Critical evaluation of the role of the Toll-like receptor 18-Wheeler in the host defense of Drosophila

Essential aspects of innate immune responses to microbial infections appear to be conserved between insects and mammals. In particular, in both groups, transmembrane receptors of the Toll superfamily play a crucial role in activating immune defenses. The Drosophila Toll family member 18-Wheeler had been proposed to sense Gram-negative infection and direct selective expression of peptides active against Gram-negative bacteria. Here we re-examine the role of 18-Wheeler and show that in adults it is dispensable for immune responses. In larvae, 18wheeler is required for normal fat body development, and in mutant larvae induction of all antimicrobial peptide genes, and not only of those directed against Gram-negative bacteria, is compromised. 18-Wheeler does not qualify as a pattern recognition receptor of Gram-negative bacteria.     

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.     

Control of Drosophila Blood Cell Activation via Toll Signaling in the Fat Body

The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. Here we have studied this response in detail, and found that Toll signaling in several different tissues can activate a cellular immune defense, and that this response does not require Toll signaling in the blood cells themselves. Like in the humoral immune response, we show that Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen.     

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.     

Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila

In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.     

Alcohol resistance in Drosophila is modulated by the Toll innate immune pathway

A growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol‐related behaviors. Here, we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder. The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll‐like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila‐specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance whereas decreasing the pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.     

Cellular and humoral immune interactions between Drosophila and its parasitoids

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila–wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps’ side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.     

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.     

Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

The Drosophila Toll-9 activates a constitutive antimicrobial defense

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. We analyzed the nine Drosophila Toll proteins and found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms.     

Apoptotic caspase inhibits innate immune signaling by cleaving NF-κBs in both Mammals and Flies

Host organisms use different innate immune mechanisms to defend against pathogenic infections, while tight control of innate immunity is essential for proper immune induction and balance. Here, we reported that apoptotic induction or caspase-3 overexpression caused dramatic reduction of differently triggered cytokine signalings in human cells, murine primary cells and mouse model, while the loss of caspase-3 or inhibiting apoptosis markedly enhances these immune signalings. Furthermore, caspase-3 can mediate the cleavage of NF-κB members p65/RelA, RelB, and c-Rel via its protease activity. And the caspase-3-resistant p65/RelA, RelB, or c-Rel mutant mostly restored the caspase-3-induced suppression of cytokine production. Interestingly, we further uncovered that apoptotic induction also dramatically inhibited Toll immune signaling in Drosophila, and the Drosophila effector caspases, drICE and DCP-1, also mediated the degradation of DIF, the NF-κB of Toll signaling. Together, our findings demonstrate apoptotic effector caspases, including mammalian caspase-3 and fly drICE/DCP-1, can function as repressors of NF-κB-mediated innate immune signalings.Subject terms: Immunology, Innate immunity     

DmMyD88 controls dorsoventral patterning of the Drosophila embryo

MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. A Drosophila homologue of MyD88 (DmMyD88) was recently shown to be required for the Toll-mediated immune response. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. We found that, like Toll, DmMyD88 messenger RNA is maternally supplied to the embryo. Here we report the identification of a new mutant allele of DmMyD88, which generates a protein lacking the carboxy-terminal extension, normally located downstream of the Toll/IL-1 receptor domain. Homozygous mutant female flies lay dorsalized embryos that are rescued by expression of a transgenic DmMyD88 complementary DNA. The DmMyD88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that DmMyD88 encodes an essential component of the Toll pathway in dorsoventral pattern formation.     

The imd gene is required for local Cecropin expression in Drosophila barrier epithelia

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.     

The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated <Emphasis Type="Italic">Drosophila</Emphasis>

Background

Host sexual dimorphism is being increasingly recognized to generate strong differences in the outcome of infectious disease, but the mechanisms underlying immunological differences between males and females remain poorly characterized. Here, we used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection.

Results

We demonstrated sexual dimorphism in susceptibility to infection by a broad spectrum of Gram-positive and Gram-negative bacteria. We found that both virgin and mated females are more susceptible than mated males to most, but not all, infections. We investigated in more detail the lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster. We found that females have a higher number of phagocytes than males and that ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, we found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. We additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection.

Conclusion

Altogether, our data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection, thus identifying innate immune signaling as a determinant of sexual immune dimorphism.

     

A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway.

A hallmark of the systemic antimicrobial response of Drosophila is the synthesis by the fat body of several antimicrobial peptides which are released into the hemolymph in response to a septic injury. One of these peptides, drosomycin, is active primarily against fungi. Using a drosomycin-green fluorescent protein (GFP) reporter gene, we now show that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. We also show that, in contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway.