Toll Mutants Research Articles

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

The Toll/NF-κB signaling pathway is required for epidermal wound repair in Drosophila.

The Toll/NF-κB pathway, first identified in studies of dorsal-ventral polarity in the early Drosophila embryo, is well known for its role in the innate immune response. Here, we reveal that the Toll/NF-κB pathway is essential for wound closure in late Drosophila embryos. Toll mutants and Dif dorsal (NF-κB) double mutants are unable to repair epidermal gaps. Dorsal is activated on wounding, and Dif and Dorsal are required for the sustained down-regulation of E-cadherin, an obligatory component of the adherens junctions (AJs), at the wound edge. This remodeling of the AJs promotes the assembly of an actin-myosin cable at the wound margin; contraction of the actin cable, in turn, closes the wound. In the absence of Toll or Dif and dorsal (dl), both E-cadherin down-regulation and actin-cable formation fail, thus resulting in open epidermal gaps. Given the conservation of the Toll/NF-κB pathway in mammals and the epithelial expression of many components of the pathway, this function in wound healing is likely to be conserved in vertebrates.

2011 Nobel Prize in Physiology or Medicine to Three Immunologists

The Nobel Assembly at Karolinska Institutet has decided to award the 2011 Nobel Prize in Physiology or Medicine to three immunologists, Bruce Beutler, Jules A. Hoffman, and Ralph M. Steinman. As stated in the press release from the Nobel Committee (http://www.nobelprize.org), these scientists are recognized for having revolutionized our understanding of the immune system by discovering key principles for its activation. Immunologists had long been searching for the gatekeepers of the immune response by which man and other animals defend themselves against attack by microorganisms. Jules A. Hoffmann and Bruce Beutler discovered key receptor proteins, Toll/Toll-like receptors, which can recognize microorganisms and activate innate immune responses. Ralph Steinman discovered dendritic cells (DC) and their unique capacity to activate and regulate T lymphocytes, paving the way for activation of adaptive immune responses. Jules Hoffmann made his pioneering discovery in 1996, when he and his co-workers investigated how fruit flies combat infections [1]. They had access to flies with mutations in several different genes including Toll, a gene previously found to be involved in embryonal development. When Hoffmann infected Toll mutant fruit flies with bacteria or fungi, he discovered that the Toll mutants infected with fungi died because they could not mount an effective immune response. Bruce Beutler was searching for a receptor that could bind the bacterial product, lipopolysaccharide (LPS), which can cause septic shock, a life-threatening condition that involves overstimulation of the immune system. In 1998, Beutler and his colleagues discovered that mice that were resistant to LPS had a mutation in a gene that was similar to the Toll gene of the fruit fly [2]. This Toll-like receptor (TLR) turned out to be the elusive LPS receptor. These findings showed that mammals and fruit flies use similar molecules to activate innate immunity when encountering pathogenic microorganisms. As concluded by the Nobel Committee (http://www.nobelprize.org), the discoveries of Hoffmann and Beutler triggered an explosion of research in innate immunity. Around a dozen different TLRs have now been identified in humans and mice, and several other receptor families with similar functions have been identified. Each one of them recognizes certain types of molecules common in microorganisms. Individuals with certain mutations in these receptors carry an increased risk of infections. Ralph Steinman discovered, in 1973, a new type of cell that he called the dendritic cell (DC) [3]. He speculated that DC could be important in the immune system and went on to test whether antigens in the presence of DC could activate T lymphocytes, key cells in adaptive immunity [4]. In cell culture experiments, he showed that the presence of DC resulted in vivid responses of T cells to antigens. Findings by Steinman were initially met with some scepticism but subsequent work by Steinman demonstrated that DC had an absolutely unique capacity to activate T cells. Further studies by Steinman [5] and by other scientists went on to address how DCs decide whether or not they should activate T lymphocytes when encountering self or foreign ‘antigens’. Signals arising from the innate immune response via TLR, sensed by DC, were shown to control T cell activation. This makes it possible for T lymphocytes to react towards pathogenic microorganisms while avoiding an attack on the body’s own endogenous molecules. The discoveries that are awarded the 2011 Nobel Prize have provided novel insights into the activation and regulation of the immune system. They have made possible the development of new methods for preventing and treating disease, for instance with improved vaccines against infections and in attempts to stimulate the immune system to attack tumours. These discoveries also help us understand why the immune system can attack our own tissues, thus providing clues for novel treatment of inflammatory diseases.

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Pattern Recognition and Gestalt Psychology: The Day Nüsslein‐Volhard Shouted “Toll!”

Pattern Recognition and Gestalt Psychology: The Day Nüsslein‐Volhard Shouted “Toll!”

Drosophila Tey represses transcription of the repulsive cue Toll and generates neuromuscular target specificity

Little is known about the genetic program that generates synaptic specificity. Here we show that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. We focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. We found that Toll, which encodes a transmembrane protein with leucine-rich repeats, was preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) formed smaller synapses on M12 and instead appeared to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. We identified Tey as a negative regulator of Toll expression in M12. In tey mutants, Toll was strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue.

Candida albicansCas5, a Regulator of Cell Wall Integrity, Is Required for Virulence in Murine andTollMutant Fly Models

Candida albicans is the most common human fungal pathogen, yet the pathogenesis of C. albicans infection remains incompletely understood. We hypothesized that C. albicans has developed evolutionarily conserved mechanisms to invade disparate hosts and tested whether Toll mutant flies could serve as a model host for high-throughput screening of C. albicans virulence genes. We screened 34 C. albicans mutants defective in putative transcription factor genes (see http://www.tigr.org/tigr-scripts/e2k1/qzhao/page.cgi?num=1 ) by means of a previously established model of invasive candidiasis in Toll mutant flies. C. albicans mutants that displayed attenuated virulence in flies were subsequently tested for virulence in a mouse model of hematogenous candidiasis. Of the 34 C. albicans mutants tested, only the prototrophic cas5Delta/Delta mutant (strain VIC1186) exhibited attenuated virulence in Toll mutant flies that was restored in the complemented strain (VIC1190). Similarly, BALB/c mice infected intravenously with the cas5Delta/Delta mutant had significantly better survival and a lower fungal burden in kidneys and spleen than did those infected with the isogenic wild-type strain DAY185. CAS5 encodes a key transcriptional regulator of genes involved in cell wall integrity and lacks an orthologue in Saccharomyces cerevisiae. Our findings support the notion that Drosophila melanogaster is a promising model for large-scale studies of genes involved in the pathogenesis of C. albicans infection in mammals.

The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-kappa B-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an I kappa B-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila.

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll(10b)), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll(10b), and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

Toll-like receptors: molecular mechanisms of the mammalian immune response.

The unrelenting bombardment by microbial pathogens from the environment and their increasing resistance to medical treatments are a constant threat to the survival of all organisms on earth. Microbes are covered by molecular patterns that are common among a broad range of pathogens. These include the lipopolysaccharides (LPS) of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria, lipoproteins of bacteria and parasites, glycolipids of mycobacteria, mannans of yeast and double-stranded RNAs of viruses.1,2 Recognition of and responses to these molecules are controlled by a wide variety of cellular receptors. The best characterized receptors are the T-cell receptor and B-cell antibody receptor of the adaptive immune response, the specificity of which is randomly generated and clonally selected during the development of T and B lymphocytes.3 Unlike the receptors of the adaptive immune system, the pattern recognition receptors of the innate immune system have predetermined specificity generated early on in evolution and play an essential role in the determination of self versus non-self during the initial rapid responses to infection.1,2 As described in this review, a family of cell surface receptors, termed Toll-like receptors, are emerging as key regulators of host responses to infection.

The Drosophila Toll Gene Functions Zygotically and Is Necessary for Proper Motoneuron and Muscle Development

Toll is a maternally required Drosophila gene that encodes a transmembrane protein with an important function in embryonic dorsal-ventral patterning. The Toll protein is widely expressed zygotically, but its roles in late embryogenesis have not been described in detail. We have examined the expression of Toll protein in the late embryonic central nervous system and somatic musculature. Toll is expressed in a dynamic pattern in the musculature, initially in several muscle fibers in each hemisegment, with a later narrowing of expression to a single muscle fiber pair. Zygotic Toll mutants were used to investigate the developmental consequences of loss of Toll expression. We found that loss of one or both copies of the Toll gene leads to widespread defects in motoneuron number and muscle patterning. Loss of motoneurons prevents certain muscle fibers from receiving their wild-type innervation. Denervation in the mutants results in collateral sprouting from nearby nerve branches and leads to the appearance of ectopically placed motor endings. The limited expressivity observed suggests that Toll is only one of several genes required for proper motoneuron and muscle specification.

The Toll gene of drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein

The Toll gene of Drosophila, a maternal effect gene that plays a central role in the establishment of the embryonic dorsal-ventral pattern, has been cloned using P element tagging. A 5.3 kb poly(A) + ovarian transcript from the cloned region was purified by hybrid selection with the cloned DNA. This purified transcript complements the Toll mutant phenotype when injected into Toll − embryos, which proves that it is the Toll transcript. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains at least 15 repeats of a 24 amino acid, leucinerich sequence found in both human and yeast membrane proteins.