Molecular characterization of the Drosophila responses towards nematodes

A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above. We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity. Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.</p

Monitoring the effect of pathogenic nematodes on locomotion of Drosophila larvae

One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and Drosophila melanogaster larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing Drosophila strains. These differences were further influenced by the presence of nematodes. Given a choice, Drosophila larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction

PIP degron-stabilized Dacapo/p21Cip1 and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels

During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21 and Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCF targets phosphorylated CycE, whereas p21 and Dap are targeted by the CRL4 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ()-mediated CycE degradation, and of Dap and p21 degradation during CNS development. We find that mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21 transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels

PIP degron-stabilized Dacapo/p21(Cip)(1) and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels

During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21(Cip)(1) and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCFFoxw7/Ago targets phosphorylated CycE, whereas p21(Cip)(1) and Dap are targeted by the CRLCdf2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago (Fbxw7)-mediated CycE degradation, and of Dap and p21(Cip)(1) degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21(Cip)(1) transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.Funding Agencies|Swedish Vetenskapsradet [2017-04061]; Knut och Alice Wallenbergs Stiftelse [KAW2012.0101]; Cancerfonden [CAN2017/257]; Royal Swedish Academy of Sciences</p

Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical extracellular matrix (ECM) components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36 homolog belonging to the scavenger receptor class B protein family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad, and phosphorylated Src (p-Src). We show that Emp associates with and organizes the βH-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation

Drosophila Neuroblast Selection Is Gated by Notch, Snail, SoxB, and EMT Gene Interplay

In the developing Drosophila central nervous system (CNS), neural progenitor (neuroblast [NB]) selection is gated by lateral inhibition, controlled by Notch signaling and proneural genes. However, proneural mutants still generate many NBs, indicating the existence of additional proneural genes. Moreover, recent studies reveal involvement of key epithelial-mesenchymal transition (EMT) genes in NB selection, but the regulatory interplay between Notch signaling and the EMT machinery is unclear. We find that SoxNeuro (SoxB family) and worniu (Snail family) are integrated with the Notch pathway, and constitute the missing proneural genes. Notch signaling, the proneural, SoxNeuro, and worniu genes regulate key EMT genes to orchestrate the NB selection process. Hence, we uncover an expanded lateral inhibition network for NB selection and demonstrate its link to key players in the EMT machinery. The evolutionary conservation of the genes involved suggests that the Notch-SoxB-Snail-EMT network may control neural progenitor selection in many other systems.Funding Agencies|Swedish Research CouncilSwedish Research Council [621-2013-5258, 2017-04061]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [KAW2012.0101, KAW2017.0312]; Swedish Cancer Foundation [CAN2017/257]; University of QueenslandUniversity of Queensland</p

Monitoring the effect of pathogenic nematodes on locomotion of <i>Drosophila</i> larvae

<p>One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and <i>Drosophila melanogaster</i> larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing <i>Drosophila</i> strains. These differences were further influenced by the presence of nematodes. Given a choice, <i>Drosophila</i> larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction.</p

Antibiotic treatment, different fly medium and inhibition of apoptosis can rescue eclosion, and melanization defects after hemocyte depletion.

<p>(A) Both Hid and Grim lines showed significantly higher pupal lethality (measured as a drop in eclosure rate in %) than controls; antibiotic treatment rescued Hid and Grim induced lethality. Lethality of Hid-expressing larvae was also affected by using different fly media. SF—standard fly food (potato source). DIM—Drosophila instant medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136593#pone.0136593.s009" target="_blank">S2 Table</a> for the composition of the food, both parental lines were homozygous). (B) Coexpression of UAS-<i>grim28</i>.<i>2</i> with UAS-<i>p35</i> (caspase inhibitor) in the same larva rescued pupal lethality. Dashed lines indicate the expected frequency of eclosing flies for the crosses (25 and 50% respectively). (C) A higher melanotic spot frequency was found in adults in both Hid- and Grim-expressing lines compared to controls. Antibiotic treatment rescued melanotic mass formation in Hid- but not in Grim-expressing lines. The melanotic spot frequency was compared between Hid, Grim and controls using standard fly food (bracket 1), the influence of the food source (bracket 2, no significant differences) and the antibiotic treatment, (bracket 3, significant only for Hid-expressing larvae). Data presented are means ± SD; t test: * p<0.05; **p<0.01.</p

Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the <i>Drosophila</i> Immune System and Leads to a Pro-Inflammatory State

<div><p>Apart from their role in cellular immunity via phagocytosis and encapsulation, <i>Drosophila</i> hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemo^less) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.</p></div

Lamellocyte differentiation in the lymph gland of Hml-apo larvae.

<p>Dissected fixed lymph glands from 3^rd instar larvae were stained with DAPI and the early lamellocyte-specific antibody L1. (B and G) Control (HFP/+) lymph glands show the presence of some plasmatocytes and/or crystal cells (B) whereas no GFP signal was observed in Hid-expressing lymph gland (G), which confirms effective elimination through apoptosis. Strong and extensive L1 staining was observed in the lymph gland of Hid-expressing larvae (H) whereas no L1 staining was found in the control (C). HFP: <i>hml-</i>Gal4,UAS-<i>eGFP</i>. The scale bars represent 50 μm.</p