Expression, Purification and Characterization of Peptidoglycan Recognition Protein 1 From Manduca sexta (L.)

Insects have an effective defense system against invading microbes. Peptidoglycan recognition proteins (PGRPs) in hemolymph detect peptidoglycans (a structural component of bacterial cell walls) and trigger a serine proteinase network that mediates and coordinates various host immune responses. To elucidate its biochemical functions, we have expressed Manduca sexta PGRP-1 in the baculovirus expression system and purified the protein from the conditioned culture medium. Purified PGRP-1 has a molecular mass of 19 kDa. The recombinant PGRP-1 specifically binds to Lys- and DAP-type peptidoglycans from Gram-positive and Gram-negative bacteria, respectively and leads to the proteolytic activation of prophenoloxidase. The binding affinity is high for all polymeric peptidoglycans tested, except for the one from Staphylococcus aureus. M. sexta PGRP-1 is produced at a low, constitutive level by hemocytes and fat body, and its transcripts become highly abundant in both tissues after larvae are challenged by bacterial injection.Department of Entomology and Plant Patholog

Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development

<p>Abstract</p> <p>Background</p> <p>In eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (<it>Bombyx mori </it>L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.</p> <p>Results</p> <p>We generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of <it>B. mori </it>and obtained ~2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.</p> <p>Conclusions</p> <p>We identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.</p

Comparative genomic analysis of the Tribolium immune system

The annotation, and comparison with homologous genes in other species, of immunity-related genes in the Tribolium castaneum genome allowed the identification of around 300 candidate defense proteins, and revealed a framework of information on Tribolium immunity

Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2−5, 12 and 13 cDNAs, produced the proteins in full (PGRP2–5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects

Phylogenetic relationships of Toll-like receptors from five insect species

The sequences of nine (Tc), nine (Dm), six (Ag), five (Am), and two (Aa) Toll-related proteins are compared. Species-specific family expansion is shaded yellow for and blue for . Nodes with pink arrowheads have bootstrap values exceeding 800 from 1,000 trials, and green lines connect putative orthologs with 1:1, 1:1:1 or 1:1:1:1 relationship. Note that Toll-9 does not have a Toll/interleukin1 receptor domain.<p><b>Copyright information:</b></p><p>Taken from "Comparative genomic analysis of the immune system"</p><p>http://genomebiology.com/2007/8/8/R177</p><p>Genome Biology 2007;8(8):R177-R177.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2375007.</p><p></p

Real-time PCR analysis of expression of immunity-related genes in adults 24 h after injections of (

), (.), (.), (.), or phosphate-buffered saline (PBS). Uninjured insects (-) were used as another negative control. With green, black and red colors representing low, intermediate and high transcript levels, respectively, relative mRNA abundances were used to cluster samples by average-linker clustering.<p><b>Copyright information:</b></p><p>Taken from "Comparative genomic analysis of the immune system"</p><p>http://genomebiology.com/2007/8/8/R177</p><p>Genome Biology 2007;8(8):R177-R177.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2375007.</p><p></p

A major family expansion of serpins and their phylogenetic relationships with the serpins from other insect species

The sequences of 29 (Tc), 3 (Dm), 3 (Ag), 4 (Am) and 5 (Ms) serpins are compared. serpin2 (758 residues) and serpin26 (568 residues), much longer than a typical serpin (40-50 kDa), are excluded from the analysis. For simplicity, serpins 1b, 15a, 20b and 28a are also eliminated because they are products of alternative splicing of the genes 1a, 15b, 20a and 28b, which differ only in the region coding for reactive site loop. As shown in the tree (left panel), extensive expansion gives rise to this group of highly similar genes (shaded blue) located in a small chromosomal region (right panel). Pink arrowheads at nodes denote bootstrap values greater than 800 for 1,000 trials. Putative 1:1, 1:1:1 or 1:1:1:1 orthologous relationship is indicated by green bars connecting the group members.<p><b>Copyright information:</b></p><p>Taken from "Comparative genomic analysis of the immune system"</p><p>http://genomebiology.com/2007/8/8/R177</p><p>Genome Biology 2007;8(8):R177-R177.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2375007.</p><p></p