CHARACTERIZATION OF ANTI-MICROBIAL COMPOUNDS IN ORAL SECRECTIONS OF BEETLES

甲虫口腔分泌物中抗微生物化合物的表征

• 批准号: 7725193
• 负责人: JULIE SHAFFER
• 金额: $ 3.22万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7725193
• 关键词: Alkaline Phosphatase; Amino Acid Sequence; Antibiotic Resistance; Anus; Aspartic Endopeptidases; Bacteria; Biological Assay; Bioluminescence; Computer Retrieval of Information on Scientific Projects Database; Cysteine Protease; Enzymes; Exhibits; Fractionation; Funding; Future; Gel; Grant; High Pressure Liquid Chromatography; Insecta; Institution; Laboratories; Larva; Left; Maggots; Measures; Nature; Nebraska; North America; Oral; Order Coleoptera; Pattern; Peptide Hydrolases; Peptide Sequence Determination; Peptides; Peroxidase; Peroxidases; Proteins; Research; Research Personnel; Resources; Route; Sampling; Solutions; Source; Staging; Techniques; Tube; United States National Institutes of Health; Vibrio; antimicrobial; feeding; gel electrophoresis; member; microorganism; tricine

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The carrion beetles (Coleoptera: Silphidae) consist of two subfamilies in North America. Members of the Silphinae arrive at carcasses during the mid-stage of decay and their larvae feed on developing maggots, while members of the Nicrophorinae or burying beetles bury and tend carcasses upon which their developing larvae feed. It has been hypothesized that the anal and/or oral secretions from these insects are antimicrobial, although quantification has not been made. My laboratory has been able to quantify the antimicrobial activity of the Nicrophorinae oral secretions with the Microtox Analyzer, which measures bioluminescence of the bacterium, Vibrio fischerii. The antimicrobial activity of the Nicrophorinae appears to be proteinaceous in nature. The future research of my laboratory is to identify these proteins. We plan to use a combination of techniques. One route is to identify all of the enzymatic activity in the oral secretions. The experimental techniques used for this research is colorimetric assays. Another route to identify the antimicrobial compounds is to compare the one species of Nicrophorinae, Nicrophorus carolinus, which does not exhibit antimicrobial activity to Nicrophorus marginatus, which does have antimicrobial activity in their oral secretion and is the most common burying beetle in Nebraska. We plan on using a subtractive comparison of gel electrophoresis banding patterns to help identify possible antimicrobial compounds. Once differences are identified the proteins can be isolated from gels and identified by sequencing these proteins. There is potential that the proteins responsible for the antimicrobial activity are small peptides. If the antimicrobial compounds seem to be peptides, we will use a combination of Tris-tricine gels, HPLC, and GC mass spec to separate and identify the compounds. By identifying these antimicrobial compounds we are hopeful that these proteins may be useful in treating antibiotic-resistant microorganisms. We have almost completed the characterization of the enzymes found in the oral secretion of N. marginatus. We have used colorimetric assays and have found the presence of alkaline phosphatase, peroxidase, and peptidase. We have further characterized the peptidases and find aspartic proteases and cysteine proteases present in the oral secretion. This is not surprising as these enzymes are commonly found in insect oral secretion. Since we have completed the characterization of the enzymes, we are now beginning fractionation of the secretion. After basic fractionation using microcon spin tubes, we find the active fraction in the solution left in the tube. We now believe that the antimicrobial compound could be a larger protein rather than a peptide as first hypothesized. Our next step is to fractionate samples further with the HPLC.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 腐肉甲虫（鞘翅目：Silphidae）由北美的两个亚家族组成。 Silphinae的成员在腐烂的中期到达尸体，而幼虫则以发展为虫的幼虫，而烟虫的成员或埋葬甲虫埋葬并散发出其发育中的幼虫饲料的尸体。 据推测，这些昆虫的肛门和/或口服分泌是抗菌剂，尽管尚未进行定量。 我的实验室能够用微毒素分析仪来量化烟碱口服分泌的抗菌活性，该分析仪测量了细菌的生物发光，Vibrio fischerii。 烟脂脂的抗菌活性本质上似乎是蛋白质的。 我的实验室的未来研究是识别这些蛋白质。 我们计划使用多种技术。 一种途径是确定口服分泌物中的所有酶活性。 该研究使用的实验技术是比色测定。 鉴定抗菌化合物的另一种途径是比较烟果果仁酸果仁果的一种一种物种，它不会表现出对杆菌症状的抗菌活性，它们确实具有口腔分泌物中的抗菌活性，并且是最常见的内布拉斯加州囊中的抗菌活性。我们计划使用凝胶电泳带模式的减法比较，以帮助鉴定可能的抗菌化合物。 一旦鉴定出差异，就可以从凝胶中分离蛋白质，并通过对这些蛋白质进行测序来鉴定。 有可能导致抗菌活性的蛋白质是小肽。 如果抗微生物化合物似乎是肽，我们将使用Tris-tricine凝胶，HPLC和GC质量规格的组合来分离并识别化合物。 通过鉴定这些抗菌化合物，我们希望这些蛋白质可能有助于治疗抗生素耐药的微生物。 我们几乎已经完成了在Marginatus口服分泌中发现的酶的表征。 我们使用了比色测定法，并发现存在碱性磷酸酶，过氧化物酶和肽酶。 我们进一步表征了肽酶，并发现口服分泌中存在的天冬氨酸蛋白酶和半胱氨酸蛋白酶。 这并不奇怪，因为这些酶在昆虫口腔分泌中常见。 由于我们已经完成了酶的表征，因此我们现在开始分泌分泌。 使用微孔自旋管基本分馏后，我们在管中左侧的溶液中找到了活性分数。 我们现在认为，抗菌化合物可能是较大的蛋白质，而不是首先假设的肽。 我们的下一步是用HPLC进一步分馏样品。