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: 7381529
• 负责人: JULIE SHAFFER
• 金额: $ 5.34万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381529
• 关键词: CHARACTERIZATION; ANTI; MICROBIAL; COMPOUNDS; ORAL

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。腐肉甲虫（鞘翅目：食甲虫科）由北美洲的两个亚科组成。 Silphinae的成员在腐烂的中期到达尸体，它们的幼虫以发育中的蛆为食，而Nicrophorinae或埋葬甲虫的成员则埋葬和照料尸体，其发育中的幼虫以发育中的幼虫为食。据推测，这些昆虫的肛门和/或口腔分泌物具有抗菌作用，但尚未进行量化。我的实验室已经能够使用 Microtox 分析仪量化 Nicrophorinae 口腔分泌物的抗菌活性，该分析仪可测量费氏弧菌的生物发光。 Nicrophorinae 的抗菌活性似乎本质上是蛋白质的。我实验室未来的研究就是鉴定这些蛋白质。我们计划使用多种技术的组合。一种途径是鉴定口腔分泌物中的所有酶活性。本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 Nicrophorus carolinus 的一种，它不表现出抗菌活性，而 Nicrophorus marginatus 的口腔分泌物确实具有抗菌活性，是内布拉斯加州最常见的埋葬甲虫。我们计划使用凝胶电泳条带图案的减色比较来帮助识别可能的抗菌化合物。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。负责抗菌活性的蛋白质有可能是小肽。如果抗菌化合物似乎是肽，我们将结合使用 Tris-tricine 凝胶、HPLC 和 GC 质谱来分离和鉴定该化合物。通过鉴定这些抗菌化合物，我们希望这些蛋白质可用于治疗耐抗生素微生物。 我们几乎完成了边缘猪笼草口腔分泌物中酶的表征。我们使用比色测定法发现碱性磷酸酶、过氧化物酶和肽酶的存在。我们进一步表征了肽酶，并发现口腔分泌物中存在天冬氨酸蛋白酶和半胱氨酸蛋白酶。这并不奇怪，因为这些酶常见于昆虫口腔分泌物中。由于我们已经完成了酶的表征，我们现在开始对分泌物进行分级分离。使用微型旋转管进行基本分馏后，我们在管中留下的溶液中找到了活性部分。我们现在相信，抗菌化合物可能是一种更大的蛋白质，而不是最初假设的肽。我们的下一步是使用 HPLC 进一步分级样品。