Christine Kappler

Drosophila Immune Deficiency (IMD) Is a Death Domain Protein that Activates Antibacterial Defense and Can Promote Apoptosis

We report the molecular characterization of the immune deficiency (imd) gene, which controls antibacterial defense in Drosophila. imd encodes a protein with a death domain similar to that of mammalian RIP (receptor interacting protein), a protein that plays a role in both NF-kappaB activation and apoptosis. We show that imd functions upstream of the DmIKK signalosome and the caspase DREDD in the control of antibacterial peptide genes. Strikingly, overexpression of imd leads to constitutive transcription of these genes and to apoptosis, and both effects are blocked by coexpression of the caspase inhibitor P35. We also show that imd is involved in the apoptotic response to UV irradiation. These data raise the possibility that antibacterial response and apoptosis share common control elements in Drosophila.

Ecdysone titre and metabolism in relation to cuticulogenesis in embryos of Locusta migratoria

Abstract Eggs of Locusta migratoria contain remarkably high concentrations of ecdysone and several other ecdysteroids. During the time-span of embryonic development (11 days) 4 distinct peaks of ecdysone concentration (up to 8 μM) are observed in the egg, demonstrating the ecdysiosynthetic capacity of the embryo. Only during postblastokinetic development, is ecdysone efficiently hydroxylated to 20-hydroxyachieved through conjugation. On the basis of optical and electron microscopic observations, we have been able to correlate precisely each of the four peaks of ecdysone concentration in the egg with the time of deposition of a cuticle by the embryonic tissues (peak 1: serosal cuticle; peak 2: first embryonic cuticle; peak 3: second embryonic cuticle; peak 4: third embryonic cuticle).

Insect immunity. Two 17 bp repeats nesting a kappa B-related sequence confer inducibility to the diptericin gene and bind a polypeptide in bacteria-challenged Drosophila.

The Drosophila diptericin gene codes for a 9 kDa antibacterial peptide and is rapidly and transiently expressed in larvae and adults after bacterial challenge. It is also induced in a tumorous Drosophila blood cell line by the addition of lipopolysaccharide (LPS). The promoter of this gene contains two 17 bp repeats located closely upstream of the TATA‐box and harbouring a decameric kappa B‐related sequence. This study reports that the replacement of the two 17 bp repeats by random sequences abolishes bacteria inducibility in transgenic fly lines. In transfected tumorous blood cells, the replacement of both or either of the 17 bp motifs reduces dramatically LPS inducibility, whereas multiple copies significantly increase the level of transcriptional activation by LPS challenge. A specific DNA‐protein binding activity is evidenced in cytoplasmic and nuclear extracts of induced blood cells and fat body. It is absent in controls. It is proposed that induction of the diptericin gene mediated by the two 17 bp repeats occurs via a mechanism similar to that of mammalian NF‐kappa B.

The Drosophila Immune Defense against Gram-Negative Infection Requires the Death Protein dFADD

Drosophila responds to Gram-negative infections by mounting an immune response that depends on components of the IMD pathway. We recently showed that imd encodes a protein with a death domain with high similarity to that of mammalian RIP. Using a two-hybrid screen in yeast, we have isolated the death protein dFADD as a molecule that associates with IMD. Our data show that loss of dFADD function renders flies highly susceptible to Gram-negative infections without affecting resistance to Gram-positive bacteria. By genetic analysis we show that dFADD acts downstream of IMD in the pathway that controls inducibility of the antibacterial peptide genes.

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (insecta, orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.

INSECT IMMUNITY. A TRANSGENIC ANALYSIS IN DROSOPHILA DEFINES SEVERAL FUNCTIONAL DOMAINS IN THE DIPTERICIN PROMOTER

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single‐copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis‐regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B‐related motifs in the proximal promoter as the sites conferring inducibility and tissue‐specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon‐gamma responsive sites in this up‐regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of ‐0.6 kb which increases the level of expression by a factor of five.

Fate of maternal conjugated ecdysteroids during embryonic development in Locusta migratoria

Abstract Newly laid eggs of Locusta migratoria contain impressively high concentrations of conjugated 2-deoxyecdysone and conjugated ecdysone of maternal origin. These molecules are metabolized during embryonic development, the changes concerning not only the ecdysteroid genins but also the conjugating moieties. In the present paper the fates of the maternal conjugates were followed during embryogenesis in the eggs. The conjugates were separated both by silica gel TLC and reverse-phase HPLC and measured, before and after hydrolysis, by RIA. Fluctuations of radioactive ecdysteroid conjugates were also investigated in eggs laid by females subjected to massive injections of tritiated cholesterol. The results are discussed in relation to recent data on identification of ecdysteroid conjugates in Locusta and a model for the sequences of metabolic events leading from maternal ecdysteroid conjugates to the embryonic ecdysteroids is proposed.

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.

Studies on the C-2 hydroxylation of 2-deoxyecdysone in Locusta migratoria

Abstract 2-Deoxyecdysone is the immediate precursor of ecdysone in several biological systems. We have used a high specific activity tritiated 2-deoxyecdysone to probe the capacity of various tissues of larvae and adults of Locusta to hydroxylate 2-deoxyecdysone to ecdysone. Not only prothoracic glands and follicle cells, but also Malpighian tubules, fat body, midgut, etc. are able to hydroxylate 2-deoxyecdysone to ecdysone efficently. Larval Malpighian tubules, which exhibited the highest conversion rate in our experiments, were used to gain biochemical information on the C-2 hydroxylase in non-endocrine tissues. The enzyme was found to have a strict mitochondrial localization; its Km is 0.4 μM. It requires aerobic conditions and is inhibited by metyrapone (I50: 60 μM) and piperonyl butoxide (I50: 10 μM). These and other results suggest that the C-2 hydroxylase is a cytochrome P-450 monooxygenase; surprisingly however CO inhibition could not be evidenced even at a CO O 2 ratio of 9:1. In the follicle cells, C-2 hydroxylation is also localized in the mitochondria.

The biosynthetic pathway of ecdysone: Studies with vitellogenic ovaries of Locusta migratoria (orthoptera)

Ovaries of adult females of Locusta migratoria synthesize impressive amounts of the steroid hormone ecdysone (and related ecdysteroids) during the late phases of vitellogenesis. The present study, aimed at elucidating the sequence of the biosynthetic steps that lead from cholesterol to ecdysone, has taken benefit of this remarkable biological model by using a double approach: (1) isolation and physico-chemical identification of endogenous biogenetic intermediates; (2) metabolic study of labelled putative precursor molecules. The data presented in this paper lead us to propose the following sequence of events: conversion of cholesterol to 3 beta-hydroxy-5 beta-cholest-7-en-6-one (via several intermediates not identified in this study) followed by 14 beta-hydroxylation to 3 beta, 14 alpha-dihydroxy-5 beta-cholest-7-en-6-one; hydroxylation on the side-chain at C-25 and C-22 (in this order) to 2-deoxyecdysone; hydroxylation at C-2 to ecdysone.