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PH. BULET and S. UTTENWEILER-jOSEPH
morphological level (Rizki and Rizki 1984) while the molecular aspects have
only been recently investigated (Franc et al. 1996, Braun et al. 1997). The
humoral defense involves, as first line, the activation of proteolytic cascades
leading to coagulation and melanization at the site of injury. Little information
is available on insect coagulation. Melanization, which has been largely investigated in Lepidopera (for review, see Ashida and Brey 1997), is poorly understood
in Drosophila as only enzymes acting at the end of this cascade were characterized (Fujimoto et al. 1993, Chosa et al. 1997). The second line of humoral defense
in insects is the rapid synthesis by the fat body (an equivalent of the mammalian
liver) and by certain blood cells of antimicrobial peptides and their release into
the hemolymph (insect body fluid). This latter aspect of insect immunity is well
documented in Drosophila where seven distinct antimicrobial peptides/polypeptides (plus isoforms) have been characterized (for review, see Hoffmann and
Reichhart 1997). In general, insect antimicrobial peptides have a broad range of
activity and are not cytotoxic. They share common features such as a low molecular mass (below 5 kDa), a positive net charge at physiological pH and for most
of them, amphiphilic a-helices or hairpin-like f3-sheets or mixed structures. The
existence of several antimicrobial pep tides with different functional properties
in association with the genetic potential of Drosophila allowed to demonstrate
the existence of two signaling pathways, referred to as the imd (for immune deficiency) and Toll pathways, leading to the expression of the antibacterial or antifungal genes, respectively (Lemaitre et al. 1995, Lemaitre et al. 1996). However,
many of the components of these two signaling cascades have yet to be identified.
Although significant progress has been made in recent years in the field of
Drosophila immunity, essential questions remain to be answered. First, no information is available to date on the mechanisms of recognition of the pathogens
that induce the immune response. At a second level, the interconnections
between the different reactions of the insect immune response described above
are unknown. Furthermore, many effectors involved in these different immune
mechanisms remain to be characterized. Finally, we cannot eliminate the possible
existence of yet unknown aspects of the immune response.
The components of Drosophila immunity which have been characterized to
date were isolated either by a biochemical approach on the basis of their activities (e.g. antimicrobial peptides) or by molecular biology (cloning of genes by
homology) or by genetics (e.g. genes involved in the imd and Toll pathways) (for
review, see Hoffmann and Reichhart 1997). Our aim was to develop a methodology to detect novel molecules involved in Drosophila humoral immunity at the
protein level, regardless of a precise biological activity and omitting any purification step that could introduce a certain selectivity. As the immune reactions can
be activated by a septic injury, a differential analysis between the hemolymph of
unchallenged and experimentally immune-challenged Drosophila can be performed to detect molecules induced during the humoral immune response. This
strategy could be operated by two-dimensional gel electrophoresis but as collecting Drosophila hemolymph is labor-intensive, a more sensitive technology is
required for analyzing the components present in the body fluid (0.1 [11) of individual flies.
PH. BULET and S. UTTENWEILER-jOSEPH
morphological level (Rizki and Rizki 1984) while the molecular aspects have
only been recently investigated (Franc et al. 1996, Braun et al. 1997). The
humoral defense involves, as first line, the activation of proteolytic cascades
leading to coagulation and melanization at the site of injury. Little information
is available on insect coagulation. Melanization, which has been largely investigated in Lepidopera (for review, see Ashida and Brey 1997), is poorly understood
in Drosophila as only enzymes acting at the end of this cascade were characterized (Fujimoto et al. 1993, Chosa et al. 1997). The second line of humoral defense
in insects is the rapid synthesis by the fat body (an equivalent of the mammalian
liver) and by certain blood cells of antimicrobial peptides and their release into
the hemolymph (insect body fluid). This latter aspect of insect immunity is well
documented in Drosophila where seven distinct antimicrobial peptides/polypeptides (plus isoforms) have been characterized (for review, see Hoffmann and
Reichhart 1997). In general, insect antimicrobial peptides have a broad range of
activity and are not cytotoxic. They share common features such as a low molecular mass (below 5 kDa), a positive net charge at physiological pH and for most
of them, amphiphilic a-helices or hairpin-like f3-sheets or mixed structures. The
existence of several antimicrobial pep tides with different functional properties
in association with the genetic potential of Drosophila allowed to demonstrate
the existence of two signaling pathways, referred to as the imd (for immune deficiency) and Toll pathways, leading to the expression of the antibacterial or antifungal genes, respectively (Lemaitre et al. 1995, Lemaitre et al. 1996). However,
many of the components of these two signaling cascades have yet to be identified.
Although significant progress has been made in recent years in the field of
Drosophila immunity, essential questions remain to be answered. First, no information is available to date on the mechanisms of recognition of the pathogens
that induce the immune response. At a second level, the interconnections
between the different reactions of the insect immune response described above
are unknown. Furthermore, many effectors involved in these different immune
mechanisms remain to be characterized. Finally, we cannot eliminate the possible
existence of yet unknown aspects of the immune response.
The components of Drosophila immunity which have been characterized to
date were isolated either by a biochemical approach on the basis of their activities (e.g. antimicrobial peptides) or by molecular biology (cloning of genes by
homology) or by genetics (e.g. genes involved in the imd and Toll pathways) (for
review, see Hoffmann and Reichhart 1997). Our aim was to develop a methodology to detect novel molecules involved in Drosophila humoral immunity at the
protein level, regardless of a precise biological activity and omitting any purification step that could introduce a certain selectivity. As the immune reactions can
be activated by a septic injury, a differential analysis between the hemolymph of
unchallenged and experimentally immune-challenged Drosophila can be performed to detect molecules induced during the humoral immune response. This
strategy could be operated by two-dimensional gel electrophoresis but as collecting Drosophila hemolymph is labor-intensive, a more sensitive technology is
required for analyzing the components present in the body fluid (0.1 [11) of individual flies.