CHARACTERIZATION OF ANTI-MICROBIAL COMPOUNDS IN ORAL SECRECTIONS OF BEETLES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。北美腐肉甲虫(鞘翅目：狮蛛科)由两个亚科组成。Silphinae的成员在腐烂的中期到达身体，它们的幼虫以发育中的蚯蚓为食，而Nmicrophorinae或埋葬甲虫的成员埋葬和照料它们发育的幼虫赖以生存的身体。据推测，这些昆虫的肛门和/或口腔分泌物具有抗菌作用，尽管还没有进行量化。我的实验室已经能够用Microtox Analyzer来量化Nmicrophorinae口腔分泌物的抗菌活性，它测量了费氏弧菌的生物发光。夜蛾的抗菌活性似乎是蛋白质性质的。本实验室未来的研究方向是对这些蛋白质进行鉴定。我们计划使用多种技术的组合。一种方法是鉴定口腔分泌物中的所有酶活性。这项研究使用的实验技术是比色法。鉴定抗菌化合物的另一种方法是比较夜甲亚科的一种--卡罗琳夜蛾，它对边缘夜蛾没有抗菌活性，而边缘夜蛾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带模式的减法比较来帮助识别可能的抗菌化合物。一旦确定了差异，就可以从凝胶中分离出这些蛋白质，并通过对这些蛋白质进行测序来鉴定它们。有可能负责抗菌活性的蛋白质是小肽。如果抗菌化合物看起来是多肽，我们将使用Tris-tricine凝胶、高效液相色谱和GC-MS相结合的方法来分离和鉴定化合物。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耐药微生物。我们几乎已经完成了对边缘毛虫口腔分泌物中发现的酶的表征。我们使用了比色法，发现了碱性磷酸酶、过氧化物酶和肽酶的存在。我们进一步鉴定了这些多肽酶，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。这并不令人惊讶，因为这些酶通常存在于昆虫的口腔分泌物中。既然我们已经完成了酶的表征，我们现在就开始对分泌物进行分级。使用MicroCon旋转管进行基本分馏后，我们发现溶液中的活性组份留在了旋转管中。我们现在认为，这种抗菌化合物可能是一种更大的蛋白质，而不是最初假设的多肽。我们的下一步是用高效液相色谱进一步分离样品。

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.