Dataset Information

Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

ABSTRACT: Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since G. mellonella was recently established as a valuable pathogenicity model for Cryptococcus neoformans infection, the present results add to our understanding of the mechanisms of immune responses in G. mellonella. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.

SUBMITTER: Altincicek B

PROVIDER: S-EPMC1828416 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

Altincicek Boran B Linder Monica M Linder Dietmar D Preissner Klaus T KT Vilcinskas Andreas A

Infection and immunity 20061030 1

Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report th ...[more]

PMID: 17074843

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Project description:BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that act as key players in the post-transcriptional regulation of protein synthesis. Although little is known about their role in complex physiological processes such as development and immunity, our knowledge is expanding rapidly, thanks to the use of model systems. The larvae of the greater wax moth Galleria mellonella are now established as model hosts for pathogens that infect insects or humans. To build on our previously-reported comprehensive G. mellonella transcriptome, here we describe the identification and analysis of development and immunity-related miRNAs, thus providing valuable additional data to promote the use of this model host for the analysis of complex processes.ResultsTo screen for miRNAs that are differentially expressed in G. mellonella (1) during metamorphosis or (2) following infection with the entomopathogenic bacterium Serratia entomophila or (3) with the parasitic fungus Metarhizium anisopliae, we designed a microarray containing more than 2000 insect miRNA probe sequences. We identified miRNAs that were significantly expressed in pre-pupae (16), pupae (22) and last-instar larvae infected with M. anisopliae (1) in comparison with untreated last-instar larvae which were used as a reference. We then used our transcriptomic database to identify potential 3' untranslated regions that form miRNA-mRNA duplexes by considering both base pair complementarity and minimum free energy hybridization. We confirmed the co-expression of selected miRNAs (such as miR-71, miR-263a and miR-263b) with their predicted target mRNAs in last-instar larvae, pre-pupae and pupae by RT-PCR. We also identified miRNAs that were expressed in response to infection with bacterial or fungal pathogens, and one miRNA that may act as a candidate mediator of trans-generational immune priming.ConclusionsThis is the first study to identify miRNAs that are predicted to regulate genes expressed during metamorphosis or in response to infection in the lepidopteran model host G. mellonella.

Project description: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.

Dataset Information

Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

Publications

Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

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