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      Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.)

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          Abstract

          Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

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          Reduction of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis in chicken manure by larvae of the black soldier fly.

          MARILYN C. ERICKSON, MAHBUB ISLAM, CRAIG SHEPPARD … (2004)
          Green fluorescent protein-labeled Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were inoculated at 10(7) CFU/g into cow, hog, or chicken manure. Ten- or 11-day-old soldier fly larvae (Hermetia illucens L.) (7 to 10 g) were added to the manure and held at 23, 27, or 32 degrees C for 3 to 6 days. Soldier fly larvae accelerated inactivation of E. coli O157:H7 in chicken manure but had no effect in cow manure and enhanced survival in hog manure. The initial pH values of the hog and chicken manure were 6.0 to 6.2 and 7.4 to 8.2, respectively, and it is surmised that these conditions affected the stability of the larval antimicrobial system. Reductions of E. coli O157:H7 populations in chicken manure by larvae were affected by storage temperature, with greater reductions in samples held for 3 days at 27 or 32 degrees C than at 23 degrees C. Pathogen inactivation in chicken manure by larvae was not affected by the indigenous microflora of chicken manure, because Salmonella Enteritidis populations in larvae-treated samples were approximately 2.5 log lower than control samples without larvae when either autoclaved or nonautoclaved chicken manure was used as the contaminated medium during 3 days of storage. Extending the storage time to 6 days, larvae again accelerated the reduction in Salmonella Enteritidis populations in chicken manure during the first 4 days of storage; however, larvae became contaminated with the pathogen. After 2 days of feeding on contaminated manure, Salmonella Enteritidis populations in larvae averaged 3.3 log CFU/g. Populations decreased to 1.9 log CFU/g after 6 days of exposure to contaminated chicken manure; however, the absence of feeding activity by the maggots in later stages of storage may be responsible for the continued presence of Salmonella Enteritidis in larvae. Transfer of contaminated larvae to fresh chicken manure restored feeding activity but led to cross-contamination of the fresh manure.
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            Innate immunity of insects.

            Jules Hoffmann (1995)
            Insects are particularly resistant to microorganisms. Their host-defense system relies on several innate reactions: upon injury, the immediate onset of two proteolytic cascades leading to localized blood clotting and to melanization, the latter process involving production of cytotoxic molecules (namely reactive oxygen intermediates); the phagocytosis of bacteria and the encapsulation of larger parasites by blood cells; the induced synthesis by the fat body of a battery of potent antimicrobial peptides/polypeptides which are secreted into the hemolymph where they act synergistically to kill the invading microorganisms. The insect host defence system shares many of the basic characteristics of the mammalian acute phase response, especially at the level of the coordinate control of gene expression, where similar cis-regulatory and inducible transactivators appear to play key functions. The powerful techniques developed to study the genetics of Drosophila provide a unique opportunity to dissect the development and differentiation of this primordial immune system and may contribute to our understanding of the innate immune response in higher organisms.
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              Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae

              Soon-Ik Park, Jong-Wan Kim, Sung Yoe (2015)
              In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Gram-positive bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI-TOF and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization.
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                Author and article information

                Contributors
                Humberto Lanz-Mendoza: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 5 January 2017
                Publication date Collection: 2017
                Volume: 12
                Issue: 1
                Electronic Location Identifier: e0169582
                Affiliations
                [1 ]State Key Laboratory of Agricultural Microbiology, National Engineering Research Center of Microbial Pesticides, College of Life Science and Technology, Huazhong Agricultural University- Wuhan, China
                [2 ]Faculty of Science and Technology, Omdurman Islamic University, Khartoum, Sudan
                Instituto Nacional de Salud Pública, MEXICO
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: OE DZ JZ.

                • Funding acquisition: JZ.

                • Investigation: OE DZ.

                • Methodology: OE DZ.

                • Project administration: LZ ZY.

                • Resources: QS AAS.

                • Supervision: ZY JZ.

                • Validation: ZY JZ.

                • Visualization: OE DZ.

                • Writing – original draft: OE DZ.

                • Writing – review & editing: MC QS AAS.

                Article
                Publisher ID: PONE-D-16-36355
                DOI: 10.1371/journal.pone.0169582
                PMC ID: 5215879
                PubMed ID: 28056070
                SO-VID: 66115787-2549-4c52-895c-17610f85601a
                Copyright © © 2017 Elhag et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 10 September 2016
                Date accepted : 19 December 2016
                Page count
                Figures: 5, Tables: 4, Pages: 15
                Funding
                Funded by: Special Fund for Agro-scientific Research in the Public Interest
                Award ID: 201303094
                Award Recipient :
                Funded by: Natural Science Foundation of Hubei Province, China
                Award ID: 2010CBB02002
                Award Recipient :
                This research was funded by the Special Fund for Agro-scientific Research in the Public Interest, China (201303094) and Natural Science Foundation of Hubei Province, China (2010CBB02002).
                Categories
                Subject: Research Article
                Subject: Medicine and Health Sciences
                Subject: Pharmacology
                Subject: Drugs
                Subject: Antimicrobials
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Microbial Control
                Subject: Antimicrobials
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Animals
                Subject: Invertebrates
                Subject: Arthropoda
                Subject: Insects
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Fungal Pathogens
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Fungal Pathogens
                Subject: Biology and Life Sciences
                Subject: Mycology
                Subject: Fungal Pathogens
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Research and Analysis Methods
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Subject: Biology and life sciences
                Subject: Molecular biology
                Subject: Molecular biology techniques
                Subject: DNA construction
                Subject: Plasmid Construction
                Subject: Research and analysis methods
                Subject: Molecular biology techniques
                Subject: DNA construction
                Subject: Plasmid Construction
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Fungi
                Subject: Research and analysis methods
                Subject: Database and informatics methods
                Subject: Bioinformatics
                Subject: Sequence analysis
                Subject: DNA sequence analysis
                Custom metadata
                Data Availability All relevant data are within the paper and its Supporting Information file.

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