Interactive Fly, Drosophila

Putative regulatory regions of the slit and Toll genes have been dissected to identify CNS midline-restricted transcriptional enhancers. This analysis has uncovered DNA regions able to drive expression in most tissues in which embryonic slit and Toll are expressed, including three separable CNS midline-conferring regions: one in the Toll gene expressed early in all of the CNS midline precursors, and two in the slit gene expressed later in the midline glia (Wharton, 1993).

Early heart development in Drosophila and vertebrates involves the specification of cardiac precursor cells within paired progenitor fields, followed by their movement into a linear heart tube structure. The latter process requires coordinated cell interactions, migration, and differentiation as the primitive heart develops toward status as a functional organ. In the Drosophila embryo, cardioblasts emerge from bilateral dorsal mesoderm primordia, followed by alignment as rows of cells that meet at the midline and morph into a dorsal vessel. Genes that function in coordinating cardioblast organization, migration, and assembly are integral to heart development, and their encoded proteins need to be understood as to their roles in this vital morphogenetic process. The Toll transmembrane protein is expressed in a secondary phase of heart formation, at lateral cardioblast surfaces as they align, migrate to the midline, and form the linear tube. The Toll dorsal vessel enhancer has been characterized, with its activity controlled by Dorsocross and Tinman transcription factors. Consistent with the observed protein expression pattern, phenotype analyses demonstrate Toll function is essential for normal dorsal vessel formation. Such findings implicate Toll as a critical cell adhesion molecule in the alignment and migration of cardioblasts during dorsal vessel morphogenesis (Wang, 2005).

At the time dorsal-ventral polarity is established during early Drosophila development, Toll is associated with the plasma membrane around the entire syncytial blastoderm embryo. Thereafter, Toll exhibits zygotic expression on several cell surfaces, including a specific dorsal cell type in late-stage embryos. These were identified at first as leading-edge cells of the two-epidermal sheets moving toward the dorsal midline. Toll expression in dorsal aspects of the embryo has been reevaluated and, to the contrary, it has now been concluded the gene is expressed in cardioblasts of the developing and formed dorsal vessel (Wang, 2005).

Initially, Toll mRNA accumulation was analyzed by in situ hybridization, with gene transcripts first detected in dorsal cell populations in stage 12 embryos and later in two converging rows of cells during the process of dorsal closure. The likelihood of the Toll-positive cells being cardioblasts was strongly implied by the pattern of mRNA accumulation in stage 16 embryos. Toll expression was detected in roughly 50 cell pairs, and the organization of said cells was reminiscent of cardioblasts within structurally identifiable aorta and heart regions of the assembled dorsal vessel. The pattern of Toll protein expression was also investigated, with results comparable to those obtained in the RNA analysis. The transmembrane protein was detected in dorsal cells in late stage 12/early stage 13 embryos. Thereafter, it showed a clear presence on lateral surfaces of all cells aligned within two contiguous rows as they migrate toward the dorsal midline. By stage 16, the Toll-positive cells populate the core of the dorsal vessel, again within defined aorta and heart subregions. Toll was found exclusively on cardioblast surfaces, while organ-associated pericardial, lymph gland, and ring gland cells failed to express the protein. High-resolution analysis by confocal microscopy demonstrated Toll presence at lateral points of contact between all cardioblasts of the mature dorsal vessel (Wang, 2005).

Toll zygotic transcription is complex based on the numerous cell and tissue types that express the gene. Through efforts to identify a regulatory sequence controlling Toll expression in central nervous system (CNS) midline glial cells, Wharton (1993) located three regions upstream of the gene that possessed transcriptional enhancer activity. Relevant to the demonstration of Toll expression in the dorsal vessel, a 6.5-kb DNA was fortuitously found to direct lacZ reporter expression in all cardioblasts, and in pharyngeal and body wall muscles as well. Due to the interest in understanding how this expression might be regulated, the Toll cardioblast enhancer was delimited within the defined upstream region. At first, the analysis involved testing Toll 5'-flanking DNAs for the ability to drive lacZ expression in embryos of transgenic strains. A 7.1-kb region located between ~9.3 and ~2.2 upstream of the gene showed strong enhancer function in all cardioblasts of the dorsal vessel. The DNA was subdivided into five overlapping segments, and only the most distal 1.7-kb DNA maintained cardioblast activity. Subsequently, five fragments spanning this 1.7-kb interval were tested for enhancer function, and dorsal vessel activity was mapped to a 305-bp sequence located between ~8.3 and ~8.0 relative to the Toll transcription start site. Consistent with the timing of Toll mRNA and protein accumulation in cardioblasts, the 305-bp enhancer becomes active during stage 12 and maintains its activity through all subsequent events of dorsal vessel morphogenesis. It is noteworthy that this small DNA also functions in amnioserosa cells from stage 11 through stage 15 (Wang, 2005).

Since Toll encodes a transmembrane protein with leucine-rich repeats in its extracellular domain, a prediction was made that Toll could function as a homophilic cell adhesion molecule, in addition to its well-characterized role as a signal-transducing receptor. In support of this hypothesis, induced expression of the protein in the nonadhesive Schneider 2 cell line causes cellular aggregation, with Toll accumulating at sites of cell-cell interaction. Such a localization property is characteristic of cellular adhesion molecules. Given the highly specialized localization, and structural and functional features of the protein, it is likely that Toll contributes prominently to the molecular environment that aligns and stabilizes cardioblasts on their path toward assembly within the dorsal vessel (Wang, 2005).

The observation of structurally defective dorsal vessels within Toll mutant embryos is consistent with the pattern of Toll expression in cardioblasts. D-MEF2 serves as a marker for all cardioblasts, from their early appearance through their organization within the mature organ. Based on D-MEF2 staining, it appears appropriate numbers of cardioblasts are specified in mutant embryos, but deviations are observed from the normal process of cardioblast alignment and synchronous migration as two contiguous rows of 52 cells. Several other markers for the formed dorsal vessel identified random gaps in the linear organ due to missing and/or abnormally located cardioblasts. Such cardiac phenotypes are reminiscent of those presented by faint sausage (fas) mutant embryos; mutations of the immunoglobulin-like cell adhesion molecule also led to cardioblast alignment problems. Whether Toll and Fas work in combination for the proper alignment and migration of these cells remains to be investigated. Additionally, while structural and phenotypic properties are consistent with its role as a cardioblast adhesion molecule, a function for Toll in mediating signaling events between neighboring cardiac cells cannot be ruled out. So far, no indicators exist for the latter possibility; it was not possible to demonstrate expression of potential Toll transcriptional effectors (Dorsal and Dif) in cells of the dorsal vessel. Either way, these molecular and genetic findings identify Toll as a vital player in dorsal vessel formation (Wang, 2005).

The regulation of Toll expression in cardioblasts was pursued due to an interest in further defining the transcriptional network controlling heart development in Drosophila. The studies demonstrated Toll heart expression is controlled by a 305-bp DNA located 8.0 kb upstream of the transcription start site. This regulatory module contains multiple binding sites for Doc T-box proteins and a single recognition site for the Tin homeodomain protein. The Toll dorsal vessel enhancer contains a single TCAAGTG sequence at nucleotides 163 to 169. The evidence is strong for the transcriptional enhancer being regulated by both of these cardiogenic factors. Doc and Tin are expressed in adjacent but nonoverlapping sets of cardioblasts within segments of the dorsal vessel; together, they make up the complete population of inner cardiac cells. A deletion of the distal part of the Toll 305-bp enhancer that removes the strong Doc-A footprint sequence, which likely binds multiple Doc molecules through T-box domain recognition of GTG motifs, eliminates enhancer function in Svp/Doc cells while maintaining activity in Tin cells. Systematically adding back T-box core binding elements to partially, then fully, reestablish the Doc-A binding site restores enhancer function in the Svp/Doc population (Wang, 2005).

As for Tin, mutation of its recognition element in the Toll 305 DNA leads to decreased and variable enhancer activity in both Tin and Svp/Doc cardioblasts. This result suggests that Tin is required not only for the activation of Toll expression in the four cardioblasts per hemisegment that are Tin positive after stage 12 but also for its initiation in all six cardioblasts in each hemisegment during early stage 12. The residual activity of the mutated Toll 305 DNA may reflect some degree of Tin regulation through cryptic, low-affinity binding sites present in the enhancer. Indeed, perusal of the Toll sequence identifies three candidate Tin elements that match the binding consensus at six of seven nucleotide pairs, and other Tin-regulated enhancers of genes such as D-mef2, ß3-tubulin, and pnr also employ more than one Tin binding site (Wang, 2005).

In contrast to the Toll 305 enhancer element, mutation of the exact Tin site in the Toll 258 DNA completely silenced the enhancer in the normally Tin-active cells. This result strongly implies that Tin, and at least one other factor working through the distal 47 bp of DNA, are required for activating the Toll gene. Candidates for such factors are the Doc T-box proteins, which are initially expressed in all cardioblast progenitors during mid stage 12 , as well as products of the T-box genes H15 and Midline (Mid), which are expressed in all cardioblasts from mid stage 12 onward. Mid can bind to the same regions of Toll DNA as Doc, although the relevance of such interactions remains to be investigated. A combinatorial requirement for T-box proteins and Tin during the initiation and/or maintenance of Toll expression is further supported by the observation that derivatives of the enhancer containing only the Doc-A sequences fail to show activity in Svp/Doc cells. Together, these molecular data point to a mechanism wherein T-box proteins, in combination with Tin, initially activate the Toll gene in all cardioblast progenitors. After stage 12, Doc and Tin (perhaps in cooperation with H15 and/or Mid) activate Toll in two complementary subsets of cardioblasts of the dorsal vessel (Wang, 2005).

Unfortunately, a genetic requirement for these two factors in the regulation of the Toll enhancer cannot be proven at this time since Doc and tin mutant embryos fail to produce cardioblasts. Such an analysis could be attempted with the generation of specialized Doc or tin genetic backgrounds that allow for cardioblast specification early on, while lacking protein functions in later stages of dorsal vessel formation. However, forced-expression studies have demonstrated that individual expression of Tin or Doc2 leads to expanded enhancer activity, while simultaneous expression of the cardiac factors results in a robust activation of Toll transcription. These findings convincingly support the model of Doc and Tin being positive transcriptional regulators of the Toll dorsal vessel enhancer (Wang, 2005).

In addition to the demonstration of Doc and Tin as activators of Toll expression in the dorsal vessel, the regulatory analysis has generated important reagents that should facilitate the discovery of novel cardiac-functioning genes of Drosophila. That is, the Toll-cGFP and Toll-nGFP transgenes serve as sensitive markers for assessing distinct aspects of dorsal vessel morphogenesis in living animals. In stage 16 to 17 embryos and thereafter, Toll-cGFP expression can be used to monitor the formation and function of the three pairs of valvelike ostia within the heart region of the dorsal vessel. Likewise, Toll-nGFP can be used to determine the exact number and diversification status of cardioblasts, as larger nuclei are present within Tin-determined cells while smaller nuclei are found in Svp/Doc-determined cells. Such sensitive and easy-to-use reagents will be valuable in genomewide screens to discover new genes involved in Drosophila heart development (Wang, 2005).

Transcriptional Regulation

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. In sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F. The sim mutant CNS defect may be largely due to loss of midline slit expression. The snail gene is required to repress sim and other midline genes in the presumptive mesoderm (Nambu, 1990).

Targets of Activity

Increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of Dorsal. In contrast, activation of endogenous protein kinase A and expression of wild-type Toll receptors, have only a marginal effect on the cellular distribution of the Dorsal protein. Treatment of cells with activators of Protein kinase C and radical oxygen intermediates, both of which activate vertebrate Nuclear factor kappa B, also have little effect on Dorsal protein localization (Kubota, 1993).

Dorsal is an embryonic phosphoprotein; its phosphorylation state is regulated by an intracellular signaling pathway initiated by the transmembrane receptor Toll. The phosphorylation state of Dorsal is altered during the time period during which Toll is activated. Mutations that constitutively activate Toll stimulate Dorsal phosphorylation, while mutations that block Toll activation reduce the level of Dorsal phosphorylation. Signal-dependent Dorsal phosphorylation is modulated by three intracellular proteins: Pelle, Tube, and Cactus. Free Dorsal is a substrate for a signal-independent kinase activity. Dorsal appears to be a substrate for a Toll-dependent kinase. (Gillespie, 1994).

The Torso receptor tyrosine kinase modulates DL activity. The Torso pathway selectively masks the ability of DL to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of Torso, such as D-raf and Rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and Torso pathways are both active only at the embryonic poles, and consequently, zen and dpp, target genes otherwise repressed in middle body regions, are expressed at these sites. Constitutive activation of the Torso pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of DL target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor (Rusch, 1994).

Drosophila dorsoventral patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (Dorsal, Nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid NEE, a 300-bp region of the rho promoter, sufficient for neuroectoderm expression, and kappa light chain enhancer, which contain similar arrangements of activator and repressor binding sites. The kappa enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the kappa enhancer, including the kappa B site and kappa E boxes. This work suggests that enhancers are able to couple conserved signaling pathways to divergent gene functions (Gonzalez-Crespo, 1994).

In addition to its function in embryonic development dorsal is expressed in larval and adult fat body where its RNA expression is enhanced upon injury. Injury also leads to a rapid nuclear translocation of DL from the cytoplasm in fat body cells. The nuclear localization of DL during the immune response is controlled by the Toll signaling pathway, comprising gene products that participate in the intracellular part of the embryonic dorsoventral pathway.In mutants such as Toll or cactus,exhibiting melanotic tumor phenotypes, DL is constitutively nuclear. Although DL has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, injury-induced expression of diptericin can occur in the absence of DL. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. Like other rel proteins, dl can control the level of its own transcription (Lemaitre, 1995).

Toll is subject to glycosylation. A constitutively activated mutant receptor, Toll10B, is processed into a distinct isoform of slower electrophoretic mobility when compared with the wild type molecule in cell lines and in the embryo. The wild type protein can also be processed into this form if over-expressed but in the embryo is present as the smaller species. The decrease in the mobility of Toll10B and over-expression of wild type receptors is caused by altered patterns of N-linked glycosylation. It is probable that the Toll10B receptor is unable to associate with a limiting co-factor that masks supplementary N-linked glycosylation sites (Kubota, 1995).

The possible involvement of Toll in the Drosophila immune response was investigated by overexpressing Toll10B, a constitutively active mutant protein, in the Drosophila blood cell line mbn-2. Induction of the Cecropin A1 (CecA1) gene, coding for a bactericidal peptide, was used as an indicator for the immune response. Toll10B was found to increase CecA1 transcription. This effect depends on the presence of a kappa B-like site in the CecA1 promoter. The endogenous Toll gene is expressed in mbn-2 cells, indicating that this gene may normally play a role in Drosophila blood cells (Rosetto, 1995).

RNA localization and post-transcriptional regulation

Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs whose protein products determine cell fate. Three mRNAs that dictate anterior, dorsoventral, and terminal specification--Bicoid, Toll, and Torso, respectively--show increases in polyadenylate [poly(A)] tail length concomitant with translation. These experiments identify a regulatory pathway that can coordinate initiation of maternal pattern formation systems in Drosophila (Salles, 1994).

Maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of Bicoid and Toll mRNAs. cortex mutant embryos contain a Bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the Bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly[A]) tail. Injection of polyadenylated Bicoid mRNA into mutant cortex embryos allows translation demonstrating that insufficient polyadenylation prevents endogenous Bicoid mRNA translation. In contrast Nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in mutant cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. cortex eggs are fertile, but embryos arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development (Lieberfarb, 1996).

Toll mRNA is maternally provided and is found in the ovary and early embryo. Toll protein does not accumulate in the embryo, however, until the syncytial blastoderm stage. Toll protein is not detectable in ovaries and protein levels substantially increase during the first 2 hours of embryogenesis. Toll protein expression peaks at the syncytial blastoderm stage, consistent with whole-mount staining results. These results are consistent with translational activation of Toll mRNA during early embryogenesis or with Toll protein instability during the first hour of development. No evidence for differential protein stability is found during the first few hours of embryogenesis. Therefore, the favored interpretation is that Toll mRNA is translationally activated during early development. It has been suggested that the accumulation of Toll protein in the embryo temporally correlates with elongation of the poly (A) tail of the message. Toll mRNA is translationally activated by regulated cytoplasmic polyadenylation. A 192 nucleotide regulatory element has been identified in the Toll 3' UTR that is necessary for robust translational activation of Toll mRNA and that also regulates polyadenylation. UV crosslinking analyses suggest that two proteins bind specifically to the 192 nucleotide element. One or both of these proteins may be factors that are required for translational regulation or cytoplasmic polyadenylation. These studies demonstrate that regulated polyadenylation plays a critical role in the Drosophila dorsal-ventral patterning system (Schisa, 1998).

It will be informative to determine the identity of these proteins to aid in understanding how polyadenylation promotes translational activation. One candidate protein might be a homolog of the Xenopus CPEB (CPE-binding protein). A Drosphila protein, Orb, has been identified that has homology to CPEB. The major defect in severe orb alleles is lack of localization of several mRNAs during oogenesis. The Orb protein may have a role in polyadenylation of mRNAs during oogenesis. One of the proteins identified here, of apparent molecular mass 101 kDa, may be Orb, which is 97-99 kDa. Examination of Toll polyadenylation in orb mutants or immunoprecipitation of the cross-linked proteins with an orb antibody may be useful in determining whether any of the Toll-binding proteins are, in fact, orb. A question that remains unanswered is why the embryo temporally regulates expression of Toll. One possibility is that while ventral-specific activation of the Toll ligand (Spatzle) acts as the spatial restrictor of the dorsal-ventral signal, Toll functions as a temporal modulator. Toll is not spatially restricted at any point of development; rather it is expressed ubiquitously in the plasma membrane. If Toll protein were generated earlier in oogenesis or embryogenesis, there could be adverse developmental consequences. For instance, Toll is known to have more than one functional ligand. If such a ligand were present in the oocyte or early embryo and Toll were also expressed at this time, there could be deleterious consequences for the embryo. Alternatively, since Toll is a transmembrane protein, expression during oogenesis could result in the protein being trapped in the nurse cells and not deposited into the oocyte. Some maternal mRNAs are known to be degraded soon after translation and the current data suggests that translated Toll mRNA is slightly less stable than truncated, untranslated forms of Toll mRNA. Thus if Toll protein were translated but not transported to the oocyte, there might not be sufficient mRNA remaining in the oocyte and a lethal Toll null phenotype would result (Schisa, 1998 and references).

The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila

From a forward genetic screen for phagocytosis mutants in Drosophila, a mutation was identified that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, the data suggest that the Drosophila gene PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram⁺ bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-L-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, it was found that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis (Garver, 2006).

Host receptors must recognize a microbe before any immune response can begin. After recognition, signaling pathways are activated and result in effector responses such as induction of antimicrobial peptides (AMPs), melanization, and phagocytosis, which are important for controlling the infection. Much is known about the signaling pathways important for the AMP response in Drosophila, but less is known about the regulation of the other two responses. In Drosophila, if phagocytosis is blocked in a mutant that is already impaired for the AMP response, a normally innocuous Escherichia coli infection becomes lethal. Phagocytosis is also likely to be a first line of defense, because the response involves specific interaction between microbe and host receptors and occurs within half an hour, whereas the induction of AMPs occurs over several hours and AMPs can act on diverse groups of microbes (Garver, 2006).

The peptidoglycan recognition proteins (PGRPs) are critical receptors in the Drosophila immune response that are required for the recognition of peptidoglycan, a component of bacterial cell walls (Dziarski, 2004; Steiner, 2004), and for subsequent activation of AMP gene expression (Choe, 2002; Werner, 2003). PGRPs were first characterized in the moths Bombyx mori and Trichoplusia ni and proposed to be receptors that can trigger immune responses. PGRPs have also been identified in mammals, and mutant PGRP mice have been generated, but the most comprehensive characterization of PGRPs has been performed in Drosophila (Garver, 2006).

Drosophila has 13 PGRP genes, six long (L) forms with four that are predicted to reside in the plasma membrane, and seven short forms (S) that are all predicted to be secreted. PGRPs share homology with N-acetylmuramoyl-L-alanine amidases, which cleave peptidoglycan at the lactylamide bond between the glycan backbone and the stem peptides. Some PGRPs, such as PGRP-LC, -LE -SA, and -SD, lack a critical cysteine in the catalytic pocket and are not able to cleave peptidoglycan. PGRP-LC, -LE, and -SA have been demonstrated to bind peptidoglycan and are necessary for expression of AMP genes, supporting the hypothesis that PGRPs directly recognize bacteria and activate immune responses. The identification of mutations in PGRP-SA (seml) and PGRP-LC (ird7 or totem) indicated that these genes are necessary for activation of the two signaling pathways regulating AMP gene expression, the Toll pathway that responds to Gram⁺ bacteria and fungi and the Imd pathway that responds to Gram^– bacteria. Drosophila uses PGRP-SA and PGRP-LC to distinguish between Gram⁺ and Gram^– PGN for activation of the Toll and Imd signaling pathways (Leulier, 2003). Gram⁺ PGN and Gram^– PGN differ in the stem peptide portion; typical Gram⁺ bacteria have a lysine as the third amino acid, whereas Gram^– bacteria and the Gram⁺ Bacillus have a diaminopimelic acid (DAP) in that position. Two other noncatalytic PGRPs, PGRP-LE and PGRP-SD, also play a role in activation of the Imd and Toll pathways, respectively. PGRP-LC, PGRP-LE double mutants show a more dramatic phenotype to Bacillus and Gram^– bacterial infection than either mutation alone, suggesting that PGRP-LE acts with PGRP-LC in the recognition of Gram^– DAP-type peptidoglycan for the activation of the Imd pathway. PGRP-SD may be playing a similar role with PGRP-SA for activation of the Gram⁺/Toll pathway (Garver, 2006 and references therein).

Catalytic PGRPs, such as PGRP-SC1a and -SC1b, include this cysteine residue in the active site, and are potent enzymes that cleave peptidoglycan between the N-acetylmuramic acid of the backbone and the L-alanine in the stem peptide. After digestion with PGRP-SC1b, staphylococcal peptidoglycan exhibits less activation of the AMP genes in a Drosophila blood cell line, so it was hypothesized that catalytic PGRPs may act as scavengers to limit an inflammatory response to free peptidoglycan (Mellroth, 2003). This may not be absolute, since PGRP-SC1b-digested Gram^– peptidoglycan is still able to activate the AMP response in S2 cells, albeit at a higher dose. However, it was of interest to examine the role of these genes in vivo (Garver, 2006).

In a genetic screen for phagocytosis mutants, a novel mutant, picky was identified. picky flies fail to phagocytose S. aureus particles but can phagocytose E. coli, Bacillus subtilis, and Saccharomyces cerevisiae zymosan particles. picky mutants have defects in the activation of the Toll pathway. picky maps to the location of the PGRP-SC1a, -SC1b, and -SC2 genes, and picky flies express significantly less PGRP-SC1a. The picky defects can be rescued by expression of PGRP-SC1a, indicating that PGRP-SC1a is important for phagocytosis and activation of AMP responses. These results differ from the prevailing model of catalytic PGRPs as scavengers and suggest that in vivo, PGRP-SC1a is required for initiating immune pathways. By comparing a wild-type PGRP-SC1a with a cysteine-serine mutant PGRP-SC1a for rescue of picky phenotypes, it was found that the catalytic activity is essential for phagocytosis of live S. aureus, but not for activation of the Toll pathway (Garver, 2006).

To identify genes important for phagocytosis, a collection of ethylmethane sulfonate-induced adult viable Drosophila mutants was screened using an in vivo phagocytosis assay. To determine what a mutant might look like, the PGRP-LC (ird7) and PGRP-SA (seml) mutants were examined. PGRP-LC has been reported to be a recognition receptor for phagocytosis of E. coli in S2 cells. However, this study contradicted another report citing that ird7 blood cells can phagocytose bacteria (Choe, 2002). The current study found that ird7 mutants are able to phagocytose both E. coli and S. aureus particles. This finding suggested that phagocytosis of Gram^– bacteria may require other receptors in addition to PGRP-LC in vivo. In contrast, it was found that seml mutants are specifically impaired in their ability to phagocytose S. aureus but not E. coli. 94% of the seml mutant flies were able to phagocytose E. coli, but only 25% were able to phagocytose S. aureus. This finding suggests that PGRP-SA may be important for recognition of Gram⁺ bacteria for phagocytosis in addition to its role in activating the Toll pathway. In contrast, flies with mutations affecting other Toll pathway components, spatzle (the Toll ligand) and Dif (an NF-kappaB) were still able to phagocytose S. aureus, indicating that the phagocytosis defect is not a secondary effect from loss of Toll signaling (Garver, 2006).

From a pilot screen, one mutation was identified that was named picky eater^Z2–4761 (picky) because the mutants fail to phagocytose S. aureus particles, but can still phagocytose E. coli and Saccharomyces cerevisiae zymosan particles. Only 25% of picky mutant flies showed any phagocytosis response to S. aureus, but 83% of those same flies were still able to phagocytose E. coli. To investigate whether the recognition involved the difference in peptidoglycan between Gram⁺ and Gram^– bacteria, phagocytosis was examined of a B. subtilis-GFP strain. picky mutants are able to phagocytose B. subtilis. This finding suggests that the picky gene product is important for the specific recognition of the Lys-type peptidoglycans, typical of most Gram⁺ bacteria, but not found in Bacillus spp (Garver, 2006).

Because picky flies appear to be impaired in the recognition of S. aureus for phagocytosis and survival, it was possible that the mutation might also affect activation of the Toll or Imd pathways. The induction of Drosomycin and Diptericin AMP genes is often used to assess activation of the Toll/Gram⁺ and Imd/Gram^– signaling pathways, respectively. The AMP responses in picky were compared with those of the known Toll pathway components, seml and Dif, and to an Imd pathway component, ird7. picky flies showed no induction of Drosomycin in response to S. aureus, E. coli, or Micrococcus luteus at either 2 or 24 h. All of the S. aureus-infected picky flies were dead at 24 h, so the Drosomycin expression in this sample was not assessed. seml and Dif both show some induction of Drosomycin at 2 and 24 h. In comparison, picky has a more consistent and dramatic effect on Drosomycin expression and likely encodes an essential component of the Toll pathway. In contrast, the expression of Diptericin in response to bacterial infection in picky mutants was higher than that seen in wild type. Therefore, the defect in picky appears to be selective for the Toll pathway. To determine where in the Toll pathway picky might lie, the picky mutation was crossed into a Toll^10b gain of function mutant. picky was not able to suppress the constitutive expression of Drosomycin in a Toll^10b mutant. This epistasis analysis places picky either upstream of Toll, which would be consistent with a role in bacterial recognition or in a parallel pathway (Garver, 2006).

The peptidoglycan recognition protein family is important for allowing the Drosophila immune response to distinguish between Gram⁺ and Gram^– bacteria for the activation of specific AMP signaling pathways (Choe, 2002; Werner, 2003). The current data indicate that phagocytosis also relies on PGRPs to distinguish between Gram⁺ and Gram^– bacterial peptidoglycan. A catalytic PGRP, PGRP-SC1a, is essential for the phagocytosis of S. aureus and the activation of AMP responses. The fact that PGRP-SC1a mutants have a striking phagocytosis defect indicates that it is absolutely required and that other PGRPs are not able to substitute for its loss of function. PGRP-SC1a differs from the existing PGRP mutants in that it has catalytic activity that is essential for efficient phagocytosis and ultimately limiting the infection process. Hence, noncatalytic PGRPs, such as PGRP-SA, may not be able to substitute for PGRP-SC1a function. Because PGRP-SC1a is likely present in the hemolymph, it may be acting as an opsonin to bind bacteria, with the PGRP-bacterial complex afterwards being recognized by transmembrane phagocytosis receptors that complete the uptake into the phagocyte. An alternative model is that PGRP-SC1a may generate peptidoglycan cleavage products that function as immune modulators to stimulate phagocytic activity (Garver, 2006).

For S. aureus, PGRP-SC1a is necessary for recognition of bacteria for both the phagocytosis and AMP responses. However, for E. coli, PGRP-SC1a is similar to PGRP-LC in that it is not necessary for recognition for phagocytosis, but is necessary for the activation of an AMP response. This finding raises the interesting issue of the role PGRPs play in recognizing Gram^– bacteria. In Gram^– bacteria, the peptidoglycan is buried under an outer membrane, so it has been proposed that the small amounts of PGN that are shed may be sufficient to trigger the AMP response (Leulier, 2003). Because the cell wall PGN is not easily accessible, it makes sense that the PGRPs are not playing a direct role in recognition for phagocytosis. An alternate possibility is that the exposure of bacterial peptidoglycan occurs after the initial uptake into phagocytes, and at that later point, is then recognized by PGRP-LC and PGRP-SC1a to trigger AMP responses. There is precedence for intracellular recognition in the phagosome in mammalian immune responses, with studies indicating that mammalian short PGRPs function in the phagosome to inhibit bacterial growth and the observation that Toll-like receptor recognition of bacteria in the phagosome can influence the rate of phagosome maturation. A related possibility is that some basal level of activation of PGRP-SC1a and the Toll pathway may be required for any expression of Drosomycin. picky mutants show much lower expression of Drosomycin than wild type in the absence of infection, so perhaps an induction may not be detectable (Garver, 2006).

It was also found that seml mutants are defective in the phagocytosis of S. aureus but not E. coli. Recent reports have indicated that the processing of peptidoglycan into monomers, lactyltetrapeptides, or muropeptides, can yield potent activators of the AMP response for both the Imd and the Toll pathway. It was further suggested for the Toll pathway that processing of peptidoglycan may be a prerequisite step upstream of PGRP-SA/seml recognition and activation of the Toll AMP pathway. This model would be consistent with catalytic PGRPs, such as PGRP-SC1a, playing a supporting role in the processing of peptidoglycan for initiating recognition events (Garver, 2006).

Since many PGRPs are clearly required for activation of immune effector responses, the issue arises as to the specificity of PGRPs for their substrates. Work from several groups indicates that alternative splice variants (in PGRP-LC) or small differences in amino acid sequences (from PGRP-LB and human PGRP-Ialpha structural analyses) may result in the ability of different PGRPs to have distinct recognition potential. It will be interesting to explore the range of bacterial types recognized by the 17 Drosophila PGRP proteins and to determine how subtle differences in recognition may be reflected by specific amino acid sequences. There may also be genetic interactions or antagonism in their function, so using the genetic tools available in Drosophila may be necessary for ultimately determining both their unique and redundant roles (Garver, 2006).

This work demonstrates the utility of a forward genetic approach to understanding the recognition of pathogen types for phagocytosis and activation of AMP responses. It is hoped that identification of genes important for recognition of bacteria in Drosophila should increase understanding of how the process works in the immune system (Garver, 2006).

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