Chromatographic and electrophoretic resolution of proteins and protein complexes from the larval midgut microvilli of Manduca sexta

Pauchet, Yannick; Muck, Alexander; Svatoš, Aleš; Heckel, David G.

doi:10.1016/j.ibmb.2009.05.001

Cited by 24 publications

(21 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Gram negative bacteria binding proteins (GNBPs) and β-1,3-glucan recognition proteins (βGRPs) have been extensively studied as pattern recognition proteins in Lepidoptera [26-28]. Most of these proteins are produced in the fat body and secreted into the caterpillar's hemolymph.…”

Section: Resultsmentioning

confidence: 99%

“…Some are constitutively present whereas others are induced upon microbial infection. We have identified five different ßGRPs in the Galleria EST data collection, including one most similar to the midgut-specific short ßGRP with glucanase activity as previously described [26]. To further examine the relationships among βGRP proteins across insects and the ßGRPs identified in Galleria , a total of 45 sequences from 24 species, including many proteins that had previously been found in insect hemolymph, were collected and used to construct a Bayesian phylogeny (Additional File 3).…”

Section: Resultsmentioning

confidence: 99%

“…The phylogenetic analysis revealed that these sequences clustered in two distinct clades. One of these clades is clearly separated from the other clades by a high posterior probability and contains the Helicoverpa armigera Glucanase-1 protein (described in Pauchet et al [26]), and sequences from cDNA libraries made from midgut tissue of different Lepidoptera species, including one Galleria sequence. This phylogeny suggests that Galleria does have all of the ßGRPs found in more derived Lepidopteran species, including the gene coding for a protein with glucanase activity.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella

et al. 2011

View full text Add to dashboard Cite

BackgroundThe larvae of the greater wax moth Galleria mellonella are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in Galleria, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing.ResultsWe performed a Galleria transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (E ≤ e-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis.ConclusionHere, we have developed extensive transcriptomic resources for Galleria. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in Galleria and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella

et al. 2011

View full text Add to dashboard Cite

show abstract

“…To address this, we combined a proteomics approach to transcriptome sequencing to identify the proteins responsible for these enzymatic activities. Initially, we separated gut content proteins by anion exchange chromatography similar to our previous work [30], considering this step as the ‘first dimension’ of a two-dimensional approach. A large portion of these proteins bound to the column and were eluted with NaCl concentration ranging from 40 to 440 mM.…”

Section: Resultsmentioning

confidence: 99%

Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

et al. 2012

Self Cite

View full text Add to dashboard Cite

BackgroundThe primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae.ResultsUsing a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (β-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level.ConclusionsCombining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.

show abstract

“…Proteomic analyses of insect midguts have characterized either total BBMV proteomes 51, 52 or a proteome subset such as Bt toxin binding proteins 28, 53, 54 . Our geLC-MS/MS analysis of the DRM sub-proteome identified lipid raft marker proteins flotillin-1, flotillin-2, APN and ALP and many other proteins reported in similar analyses of DRMs 55–59 .…”

Section: Discussionmentioning

confidence: 99%

Proteome Analysis of Cry4Ba Toxin-interacting Aedes aegypti Lipid Rafts using geLC–MS/MS

et al. 2012

View full text Add to dashboard Cite

Lipid rafts are microdomains in the plasma membrane of eukaryotic cells. Among their many functions, lipid rafts are involved in cell toxicity caused by pore forming bacterial toxins including Bacillus thuringiensis (Bt) Cry toxins. We isolated lipid rafts from brush border membrane vesicles (BBMV) of Aedes aegypti larvae as a detergent resistant membrane (DRM) fraction on density gradients. Cholesterol, aminopeptidase (APN), alkaline phosphatase (ALP) and the raft marker flotillin were preferentially partitioned into the lipid raft fraction. When mosquitocidal Cry4Ba toxin was pre-incubated with BBMV, Cry4Ba localized to lipid rafts. A proteomic approach based on one dimensional gel electrophoresis, in-gel trypsin digestion, followed by liquid chromatography-mass spectrometry (geLC-MS/MS) identified a total of 386 proteins. Of which many are typical lipid raft marker proteins including flotillins and glycosylphosphatidylinositol (GPI)-anchored proteins. Identified raft proteins were annotated in silico for functional and physicochemical characteristics. Parameters such as distribution of isoelectric point, molecular mass, and predicted post-translational modifications relevant to lipid raft proteins (GPI anchorage and myristoylation or palmitoylation) were analyzed for identified proteins in the DRM fraction. From a functional point of view, this study identified proteins implicated in Cry toxin interactions as well as membrane-associated proteins expressed in the mosquito midgut that have potential relevance to mosquito biology and vector management.

show abstract

Chromatographic and electrophoretic resolution of proteins and protein complexes from the larval midgut microvilli of Manduca sexta

Cited by 24 publications

References 35 publications

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella

Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

Proteome Analysis of Cry4Ba Toxin-interacting Aedes aegypti Lipid Rafts using geLC–MS/MS

Contact Info

Product

Resources

About