated in three models of resistance of B. glabrata to infection with Schistosoma mansoni or Echinostoma paraensei, and functions as an opsonin favoring phagocytosis by hemocytes. Knock-down of FREP3 in resistant snails using siRNA-mediated interference resulted in increased susceptibility to E. paraensei, providing a direct link between a gastropod immune molecule and resistance to trematodes. FREP3 up-regulation is also associated with heightened responsiveness following priming with attenuated digenetic trematodes (acquired resistance) in this model invertebrate immune system.

Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata

Upon exposure to infection with digenetic trematodes such as Echinostoma paraensei, the freshwater snail Biomphalaria glabrata produces increased quantities of hemolymph lectins, some of which are unique polypeptides containing both immunoglobulin superfamily (IgSF) and fibrinogen domains. These unusual lectins have been termed fibrinogen-related proteins (FREPs), and recognize and precipitate digenean antigens. We here report 11 distinct FREP-encoding sequences from B. glabrata, and provide the complete genomic sequence for two of the most frequently recovered FREPs. The unique juxtaposition of IgSF and fibrinogen domains, previously known only from incomplete cDNAs, is confirmed. Sequences corresponding to known peptides derived from FREPs from hemolymph were found in one of these genes. Both genes contain four exons, the first encodes a putative signal peptide, the second and third a portion of an IgSF-type loop, and the fourth a fibrinogen domain. Cysteines, postulated to form an intrachain loop, are present in the IgSF domain and are separated from one another by 78 or 79 residues. The IgSF sequences most closely resemble V (variable)-type Ig domains, based on canonical and hydrophobic residues and predicted secondary structure. Some minor differences in genomic fragments isolated for each of the two sequences were noted and may represent allelic variants. The results may be of relevance in understanding the role of B. glabrata in transmission of Schistosoma mansoni, a digenean parasite that infects nearly 100 million people in the tropics.

Parasite-responsive IgSF members in the snail Biomphalaria glabrata: characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain

Abstract. Two novel genes of the immunoglobulin superfamily (IgSF), FREP3 and FREP7, are reported from the snail Biomphalaria glabrata, a prominent intermediate host of the human parasite Schistosoma mansoni. They resemble other B. glabrata genes that encode fibrinogen-related proteins (FREPs), but differ in that they encode proteins with two tandemly arranged IgSF domains followed by a C-terminal fibrinogen domain. FREPs are hemolymph proteins that increase in abundance following exposure to a digenetic trematode, Echinostoma paraensei, and that bind to and precipitate parasite antigens. Within each gene, the two IgSF-coding regions are dissimilar from one another: the N-terminal IgSF1 domain is encoded by a single exon whereas the downstream IgSF2 domain is encoded by three exons. For both FREPs 3 and 7, the IgSF2 domain belongs to the variable (V) type, whereas the IgSF1 domain is not easily classified with respect to IgSF type. The fibrinogen-encoding region in both genes is relatively conserved and lacks introns. FREP3 exhibits extensive variation in the IgSF1 region. A ratio of nonsynonymous versus synonymous substitutions of 2.56 suggests that this region is under positive selection. A genomic fragment identifiable as FREP7 but lacking an exon was also found, further suggestive of variability within FREP IgSF-encoding regions. Insofar as FREPs are hypothesized to function in nonself recognition, the identification of additional novel FREP genes as part of a growing gene family in B. glabrata is of interest. Such genes, particularly given their variable nature, serve as a model to study the complexity of invertebrate defense responses.

Expression profiling and binding properties of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail host Biomphalaria glabrata

A growing body of evidence suggests an important role for fibrinogen-like proteins in innate immunity in both vertebrates and invertebrates. It has been shown that fibrinogen-related proteins (FREPs), plasma proteins present in the freshwater snail Biomphalaria glabrata, the intermediate host for the human blood fluke Schistosoma mansoni, are diverse and involved in snail innate defense responses. To gain further insight into the functions of FREPs, recombinant FREP proteins (rFREPs) were produced in Escherichia coli and antibodies (Abs) were raised against the corresponding rFREPs. We first show that most FREP proteins exist in their native conformation in snail hemolymph as multimeric proteins. Western blot analyses reveal that expression of multiple FREPs including FREP4 in plasma from M line and BS-90 snails, which are susceptible and resistant to S. mansoni infection, respectively, is up-regulated significantly after infection with the trematode Echinostoma paraensei. Moreover, our assays demonstrate that FREPs are able to bind E. paraensei sporocysts and their secretory/excretory products (SEPs), and a variety of microbes (Gram-positive and Gram-negative bacteria and yeast). Furthermore, this binding capability shows evidence of specificity with respect to pathogen type; for example, 65—75-kDa FREPs (mainly FREP4) bind to E. paraensei sporocysts and their SEPs whereas 95-kDa and 125-kDa FREPs bind the microbes assayed. Our results suggest that FREPs can recognize a wide range of pathogens, from prokaryotes to eukaryotes, and different categories of FREPs seem to exhibit functional specialization with respect to the pathogen encountered.

Fibrinogen-bearing protein genes in the snail Biomphalaria glabrata: Characterization of two novel genes and expression studies during ontogenesis and trematode infection

All fibrinogen (FBG)-bearing proteins documented to date in the freshwater snail Biomphalaria glabrata, the intermediate host of the human blood fluke Schistosoma mansoni, possess the same molecular structure; one or two immunoglobin superfamily (IgSF) domains at the N-terminus and a FBG domain at the C-terminus (named as FBG-related protein (FREP)). Here we report two novel genes that encode FBG-bearing proteins from B. glabrata. Different from all known FREPs, the first gene encodes a protein (657 amino acids (aa)) composed of a long N-terminal region with no sequence homology to any known protein, a middle epidermal growth factor (EGF) repeat region and a C-terminal FBG domain, designated FBG-related molecule (FReM). Differential expression at 2 days post-exposure (dpe) to the trematode S. mansoni or Echinostoma paraensei was found in the S. mansoni susceptible M line and resistant BS-90 snail strains. The second gene is a new member of the FREP family, designated FREP14, which encodes a 399 aa putative secreted protein. FREP14 is different from known FREPs in that it is encoded by a single locus and is not upregulated in early or late stage S. mansoni exposure, but is upregulated in late stage E. paraensei infection. Furthermore, gene expression during the snails ontogenesis and at a late stage of trematode infection (52 dpe) has been investigated in the two newly identified genes (FReM and FREP14) described in this paper and five representative members of known FREPs (FREPs 2, 3, 4, 12, and 13). A variety of expression patterns were observed, suggestive of functional diversity among the members of FBG-bearing proteins. Our findings further broaden our understanding of the diversity and function of the FBG-bearing protein encoded genes in B. glabrata.

Schistosomin from the snail Biomphalaria glabrata: expression studies suggest no involvement in trematode-mediated castration.

By inhibiting reproductive hormones, the neuropeptide schistosomin produced by the snail Lymnaea stagnalis plays an essential role in parasitic castration mediated by the schistosome parasite Trichobilharzia ocellata during late stage infection. Here we report on the presence and expression of schistosomin in the snail Biomphalaria glabrata, a prominent intermediate host of the parasite Schistosoma mansoni, one of the causative agents of human schistosomiasis. The deduced amino acid (aa) sequences from complementary DNAs (cDNAs) from B. glabrata contain a 17 aa signal peptide and a 79 aa mature peptide with 62 -- 64% identity to schistosomin from L. stagnalis. Ontogenic expression at the protein and mRNA levels showed that schistosomin was in higher abundance in embryos and juveniles relative to mature snails, suggesting that schistosomin is likely involved in developmental processes, not in reproduction. Moreover, expression data demonstrated that infection with two different digenetic trematodes, S. mansoni and Echinostoma paraensei, did not provoke elevated expression of schistosomin in B. glabrata from early stage infection (4 days post-exposure; dpe) to patent infection (up to 60dpe), by which time parasitic castration has been accomplished. In conclusion, our data suggest that a role of schistosomin in parasitic castration cannot be established in B. glabrata infected with either of two trematode species.

Altered Gene Expression in the Schistosome-Transmitting Snail Biomphalaria glabrata following Exposure to Niclosamide, the Active Ingredient in the Widely Used Molluscicide Bayluscide.

In view of the call by the World Health Organization (WHO) for elimination of schistosomiasis as a public health problem by 2025, use of molluscicides in snail control to supplement chemotherapy–based control efforts is likely to increase in the coming years. The mechanisms of action of niclosamide, the active ingredient in the most widely used molluscicides, remain largely unknown. A better understanding of its toxicology at the molecular level will both improve our knowledge of snail biology and may offer valuable insights into the development of better chemical control methods for snails. We used a recently developed Biomphalaria glabrata oligonucleotide microarray (31K features) to investigate the effect of sublethal exposure to niclosamide on the transcriptional responses of the snail B. glabrata relative to untreated snails. Most of the genes highly upregulated following exposure of snails to niclosamide are involved in biotransformation of xenobiotics, including genes encoding cytochrome P450s (CYP), glutathione S-transferases (GST), and drug transporters, notably multi-drug resistance protein (efflux transporter) and solute linked carrier (influx transporter). Niclosamide also induced stress responses. Specifically, six heat shock protein (HSP) genes from three super-families (HSP20, HSP40 and HSP70) were upregulated. Genes encoding ADP-ribosylation factor (ARF), cAMP response element-binding protein (CREB) and coatomer, all of which are involved in vesicle trafficking in the Golgi of mammalian cells, were also upregulated. Lastly, a hemoglobin gene was downregulated, suggesting niclosamide may affect oxygen transport. Our results show that snails mount substantial responses to sublethal concentrations of niclosamide, at least some of which appear to be protective. The topic of how niclosamide’s lethality at higher concentrations is determined requires further study. Given that niclosamide has also been used as an anthelmintic drug for decades and has been found to have activity against several types of cancer, our findings may be of relevance in understanding how both parasites and neoplastic cells respond to this compound.

Transcriptomic responses of Biomphalaria pfeifferi to Schistosoma mansoni: Investigation of a neglected African snail that supports more S. mansoni transmission than any other snail species

Background Biomphalaria pfeifferi is highly compatible with the widespread human-infecting blood fluke Schistosoma mansoni and transmits more cases of this parasite to people than any other snail species. For these reasons, B. pfeifferi is the world’s most important vector snail for S. mansoni, yet we know relatively little at the molecular level regarding the interactions between B. pfeifferi and S. mansoni from early-stage sporocyst transformation to the development of cercariae. Methodology/Principal findings We sought to capture a portrait of the response of B. pfeifferi to S. mansoni as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control B. pfeifferi and three intramolluscan developmental stages (1- and 3-days post infection and patent, cercariae-producing infections) using field-derived west Kenyan specimens. A high-quality, well-annotated de novo B. pfeifferi transcriptome was assembled from over a half billion non-S. mansoni paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized S. mansoni reads and showed different patterns of transcriptional response than others, an indication that the ability of field-derived snails to support and respond to infection is variable. Alterations in transcripts associated with reproduction were noted, including for the oviposition-related hormone ovipostatin and enzymes involved in metabolism of bioactive amines like dopamine or serotonin. Shedding snails exhibited responses consistent with the need for tissue repair. Both generalized stress and immune factors immune factors (VIgLs, PGRPs, BGBPs, complement C1q-like, chitinases) exhibited complex transcriptional responses in this compatible host-parasite system. Significance This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. We have identified B. pfeifferi targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.

Transcriptional responses of Biomphalaria pfeifferi and Schistosoma mansoni following exposure to niclosamide, with evidence for a synergistic effect on snails following exposure to both stressors

Background Schistosomiasis is one of the world’s most common NTDs. Successful control operations often target snail vectors with the molluscicide niclosamide. Little is known about how niclosamide affects snails, including for Biomphalaria pfeifferi, the most important vector for Schistosoma mansoni in Africa. We used Illumina technology to explore how field-derived B. pfeifferi, either uninfected or harboring cercariae–producing S. mansoni sporocysts, respond to a sublethal exposure of niclosamide. This study afforded the opportunity to determine if snails respond differently to biotic or abiotic stressors, and if they reserve unique responses for when presented with both stressors in combination. We also examined how sporocysts respond when their snail host is exposed to niclosamide. Principal Findings Cercariae-producing sporocysts within snails exposed to niclosamide express ~68% of the genes in the S. mansoni genome, as compared to 66% expressed by intramolluscan stages of S. mansoni in snails not exposed to niclosamide. Niclosamide does not disable sporocysts nor does it seem to provoke from them distinctive responses associated with detoxifying a xenobiotic. For B. pfeifferi, niclosamide treatment alone increases expression of several features not up-regulated in infected snails including particular cytochrome p450s and heat shock proteins, glutathione-S-transferases, antimicrobial factors like LBP/BPI and protease inhibitors, and also provokes strong down regulation of proteases. Exposure of infected snails to niclosamide resulted in numerous up-regulated responses associated with apoptosis along with down-regulated ribosomal and defense functions, indicative of a distinctive, compromised state not achieved with either stimulus alone. Conclusions/Significance This study helps define the transcriptomic responses of an important and under-studied schistosome vector to S. mansoni sporocysts, to niclosamide, and to both in combination. It suggests the response of S. mansoni sporocysts to niclosamide is minimal and not reflective of a distinct repertoire of genes to handle xenobiotics while in the snail host. It also offers new insights for how niclosamide affects snails. Author’S Summary Schistosomaisis control programs often employ the use of chemical molluscicides, such as niclosamide, to control the obligatory intermediate snail hosts. Despite its widespread use, we know little about how niclosamide affects snails like Biomphalaria pfeifferi, the most important vector Schistosoma mansoni in Africa. By sequencing the transcriptomes of uninfected and S. mansoni-infected B. pfeifferi exposed to niclosamide, we analyze the snail’s response to both biotic and abiotic stressors. We can also examine the response of S. mansoni to niclosamide exposure during intramolluscan development. Biomphalaria pfeifferi snails exposed only to niclosamide showed unique up-regulation of stress and defense-related transcripts not seen in snails infected with a biotic, like S. mansoni infection, alone. Schistosoma mansoni-infected B. pfeifferi exposed to niclosamide were clearly unable to regulate normal metabolic and detoxification processes. Cercariae-producing sporocysts within snails exposed to niclosamide are largely unaffected and continue to produce transcripts required for cercariae production.