Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect.

Zhang X ，He Y ，Cao X ，Gunaratna RT ，Chen YR ，Blissard G ，Kanost MR ，Jiang H ... -  《-》

A genome-wide analysis of antimicrobial effector genes and their transcription patterns in Manduca sexta.

Antimicrobial proteins/peptides (AMPs) are effectors of innate immune systems against pathogen infection in multicellular organisms. Over half of the AMPs reported so far come from insects, and these effectors act in concert to suppress or kill bacteria, fungi, viruses, and parasites. In this work, we have identified 86 AMP genes in the Manduca sexta genome, most of which seem likely to be functional. They encode 15 cecropins, 6 moricins, 6 defensins, 3 gallerimycins, 4 X-tox splicing variants, 14 diapausins, 15 whey acidic protein homologs, 11 attacins, 1 gloverin, 4 lebocins, 6 lysozyme-related proteins, and 4 transferrins. Some of these genes (e.g. attacins, cecropins) constitute large clusters, likely arising after rounds of gene duplication. We compared the amino acid sequences of M. sexta AMPs with their homologs in other insects to reveal conserved structural features and phylogenetic relationships. Expression data showed that many of them are synthesized in fat body and midgut during the larval-pupal molt. Certain genes contain one or more predicted κB binding sites and other regulatory elements in their promoter regions, which may account for the dramatic mRNA level increases in fat body and hemocytes after an immune challenge. Consistent with these strong mRNA increases, many AMPs become highly abundant in the larval plasma at 24 h after the challenge, as demonstrated in our previous peptidomic study. Taken together, these data suggest the existence of a large repertoire of AMPs in M. sexta, whose expression is up-regulated via immune signaling pathways to fight off invading pathogens in a coordinated manner.

He Y ，Cao X ，Li K ，Hu Y ，Chen YR ，Blissard G ，Kanost MR ，Jiang H ... -  《-》

Structural features, evolutionary relationships, and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta.

C-type lectins (CTLs) are a large family of Ca(2+)-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca(2+)-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.

Rao XJ ，Cao X ，He Y ，Hu Y ，Zhang X ，Chen YR ，Blissard G ，Kanost MR ，Yu XQ ，Jiang H ... -  《-》

The transcriptomic expression of pattern recognition receptors: Insight into molecular recognition of various invading pathogens in Oyster Crassostrea gigas.

Pattern recognition receptors (PRRs) are essential in recognizing specific pathogen-associated molecular patterns (PAMPs) on microbes and triggering responses to eliminate the invading pathogens. Previous genomic studies have revealed a great number of PRR genes in the Pacific oyster Crassostrea gigas, a sessile and filter-feeder marine bivalve belonging to the phylum Mollusca. On the survey of PRRs in the assembly oyster reference genome version 9, a total of 1084 PRRs were identified, which were composed of at least 12 gene families. Some of the gene families were significantly expanded, including C-type lectins (CTLs), fibrinogen-related proteins (FREPs), scavenger receptor cysteine-rich repeat protein (SRCRs), leucine-rich repeat (LRR)-only proteins (LRRops), and especially C1q domain-containing proteins (C1qDCs). The transcriptomic profiles of these abundant PRRs in response to PAMP treatments were investigated by RNA-Seq using the SOLiD EZ BeadTM system. Compared to the control library, there were 6,655, 7,273, 7,593, 6,830, 6687 and 8250 differentially expressed genes in the haemocytes of oysters in response to lipopolysaccharide (LPS) stimulation for 6 h, 12 h and 24 h, and peptidoglycan (PGN), glucan (GLU) and poly I:C (IC) stimulation for 12 h, respectively. After stimulation for 12 h, there were 134, 97, 114 and 159 genes up-regulated in the LPS, PGN, GLU and IC library, respectively. Most of the gene families involved in immune response towards PAMPs were C1qDCs, CTLs and FREPs, while only a few members of LRR and immunoglobin-containing proteins (LRRIGs), retinoic acid-inducible gene I [RIG-I]-like receptors (RLRs) and Toll like receptors (TLRs) were up-regulated. After LPS stimulation, the expression level of 258 non-redundant PRR genes in oyster haemocytes increased significantly with different expression pattern, and most of them were C1qDCs, CTLs, LRRops and FREPs. The transcriptomic analyses indicated that there was a dynamic and orchestrated specific expression regulation of numerous PRR genes in response to pathogen invasion. The expanded PRR gene family members were differentiated with more specific functional responses to certain PAMPs rather than the versatile ones. Based on the different expression pattern during the LPS stimulation, the oyster PRRs could be assigned into three consecutive steps in the response against pathogen invading. All the results would provide useful information for future studies of oyster PRRs and deep insight into the researches on invertebrate innate immunity.

Wang L ，Zhang H ，Wang M ，Zhou Z ，Wang W ，Liu R ，Huang M ，Yang C ，Qiu L ，Song L ... -  《-》

Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species.

Cao X ，He Y ，Hu Y ，Zhang X ，Wang Y ，Zou Z ，Chen Y ，Blissard GW ，Kanost MR ，Jiang H ... -  《-》