Lasco, Valentina (2014) Analisi funzionale di un nuovo gene di Drosophila melanogaster coinvolto nella risposta immunitaria di incapsulamento. [Tesi di dottorato]

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Abstract

The overall aim of my PhD thesis project was the functional characterization of a Drosophila melanogaster gene showing sequence homology with an immune gene of a Noctuid Lepidoptera species, Heliothis virescens. The insect immune system relies on molecular mechanisms conserved throughout evolution and is divided into humoral and cellular responses. Antimicrobial peptides (AMPs), produced by fat body and released into the hemolymph, are the major humoral immune effectors; the humoral immune response also entails melanization and coagulation reactions triggered by tissue wounds. The cellular immune response is mediated by blood cells, named hemocytes, freely circulating into the hemocoel. In Drosophila melanogaster, blood cells are classified into plasmatocytes that are involved in phagocytosis and killing of invading microbes; crystal cells containing cytoplasmic inclusions where precursors of the phenoloxidase enzyme, involved in melanization reactions, are localized; and lamellocytes that appear in the circulation only when large foreign indruders, such as parasitoid eggs, enter the haemocoel and are involved in the formation of immune capsules. Recently, the laboratories where I carried out my experimental work demonstrated that, in Heliothis virescens, a gene named 102 codes for a protein involved in the formation of amyloids fibrils. After immune challenge, these fibrils, localized in the cisternae of the rough endoplasmic reticulum, are released to coat foreign intruders forming an immune capsule. The coating layer formed by fibrils is the site where dangerous reactions like melanization occur. To further extend our studies on the 102 gene and shed light on the molecular mechanisms underlying its function(s), we decided to look for potential orthologues in Drosophila melanogaster. Actually, this insect model system offers a wide variety of genetic and molecular tools, which are not available in Heliothis virescens. BLAST analyses identified in the Drosophila melanogaster genome two genes displaying sequence homology with the 102 Heliothis virescens gene: the CG2145 and the CG3303 genes. In the first part of my PhD thesis work I focused on the characterization of the first gene, which is more related than the latter to the Heliothis virescens 102 gene and also shares with it a high expression level in hemocytes. First of all, I characterized the spatio-temporal expression pattern of this gene during development and showed a complex, dynamic picture. Since the first embryonic stages, the gene is expressed in all embryonic anlagen where major morphogenetic movements take place and its transcripts are detected in migrating cells, such as ventral cells that invaginate to give rise to the mesoderm layer during gastrulation. However, at the end of embryogenesis the expression profile of the CG2145 gene becomes more restricted and its transcripts are virtually present only in the immune tissues: haemocytes and fat body. Interestingly, these immune tissues actively transcribe the CG2145 gene also in larval stages. By using a polyclonal antibody directed against a GC2145 fusion protein I generated, I defined the intracellular distribution pattern of the CG2145 protein. This protein shows polarized localization in the cytoplasm of the hemocytes and accumulates in peculiar vesicles that detach from the cell surface, to be likely secreted and released into the extracellular environment. To start the functional analysis of the CG2145 gene I generated, by mobilization of a P element, a set of deletions spanning the genomic region where the gene is located. Out of 14 different mutants I obtained, 9 display a molecular lesion confined to the CG2145 gene. I focused on the largest one, named CG2145-9B, which lacks the entire coding sequence and is a null allele, as confirmed by Western Blot analyses. Homozygous individuals are viable, fertile and do not shows any evident morphological defects. Then I set up specific functional assays to test their ability to react to immune challenge. Antimicrobial response and hemolymph melanization are not compromised in the CG2145 null mutant. However, I found that the encapsulation response is impaired. In the last part of my PhD thesis work I started the characterization of the CG3303 gene, which is also expressed, at low levels, in larval Drosophila hemocytes. This gene might share with the CG2145 gene part of its functions. Indeed, while silencing of the 102 gene in Lepidoptera blocks the encapsulation response, this immune reaction is not fully inhibited in the CG2145-9B mutant. The CG3303 gene shows a spatio-temporal expression pattern during development different from the CG2145 gene. Its transcripts appear only in late embryogenesis and only in two organs, the tracheal tree and the salivary glands. I set up a strategy to obtain CG3303 mutant alleles, similar to that I used before for generating CG2145 mutants. This strategy allowed me to isolate two deletion mutants, which are viable and fertile and apparently lack morphological defects. Phenotypic analyses to test the immune performance of these mutants are currently in progress. In conclusion, the obtained data demonstrate that the Drosophila melanogaster CG2145 gene, just like its Heliothis virescens counterpart, codes for a protein that is secreted by the hemocytes and is involved in the encapsulation response. This insect immune reaction comprises several steps, including recognition of foreign intruders, activation and differentiation of immune cells as well as cell-cell interactions, all of which are poorly understood. In addition, capsule formation is often associated with the localized synthesis of melanin. The CG2145 mutant I generated is a very useful tool to investigate the conserved molecular mechanisms underlying these complex processes in Lepidoptera and Diptera and, possibly, in all insects.

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