CHARACTERIZATION OF ANTI-MICROBIAL COMPOUNDS IN ORAL SECRECTIONS OF BEETLES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。食腐甲虫（鞘翅目：Silphidae）在北美由两个亚科组成。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或埋葬甲虫的成员埋葬和照料尸体，它们发育中的幼虫以其为食。据推测，这些昆虫的肛门和/或口腔分泌物是抗菌的，但尚未进行量化。我的实验室已经能够用Microtox分析仪量化Nicrophorinae口腔分泌物的抗菌活性，该分析仪测量细菌Vibrio fischerii的生物发光。Nicrophorinae的抗微生物活性似乎是蛋白质性质的。我实验室未来的研究是鉴定这些蛋白质。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。本研究所用的实验技术是比色测定法。鉴定抗微生物化合物的另一种途径是比较Nicrophorinae的一个物种，Nicrophorus carolinus，其不表现出抗微生物活性，Nicrophorus marginatus，其在口腔分泌物中具有抗微生物活性，并且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型的减法比较，以帮助确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。有可能负责抗微生物活性的蛋白质是小肽。如果抗菌化合物似乎是肽，我们将使用Tris-tricine凝胶，HPLC和GC质谱的组合来分离和鉴定化合物。通过鉴定这些抗微生物化合物，我们希望这些蛋白质可用于治疗耐药性微生物。 我们几乎已经完成了在N.边缘。我们已经使用比色法，并已发现碱性磷酸酶，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，因为这些酶通常存在于昆虫的口腔分泌物中。由于我们已经完成了酶的表征，我们现在开始分离分泌物。在使用microcon旋转管进行基本分馏之后，我们在管中留下的溶液中发现活性级分。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。我们的下一步是用HPLC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.