Yong Hun Jo

Apolipophorin-III Mediates Antiplasmodial Epithelial Responses in Anopheles gambiae (G3) Mosquitoes

Background Apolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes. Methodology/Principal Findings We report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold. Conclusion There are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model.

Cloning, Characterization and Effect of TmPGRP-LE Gene Silencing on Survival of Tenebrio Molitor against Listeria monocytogenes Infection

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

EXPRESSED SEQUENCE TAG ANALYSIS OF PHYSA ACUTA: A FRESHWATER PULMONATE IN KOREA

ABSTRACT Physa acuta (left-handed shell) have strong natural growth activity not only in lentic waters but also in eutrophic environments. Therefore, it has been considered one of the candidate species that could evaluate the degree of water pollution by physiological and biochemical methods. In this study, we constructed a P. acuta cDNA library using the 5′ oligo capping method, and determined the sequences of 2,282 clones by 5′ end-single path sequencing. After trimming, clustering, and assembling these sequences, we finally obtained 575 distinctly available transcripts that were 718 bp in average length. These transcripts were annotated using the BLASTX search and were classified by function using KOG analysis. After comparison with biomarker genes already known in several organisms, we identified 27 potential biomarker candidates that were categorized into two groups strongly related to stress and defense genes by their functions. To the best of our knowledge, this is the first report of massive profiling of cDNA sequences and the characterizing of potential biomarker genes in P. acuta. Our study offers valuable information to scientists for developing new environmental biomonitoring markers, and for scientists studying the physiology, growth and development, immunity, genetic identification, and evolutional diversity in P. acuta.

Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor.

Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Expressed Sequence Tags (ESTs) analysis of Tenebrio molitor larvae

Tenebrio molitor has been seriously investigated as a model insect in elucidating Toll signaling pathway and related regulators of innate immunity. However, little is known with regards to the genomic information in T. molitor. In an attempt towards exploiting the rich transcriptomics data that would offer further insights into the study on insect immunity through potential screening of immune‐related genes in the model insect, we constructed a cDNA library (library titer = 5.0 × 105pfu/ml) of T. molitor larvae and analyzed expressed sequence tag (EST) sequences from 1056 clones. The base calling and quality check of obtained chromatogram files were performed by using the Phred program (trim_alt 0.05 (P‐score > 20). After removal of vector and 100 bp and less sequences, 1023 sequences were generated having an average insert size of 792 bp, including 160 clusters, 164 contigs and 387 singletons through clustering and assembling process using the TGI Clustering Tools (TGICL) package. The unique EST sequences were searched against the NCBI nr database by local BLAST (blastx, E < e−5) with 940 sequences showing significant hits. Subsequently, KOG (clusters of orthologous groups for eukaryotic complete genomes) analysis was conducted (blastx, E < e−10) towards prediction of transcriptomal functions, leading to the categorization of 638 genes. The majority of genes belonged to Z category (cytoskeleton‐related genes), R category (general function prediction), and C category (energy production and conversion related genes). These basic EST datasets and their bioinformatics analysis will be helpful in investigating the biological mechanism and molecular pathway related genes involved in innate immunity mechanisms of T. molitor.

Cloning and expression pattern of a hemolin homologue from the diamondback moth, Plutella xylostella

Hemolin has been known to play a key role in insect innate immunity. In an attempt to examine expression pattern of the Hemolin gene in the diamondback moth, Plutellea xylostella, the full-length cDNA of Hemolin was cloned using 5′-RACE PCR technique. The cDNA contained a 5′ untranslated region of 48 nucleotides and a 3′ untranslated region of 198 nucleotides, including a stop codon (TAA) and a poly (A) tail. It consists of 1,401 bp with an open reading frame of 1,245 bp, encoding 414 amino acids. The deduced amino acid sequence of PxHemolin has relatively low identities (35−42%) to various insect Hemolins. However, it has high three-dimensional structural similarity to Hemolin. Interestingly, analysis of spatial expression pattern of PxHemolin shows that it was highly expressed in the Malpighian tubule and the silk gland although it was also detected in fat body and gut. Furthermore, PxHemolin mRNA was highly induced 3 hr after immune-challenging with lipopolysaccharide and was gradually up-regulated after laminarin treatment. These data suggest that PxHemolin may play a role in innate immune responses although it remains to further elucidate the precise biological functions in P. xylostella.