Ji-Hwan Ryu

Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.

Although commensalism with gut microbiota exists in all metazoans, the host factors that maintain this homeostatic relationship remain largely unknown. We show that the intestinal homeobox gene Caudal regulates the commensal-gut mutualism by repressing nuclear factor kappa B–dependent antimicrobial peptide genes. Inhibition of Caudal expression in flies via RNA interference led to overexpression of antimicrobial peptides, which in turn altered the commensal population within the intestine. In particular, the dominance of one gut microbe, Gluconobacter sp. strain EW707, eventually led to gut cell apoptosis and host mortality. However, restoration of a healthy microbiota community and normal host survival in the Caudal-RNAi flies was achieved by reintroduction of the Caudal gene. These results reveal that a specific genetic deficiency within a host can profoundly influence the gut commensal microbial community and host physiology.

The Drosophila immune system detects bacteria through specific peptidoglycan recognition

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid–type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.

Cyanine-Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse Tissues

Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.

Peptidoglycan Molecular Requirements Allowing Detection by the Drosophila Immune Deficiency Pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.

Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

An essential complementary role of NF‐κB pathway to microbicidal oxidants in Drosophila gut immunity

In the Drosophila gut, reactive oxygen species (ROS)‐dependent immunity is critical to host survival. This is in contrast to the NF‐κB pathway whose physiological function in the microbe‐laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. We used a novel in vivo approach to reveal the physiological role of gut NF‐κB/antimicrobial peptide (AMP) system, which has been ‘masked’ in the presence of the dominant intestinal ROS‐dependent immunity. When fed with ROS‐resistant microbes, NF‐κB pathway mutant flies, but not wild‐type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re‐introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF‐κB pathway mutants in the intestine. These results imply that the local ‘NF‐κB/AMP’ system acts as an essential ‘fail‐safe’ system, complementary to the ROS‐dependent gut immunity, during gut infection with ROS‐resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS‐dependent immunity.

Proteolytic cascade for the activation of the insect Toll pathway induced by the fungal cell wall component

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation.

The Homeobox Gene Caudal Regulates Constitutive Local Expression of Antimicrobial Peptide Genes in Drosophila Epithelia

ABSTRACT In Drosophila melanogaster, although the NF-κB transcription factors play a pivotal role in the inducible expression of innate immune genes, such as antimicrobial peptide genes, the exact regulatory mechanism of the tissue-specific constitutive expression of these genes in barrier epithelia is largely unknown. Here, we show that the Drosophila homeobox gene product Caudal functions as the innate immune transcription modulator that is responsible for the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner. These results suggest that certain epithelial tissues have evolved a unique constitutive innate immune strategy by recruiting a developmental “master control” gene.

Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms

During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROS) such as hypochlorous acid (HOCl) to kill the invading pathogens. However, excess amounts of HOCl induce oxidative damage of functional biomolecules such as DNA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCl probe, as well as applications thereof, with which to detect HOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCl over other ROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2–3 d, after which it can be used for up to 5 h in the bioimaging of living cells.

Phylogenetic Characterization of Two Novel Commensal Bacteria Involved with Innate Immune Homeostasis in Drosophila melanogaster

ABSTRACT During a previous study on the molecular interaction between commensal bacteria and host gut immunity, two novel bacterial strains, A911T and G707T, were isolated from the gut of Drosophila melanogaster. In this study, these strains were characterized in a polyphasic taxonomic study using phenotypic, genetic, and chemotaxonomic analyses. We show that the strains represent novel species in the family Acetobacteraceae. Strain G707T, a highly pathogenic organism, represents a new species in the genus Gluconobacter, “Gluconobacter morbifer” sp. nov. (type strain G707 = KCTC 22116T = JCM 15512T). Strain A911T, dominantly present in the normal Drosphila gut community, represents a novel genus and species, designated “Commensalibacter intestini” gen. nov., sp. nov. (type strain A911 = KCTC 22117T = JCM 15511T).