Cloning, characterization and effect of TmPGRP-LE gene silencing on survival of Tenebrio molitor against Listeria monocytogenes infection

Hamisi Tindwa,Bharat Bhusan Patnaik,Dong Hyun Kim,Seulgi Mun,Yong Hun Jo,Bok Ruel Lee,Yong Seok Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Peptidoglycan recognition proteins (PGRPs) are 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 infections in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

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

Tindwa, Hamisi,Patnaik, Bharat Bhusan,Kim, Dong Hyun,Mun, Seulgi,Jo, Yong Hun,Lee, Bok Luel,Lee, Yong Seok,Kim, Nam Jung,Han, Yeon Soo Molecular Diversity Preservation International (MD 2013 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.14 No.11

<P>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 <I>Listeria monocytogenes</I> has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from <I>Tenebrio molitor</I> and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, <I>L. monocytogenes</I>. 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 <I>L. monocytogenes</I>, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of <I>L. monocytogenes</I> infection in <I>T. molitor</I>. These results implicate PGRP-LE as a defense protein necessary for survival of <I>T. molitor</I> against infection by <I>L. monocytogenes</I>.</P>

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes

Ju Young Noh,Bharat Bhusan Patnaik,Hamisi Tindwa,Gi Won Seo,Dong Hyun Kim,Hongray Howrelia Patnaik,Yong Hun Jo,Yong Seok Lee,Bok Ruel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

Investigation of Siderophore production and Antifungal activity against Phytophthora capsici as related to Iron (III) nutrition by Lysobacter antibioticus HS124

고현선,Hamisi Tindwa,Rong De Jin,이용성,홍성현,현해남,남이,김길용 한국토양비료학회 2011 한국토양비료학회지 Vol.44 No.4

Lysobacter antibioticus HS124 isolated from pepper rhizosphere soil produced catechol type siderophore. Purified siderophore by Diaion HP-20 and silica gel column chromatography showed several hydroxyl functional groups adjacent to benzene rings by analysis of 1H NMR spectroscopy. The strain HS124 showed different activities to suppress Phytophthora capsici with different concentrations of exogenous Fe (III) in minimal medium where antifungal activity with 100 μM Fe (III) was approximately 1.5 times higher than in absence of Fe (III). Bacterial population in this Fe (III)-amended medium was also highest with 8.9×10^8CFU ml^-1 which also corresponded to the strongest siderophore activity. When grown in rich medium (minimal medium with N, P_2O_5, K_2O and glucose), HS124 exhibited approximately 2 times stronger antifungal activity compared to minimal medium. In pot trials, treatments of bacterial culture grown in rich medium with (C1) or without (C2) 100 μM Fe (III) exhibited a high protection of pepper plants from disease, compared to medium only with (M1) or without (M2) 100 μM Fe (III). Especially, treatment C1 showed the best disease control effect of about 70 %. Thus, the strain HS124 should be recommended as a potential biocontrol agent against P. capsici in pepper.

Molecular characterization and expression analysis of target of rapamycin (TmTOR) in coleopteran insect Tenebrio molitor

조용훈,이정희,Hamisi Tindwa,Hongray Howerelia PATNAIK,성정환,김수곤,Bharat Bhusan Patnaik,오승한,이용석,한연수 한국곤충학회 2016 Entomological Research Vol.46 No.2

Target of rapamycin (TOR) is an evolutionarily conserved serine–threonine kinase that affects various cellular functions including growth, development, ageing, immunity and autophagy. Inhibition in TOR activity stimulates autophagy, a self‐eating process that ensures survival by maintaining metabolic homeostasis and energy retention at both cellular and organismal levels. To understand the interplay between TOR and autophagy during development and immunity, we screened and identified a TOR homologue from the coleopteran insect, Tenebrio molitor (TmTOR). The full‐length cDNA of TmTOR contained an open reading frame (ORF) of 7,197 bp encoding a protein of 2,398 amino acids, and a 5′‐ and 3′‐ untranslated region (UTR) of 156 and 457 bp, respectively. Deduced amino acid sequence of TmTOR shows characteristic Huntington, EF3A, ATM, TOR (HEAT) repeat, focal adhesion kinase targeting (FAT), rapamycin‐binding, phosphatidylinositol 3‐/4‐kinase and FRAP, ATM and TRRAP C‐terminal (FATC) domains known to be conserved among TOR orthologs. TmTOR showed high sequence identity (92%) with Tribolium castaneum TOR (TcTOR) and the two were placed in the same cluster of the phylogenetic tree. TmTOR shows high mRNA expression level in the fat body and integument of T. molitor larvae. Significantly, a reduced expression of TmTOR mRNA during pupation correlated to an increase in the area of autolysosomes. Intriguingly, T. molitor larvae showed an increase in TmTOR transcripts upon a challenge with various bacterial pathogens. This suggests a negative role of TmTOR in the regulation of autophagy during development.

Biocontrol of Anthracnose in Pepper Using Chitinase, β-1,3 Glucanase, and 2-Furancarboxaldehyde Produced by Streptomyces cavourensis SY224

( So Youn Lee ),( Hamisi Tindwa ),( Yong Seong Lee ),( Kyaw Wai Naing ),( Seong Hyun Hong ),( Yi Nam ),( Kil Yong Kim ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.10

A strain of Streptomyces cavourensis subsp. cavourensis (coded as SY224) antagonistic to Colletotrichum gloeosporioides infecting pepper plants was isolated. SY224 produced lytic enzymes such as chitinase, β-1,3-glucanase, lipase, and protease in respective assays. To examine for antifungal activity, the treatments amended with the nonsterilized supernatant resulted in the highest growth inhibition rate of about 92.9% and 87.4% at concentrations of 30% and 10%, respectively. However, the sterilized treatments (autoclaved or chloroform treated) gave a lowered but significant inhibitory effect of about 63.4% and 62.6% for the 10% supernatant concentration, and 75.2% and 74.8% for the of 30% supernatant concentration in the PDA agar medium, respectively, indicative of the role of a nonprotein, heat stable compound on the overall effect. This antifungal compound, which inhibited spore germination and altered hyphal morphology, was extracted by EtOAc and purified by ODS, silica gel, Sephadex LH-20 column, and HPLC, where an active fraction was confirmed to be 2-furancarboxaldehyde by GS-CI MS techniques. These results suggested that SY224 had a high potential in the biocontrol of anthracnose in pepper, mainly due to a combined effect of lytic enzymes and a non-protein, heatstable antifungal compound, 2-furancarboxaldehyde.

Investigation of Siderophore production and Antifungal activity against Phytophthora capsici as related to Iron (III) nutrition by Lysobacter antibioticus HS124

Ko, Hyun-Sun,Tindwa, Hamisi,Jin, Rong De,Lee, Yong-Seong,Hong, Seong-Hyun,Hyun, Hae-Nam,Nam, Yi,Kim, Kil-Yong Korean Society of Soil Science and Fertilizer 2011 한국토양비료학회지 Vol.44 No.4

Lysobacter antibioticus HS124 isolated from pepper rhizosphere soil produced catechol type siderophore. Purified siderophore by Diaion HP-20 and silica gel column chromatography showed several hydroxyl functional groups adjacent to benzene rings by analysis of $^1H$ NMR spectroscopy. The strain HS124 showed different activities to suppress Phytophthora capsici with different concentrations of exogenous Fe (III) in minimal medium where antifungal activity with $100{\mu}M$ Fe (III) was approximately 1.5 times higher than in absence of Fe (III). Bacterial population in this Fe (III)-amended medium was also highest with $8.9{\times}10^8\;CFU\;ml^{-1}$ which also corresponded to the strongest siderophore activity. When grown in rich medium (minimal medium with N, $P_2O_5K_2O$ and glucose), HS124 exhibited approximately 2 times stronger antifungal activity compared to minimal medium. In pot trials, treatments of bacterial culture grown in rich medium with (C1) or without (C2) $100{\mu}M$ Fe (III) exhibited a high protection of pepper plants from disease, compared to medium only with (M1) or without (M2) $100{\mu}M$ Fe (III). Especially, treatment C1 showed the best disease control effect of about 70 %. Thus, the strain HS124 should be recommended as a potential biocontrol agent against P. capsici in pepper.

Mechanisms of Structural Borrowing in Chimalaba

Mreta Abel Yamwaka,Amani Hamisi 한국외국어대학교 아프리카연구소 2012 Asian Journal of African Studies Vol.32 No.-

The paper aims to describe mechanisms through which various structural items were borrowed from Kiswahili into Chimalaba. It is an attempt to illustrate how various linguistic constraints seem to determine the borrowing of structural features in a given language contact situation. The primary data for this paper, are based on the research project by Amani (2010) on the Influence of Kiswahili in Chimalaba. There are, approximately 16 structural items borrowed from Kiswahili into Chimalaba as listed in Amani (2010: 58). The main focus is on whether the cases of structural borrowing identified are the result of direct or indirect borrowing and in displaying either of the two, what linguistic constraints are typically at work. Structural borrowing in Chimalaba reveals that Chimalaba has put a stiff resistance to direct structural borrowing, as there are only three items out of 16 structural items that have been directly borrowed from Kiswahili. This has also been possible only in the relevant sub-parts that seem to be more or less similar between Kiswahili and Chimalaba. Most of structural features in Chimalaba have been indirectly borrowed through lexical transfer, constraints of language system and blending. This paper therefore presents indirect and direct structural borrowing and the mechanisms of borrowing used in each category.

Evaluation of nutritional status of an edible grasshopper, Oxya Chinensis Formosana

현숙희,권기한,박근형,정헌천,권오석,Hamisi Tindwa,한연수 한국곤충학회 2012 Entomological Research Vol.42 No.5

This study was conducted to examine the nutritional status of the grasshopper (Oxya chinensis formosana, OCF) as human food, exploring it as an alternative edible resource. Analysis of free amino acid shows that there are various essential amino acids in addition to saturated and unsaturated fatty acids in OCF dried powder. Analysis of the mineral contents and vitamins of dried OCF indicates that it is rich in calcium, vitamin B6, and niacin. The heavy metal content of OCF recorded was low, making it safe for human consumption: OCF had plumbum at 0.01–0.03 mg/100 g, cadmium at 0.002–0.005 mg/100 g, arsenic at 0.07–0.17 mg/100 g, and mercury at 0.0003–0.0007 mg/100 g. In conclusion, given its high nutritive quality in terms of proteins and fats, coupled with lower heavy metal content, it would be recommended to use the grasshopper (OCF) as substitute to the traditional sources of protein.

Biocontrol Potential of Streptomyces griseus H7602 Against Root Rot Disease (Phytophthora capsici) in Pepper

Xuan-Hoa Nguyen,Kyaw Wai Naing,이용성,Hamisi Tindwa,이건형,정병곤,노희명,김상준,정우진,김길용 한국식물병리학회 2012 Plant Pathology Journal Vol.28 No.3

The root rot of pepper (Capsicum annuum L.) caused by Phytophthora capsici is one of the most important diseases affecting this crop worldwide. This work presents the evaluation of the capacity of Streptomyces griseus H7602 to protect pepper plants against Phytophthora capsici and establishes its role as a biocontrol agent. In this study, we isolated an actinomycete strain H7602from rhizosphere soil, identified it as Streptomyces griseus by 16S rRNA analysis and demonstrated its antifungal activity against various plant pathogens including P. capsici. H7602 produced lytic emzymes such as chitinase,β-1,3-glucanase, lipase and protease. In addition, crude extract from H7602 also exhibited destructive activity toward P. capsici hyphae. In the pot trial, results showed the protective effect of H7602 against pepper from P. capsici. Application of H7602 culture suspension reduced 47.35% of root mortality and enhanced growth of pepper plants for 56.37% in fresh root and 17.56% g in fresh shoot as compared to control, resulting in greater protection to pepper plants against P. capsici infestation. Additionally, the enzymatic activities, chitinase and β-1,3-glucanase, were higher in rhizosphere soil and roots of pepper plants treated with H7602 than other treated plants. Therefore, our results indicated a clear potential of S. griseus H7602 to be used for biocontrol of root rot disease caused by P. capsici in pepper.