Protective Signatures of Roselle (Hibiscus sabdariffa L.) Calyx Fractions against Staphylococcus aureus in Drosophila Infection Model

The rise of antibiotic-resistant Staphylococcus aureus-related clinical cases is an alarming chronicle for global communities. This research was conducted to examine the antistaphylococcal effect of roselle (Hibiscus sabdariffa L.) calyx fractions in the Drosophila model. In the infection experiment, wild-type and immunodeficient Drosophila were pricked with S. aureus and subsequently subjected to fly survivorship and colony-forming assays, in the presence or absence of roselle calyx fractions. The Involvement of immune stimulation in the host antibacterial protection was assessed in vitro using cell-based luciferase reporter assay and in vivo using RT-qPCR analysis on adult flies. A declining rate of fly survivorship and augmentation of bacterial growth were observable in S. aureus-infected wild-type flies but subject to improvement in the presence of roselle calyx fractions. Cell-based analysis revealed the absence of host immune stimulation via Drosophila Toll pathway and roselle calyx fractions-treated immune-deficient flies lacking for components in the Toll pathway were protected from infection-induced early death phenotype and harbored reduced number of S. aureus colonies. Overall, our data confirmed the in vivo anti-staphylococcal activity of roselle calyx fractions in Drosophila infection model and such protective signature was devoid of host immune stimulation

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

金沢大学医薬保健研究域薬学系In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis. © 2017 The Authors.Embargo Period 12 month

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells

金沢大学医薬保健研究域薬学系Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells. © 2009 European Molecular Biology Organization

Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection

<p>Abstract</p> <p>Background</p> <p>Gut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, <it>Erwinia carotovora carotovora 15</it>, induces a massive increase in stem cell proliferation in the gut of <it>Drosophila</it>. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal.</p> <p>Results</p> <p>To understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult <it>Drosophila </it>undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection.</p> <p>Conclusions</p> <p>This study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.</p

Skin microbiome profile in people living with HIV/AIDS in Cameroon

The presence of pathogens and the state of diseases, particularly skin diseases, may alter the composition of human skin microbiome. HIV infection has been reported to impair gut microbiome that leads to severe consequences. However, with cutaneous manifestations, that can be life-threatening, due to the opportunistic pathogens, little is known whether HIV infection might influence the skin microbiome and affect the skin homeostasis. This study catalogued the profile of skin microbiome of healthy Cameroonians, at three different skin sites, and compared them to the HIV-infected individuals. Taking advantage on the use of molecular assay coupled with next-generation sequencing, this study revealed that alpha-diversity of the skin microbiome was higher and beta-diversity was altered significantly in the HIV-infected Cameroonians than in the healthy ones. The relative abundance of skin microbes such as Micrococcus and Kocuria species was higher and Cutibacterium species was significantly lower in HIV-infected people, indicating an early change in the human skin microbiome in response to the HIV infection. This phenotypical shift was not related to the number of CD4 T cell count thus the cause remains to be identified. Overall, these data may offer an important lead on the role of skin microbiome in the determination of cutaneous disease state and the discovery of safe pharmacological preparations to treat microbial-related skin disorders

自然免疫を調節する受容体型グアニル酸シクラーゼGyc76Cのリガンド探索

私たちは受容体型グアニル酸シクラーゼGyc76Cが自然免疫を調節する新規受容体であることを明らかにしてきた。本研究では、Gyc76Cのリガンドを同定することを目標として研究を進めた。ショウジョウバエ幼虫抽出液中に抗菌ペプチド発現を誘導する活性を見いだし、単離・精製し、新規自然免疫活性化因子を同定した。今後は、この因子がGyc76Cのリガンドであるか詳細な解析を進める。We have revealed that Gyc76C, a receptor-type guanylate cyclase, mediates innate immunity. In this study, we sought to identify a ligand for Gyc76C.We found that Drosophila larval extract has an activity that induces the expression of antimicrobial peptides in Drosophila cell line. The activity was purified to homogeneity and the amino acid sequence was determined, resulting in theidentification of a new protein that activates innate immune system in Drosophila. Further analysis should be done to validate that whether the identified protein would be a ligand for Gyc76C.研究課題/領域番号:23890014, 研究期間(年度):2011-201

ショウジョウバエの腸管において腸内細菌の違いを感知する新規メカニズムの解明

金沢大学医薬保健研究域薬学系研究代表者は、ショウジョウバエ腸管における腸内細菌と宿主の相互作用について解析を進めている。本研究では、ショウジョウバエを完全に無菌化することを試みた。これは、特定の細菌のみを定着させて解析を行なうために必要な重要な技術であるが、これまでに真に無菌のショウジョウバエを確立して数世代にわたって維持することは困難であった。そこで研究代表者は、無菌マウス維持に利用される無菌アイソレータを用い、無菌ショウジョウバエの維持・作成のための方法を確立した。すなわち、完全に微生物フリーのショウジョウバエを安定して飼育することが可能になった。The intestinal tract is one of the first organs acquired by multicellular organisms in the course of evolution. Because of its essential role in the digestion and absorption of food, its nutritive environment provides an ideal niche for commensal microbes, the effects of which are considered tremendous on hosts. Drosophila melanogaster, an invertebrate model organism, certainly possesses a gut, and its microbes could be used for investigating host-microbe interactions. However, generating and maintaining “germ-free” Drosophila is rather challenging. In this study, we established a solid and perhaps the first method for establishing germ-free flies, using a vinyl isolator and fly food with a specific composition. This germ-free fly technique could provide a pivotal basis for analyzing gnotobiotic Drosophila, which will improve our understanding of evolutionarily conserved host-microbe interactions in the intestine.研究課題/領域番号:15K18855, 研究期間(年度):2015-04-01 - 2017-03-3

新規ユビキチンリガーゼSherpaによる自然免疫シグナル制御

金沢大学医薬保健研究域薬学系ショウジョウバエToll経路は、病原体や組織傷害に対する自然免疫応答をはじめ、胚発生、細胞間相互作用等重要な生理的プロセスに関与している。しかしなが ら、主に病原体を認識する哺乳動物のToll様受容体の自然免疫経路と比較して、多様な応答性を有するショウジョウバエToll受容体のシグナル伝達因子とその制御機構は完全には解明されていない。本研究では、包括的なゲノムワイドRNAiスクリーニングとシグナル伝達因子複合体プロテーム解析により、Tollシグナル伝達経路に関 与するプロテインキナーゼ等の候補分子を同定し、Toll経路の制御機構に関して新たな仮説を提唱するに至った。The Drosophila Toll pathway is involved in embryonic development, innate immunity, and cell-cell interactions. However, compared to the mammalian Toll-like receptor innate immune pathway, its intracellular signaling mechanisms are not fully understood. We have previously performed a series of ex vivo genome-wide RNAi screenings to identify genes required for the activation of the Toll pathway. In this study, we have conducted an additional genome-wide RNAi screening using the overexpression of Tube, an adapter molecule in the Toll pathway, and have performed a co-immunoprecipitation assay to identify components present in the dMyd88-Tube complex. Based on the results of these assays, we have performed a bioinformatic analysis, and describe candidate molecules and post-translational modifications that could be involved in Drosophila Toll signaling.研究課題/領域番号:17K08267, 研究期間(年度):2017-04-01 - 2020-03-31出典：「新規ユビキチンリガーゼSherpaによる自然免疫シグナル制御」研究成果報告書　課題番号17K08267（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-17K08267/17K08267seika/)を加工して作