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 ... -  《-》

Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge.

The tobacco hornworm, Manduca sexta, has been used as a biochemical model for studying insect physiological processes. While the transcriptomes of its fat body, hemocytes, midgut, and antennae have been examined in several studies, limited information is available for proteins in tissues, cells, or body fluids of this insect. In keeping pace with the M. sexta genome project, we launched a pilot study to identify differences in the peptidome of cell-free hemolymph samples from larvae injected with buffer or a mixture of bacteria. At 24 h after injection, plasma was collected and treated with 50% acetonitrile to precipitate large proteins. The supernatants, containing peptides (<25 kDa) and other stable proteins (>25 kDa), were digested with trypsin and analyzed by nano-liquid chromatography and nano-electrospray tandem mass spectrometry (nanoLC-MS/MS) on an LTQ Orbitrap XL mass spectrometer. Known M. sexta cDNA sequences and gene transcripts from the draft genome were translated in silico to generate a database of polypeptides (i.e. peptides and proteins) in this species. By searching the database, we identified 268 hemolymph polypeptides, 50 of which showed 1.67-200 fold abundance increases after the immune challenge, as judged by significant changes in normalized spectral counts between the control and induced plasma. These included a total of 33 antimicrobial peptides (attacins, cecropins, defensins, diapausins, gallerimycin, gloverin, lebocins, lysozymes), pattern recognition receptors, and proteinase inhibitors. Although there was no strong parallel (correlation coefficients: -0.13, 0.11, 0.39 and 0.62) between plasma peptide levels and their transcript levels in control or induced hemocytes or fat body, we observed the mRNA level changes in hemocytes and fat body concurred with their peptide level changes with correlation coefficients of 0.67 and 0.76, respectively. These data suggest that fat body contributed a significant portion of the plasma polypeptides involved in various aspects of innate immunity after the bacterial injection.

Zhang S ，Cao X ，He Y ，Hartson S ，Jiang H ... -  《-》

Proteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sexta.

An C ，Jiang H ，Kanost MR 《-》

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 comprehensive analysis of the Manduca sexta immunotranscriptome.

As a biochemical model, Manduca sexta has substantially contributed to our knowledge on insect innate immunity. The RNA-Seq approach was implemented in three studies to examine tissue immunotranscriptomes of this species. With the latest and largest focusing on highly regulated process- and tissue-specific genes, we further analyzed the same set of data using BLAST2GO to explore functional aspects of the larval fat body (F) and hemocyte (H) transcriptomes with (I) or without (C) immune challenge. Using immunity-related sequences from other insects, we found 383 homologous contigs and compared them with those discovered based on relative abundance changes. The major overlap of the two lists validated our previous research designed for gene discovery and transcript profiling in organisms lacking sequenced genomes. By concatenating the contigs, we established a repertoire of 232 immunity-related genes encoding proteins for pathogen recognition (16%), signal transduction (53%), microbe killing (13%) and others (18%). We examined their transcript levels along with attribute classifications and detected prominent differences in nine of the 30 level 2 gene ontology (GO) categories. The increase in extracellular proteins (155%) was consistent with the highly induced synthesis of defense molecules (e.g., antimicrobial peptides) in fat body after the immune challenge. We identified most members of the putative Toll, IMD, MAPK-JNK-p38 and JAK-STAT pathways and small changes in their mRNA levels. Together, these findings set the stage for on-going analysis of the M. sexta immunogenome.

Gunaratna RT ，Jiang H 《-》