The evolution and genetics of innate immunity.

The immune system provides protection from a wide range of pathogens. One component of immunity, the phylogenetically ancient innate immune response, fights infections from the moment of first contact and is the fundamental defensive weapon of multicellular organisms. The Toll family of receptors has a crucial role in immune defence. Studies in fruitflies and in mammals reveal that the defensive strategies of invertebrates and vertebrates are highly conserved at the molecular level, which raises the exciting prospects of an increased understanding of innate immunity.

Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity.

Thor has been identified as a new type of gene involved in Drosophila host immune defense. Thor is a member of the 4E-binding protein (4E-BP) family, which in mammals has been defined as critical regulators in a pathway that controls initiation of translation through binding eukaryotic initiation factor 4E (eIF4E). Without an infection, Thor is expressed during all developmental stages and transcripts localize to a wide variety of tissues, including the reproductive system. In response to bacterial infection and, to a lesser extent, by wounding, Thor is up-regulated. The Thor promoter has the canonical NFkappaB and associated GATA recognition sequences that have been shown to be essential for immune induction, as well as other sequences commonly found for Drosophila immune response genes, including interferon-related regulatory sequences. In survival tests, Thor mutants show symptoms of being immune compromised, indicating that Thor may be critical in host defense. In contrast to Thor, Drosophila eIF4E is not induced by bacterial infection. These findings for Thor provide the first evidence that a 4E-BP family member has a role in immune induction in any organism. Further, no gene in the translation initiation pathway that includes 4E-BP has been previously found to be immune induced. Our results suggest either a role for translational regulation in humoral immunity or a new, nontranslational function for 4E-BP type genes.

viking: identification and characterization of a second type IV collagen in Drosophila.

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila host defense.

Mammals and insects employ similar Rel/NF-kappaB signaling cascades in their humoral immune responses. The mammalian interleukin-1 type I receptor (IL-1R) is one way of activating this cascade. The Drosophila Toll protein, whose cytoplasmic domain shows striking similarity to that of the IL-1R, acts in the humoral antimicrobial response. Here we demonstrate that a second IL-1R-related Drosophila protein, 18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12%. Finally, 18-wheeler (18w) expression is induced in response to infection and, in addition to the receptor form, four immune-specific transcripts and proteins are produced.

Identification and characterization of the Cecropin antibacterial protein gene locus in Drosophila virilis.

Cecropin is a type of antibacterial peptide that is synthesized in response to infection and has been characterized in many insect species and one mammal. The Cecropin locus of Drosophila melanogaster also contains the gene Andropin, which has been identified only in this species and encodes a male-specific antibacterial peptide. As a first step in studying the molecular evolution of the cecropin and andropin genes among Drosophila species, we have isolated genomic clones that cover the Cecropin locus in Drosophila virilis. The cloned region totals approximately 25 kb, within which a 9-kb fragment contains four cecropin genes and one pseudogene. All four genes have a high level of sequence homology to D. melanogaster Cecropin, about 80% identity in the coding regions, and the intron positions are conserved. As in D. melanogaster and other insects, kappa B-related cis-regulatory elements are found upstream of these cecropin genes. An Andropin-related sequence was not identified in D. virilis; however, genome Southern hybridizations suggest that Andropin-related sequences are present in at least the melanogaster species subgroup. Analysis of 19 insect cecropin genes identifies a common ancestral Cecropin before the divergence of Diptera and Lepidoptera. In addition, D. melanogaster and D. virilis can be identified by monophyletic clades for Cecropin. In contrast, the Lepidopteran species show polyphyletic relationships for duplicated cecropin genes.

Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.

We are using Drosophila as a model system for analysis of immunity and tumor formation and have conducted two types of screens using enhancer detector strains to find genes related to these processes; genes expressed in the immune system (type A; hemocytes, lymph glands and fat body) and genes increased in expression by bacterial infection (type B). For type A, tissue-specific reporter gene activity was determined. For type B, a variation of enhancer detection was devised in which beta-galactosidase is assayed spectrophotometrically with and without bacterial infection. Because of immune system involvement in melanotic tumor formation, a third type was hypothesized to be found among types A and B; genes that, when mutated, have a melanotic tumor phenotype. Enhancer detector strains (2800) were screened for type A, 900 for B, and 11 retained for further analysis. Complementation tests, cytological mapping, P-element mobilization, and determination of lethal phase and mutant phenotype have identified six novel genes, Dorothy, wizard, toto, viking, Thor and dappled, and one previously identified gene, Collagen IV. All are associated with reporter gene expression in at least one immune system tissue. Thor has increased expression upon infection. Mutations of wizard and dappled have a melanotic tumor phenotype.

The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands.

As part of a study of the genes involved in antibacterial defense in Drosophila melanogaster, we have isolated genomic clones harboring a family of chicken-type lysozyme genes, using a lepidopteran lysozyme cDNA as probe. The locus was mapped to the cytological location 61F1-4 on the third chromosome and two of the genes at this locus, LysD and LysP, were analyzed in detail. In contrast to the bacteria-induced lysozymes in the hemolymph of many insects, the transcription levels of both Drosophila genes decrease after bacterial injections into the hemocoel. Apparently, these gene products, like the specifically adapted lysozymes in mammalian foregut fermenters, have been recruited for the digestion of bacteria present in fermenting food. The LysD gene is expressed in an anterior section of the midgut during all feeding stages of development in both larvae and adults. The LysP gene is only active in the adult where it is expressed in the salivary glands. The transcription units for both genes are very compact and they lack introns. Lysozyme D is unusual in that it is predicted to have an acidic isoelectric point whereas lysozyme P appears to be a typical basic lysozyme.

CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae.

Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.

Insect antibacterial proteins: not just for insects and against bacteria.

In response to a bacterial infection, insects launch an array of countermeasures. Among these are the antibacterial proteins, which effectively lyse bacteria or are bacteriostatic. These proteins were generally assumed to be restricted to insects, yet recent information has shown some homologous counterparts in vertebrates, including humans. Recent data have revealed that at least some of these proteins can also act against eukaryotic cells, including human infectious parasites. The latter activities have opened up new possibilities for disease control.

The andropin gene and its product, a male-specific antibacterial peptide in Drosophila melanogaster.

In our study of the cecropin locus in Drosophila we have found a gene for a new peptide, andropin, with antibacterial properties. Transcripts from this gene, Anp, could be detected in newly eclosed males and reached steady-state levels after 1 day. Transcription was strongly induced in response to mating and is strictly confined to the ejaculatory duct of adult males. The deduced peptide sequence reveals a hydrophobic amino terminus with striking similarity to the signal peptide of the cecropins. The sequence of the predicted mature andropin shows no direct homology with the cecropins, but the two peptides may have similar secondary structures. We have synthesized the predicted gene product and shown it to be antibacterial. Crude extracts from male genital tracts show a potent bactericidal activity, and electrophoretic separation revealed at least three antibacterial components, one with the same mobility as the synthetic peptide. It appears that insects have evolved a mechanism for the protection of the seminal fluid and the male reproductive tract against microbial infections.

The immune response in Drosophila: pattern of cecropin expression and biological activity.

Cecropins are antibacterial peptides, induced in Drosophila as part of the humoral immune response to a bacterial invasion. We have used the cloned Drosophila cecropin genes CecA1, A2 and B as probes to study the developmental and tissue specific regulation of this response. The genes are strongly expressed in fat body and hemocytes after injection of bacteria, the CecA genes being much more active than CecB in the fat body. All parts of the fat body and 5-10% of the hemocytes are involved in this response. CecA1 and A2 are most active in larvae and adults; CecB is preferentially active in early pupae. A small peak of constitutive cecropin expression in early pupae appears to be caused by bacteria in the food. Cecropin A, the common product of the CecA1 and A2 genes, was identified in the hemolymph of immunized flies at a concentration of 25-50 microM, enough to kill all tested bacteria except Serratia, a Drosophila pathogen. A useful in vitro system to study the immune response has been found in Schneider's line 2 cells which respond to lipopolysaccharide and laminarin by cecropin expression.

Regulation of larval cuticle protein gene expression in Drosophila melanogaster.

Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2-3 variant, which has an insertion of a copia-like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP-3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP-1 and LCP-f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer-like element, which causes altered expression of these genes. We have also investigated the cis-regulatory elements involved in expression of the LCP-2 gene in wild-type larvae. We have identified two upstream regions that may contain separate cis-regulatory elements. The region between -252 bp and -515 bp may be essential for any expression of LCP-2. Additionally, the region between -515 bp and -795 bp appears to be required for the normal level of expression of the LCP-2 gene.

The cuticle proteins of Drosophila melanogaster: genetic localization of a second cluster of third-instar genes.

Five third-instar larval cuticle protein genes are placed on the left arm of the third chromosome. For these five genes, 12 variants and two induced mutants are described. All the naturally occurring variants are codominant. One EMS-induced mutant is characterized by the codominant appearance of a new protein, and a second by a recessive mutation that codes for a modifier of third-instar larval cuticle protein 5 (L3CP-5). All but the putative modifier gene map to within less than 0.3 map units of each other on the left arm of the third chromosome at approximately 11. We propose that these genes constitute a cluster of third-instar cuticle protein genes. The induced recessive mutant maps outside of the cluster to a region also on the left arm of the third chromosome. The proteins and their genes should prove useful for both developmental and evolutionary studies.

Chromatin structure at the 44D larval cuticle gene locus in Drosophila: the effect of a transposable element insertion.

The chromatin structure of the larval cuticle gene cluster at 44D was characterized in embryos from wild-type (Oregon R) and a variant line (2/3) of Drosophila melanogaster. A major DNase I hypersensitive (DH) site was found between genes II and III in the chromatin, in a position 5' to the transcriptional start of the genes in the cluster. The introduction of a 7.3 kilobase transposable element into the cluster in the 2/3 variant enhanced the sensitivity of the major site in 2/3 chromatin but had no other effect upon the pattern of DH sites associated with the wild-type sequences. The wild-type sequences were packaged into an ordered nucleosome-like array in embryos, as revealed by digestion with the chemical cleavage reagent (methidiumpropyl-EDTA) iron (II) [MPE . Fe(II)]. Nucleolytic cleavage within the transposable element chromatin shows it to be organized in an ordered array punctuated by several DH sites. While the patterns of DNase I hypersensitivity are similar in the vicinity of the direct terminal repeats, the patterns revealed by micrococcal nuclease and MPE . Fe(II) are not, indicating a different chromatin organization of these two identical sequences.