Mechanisms of bacterial resistance

洋葱伯克霍尔德氏菌复合体(BCC)是一组土壤细菌，能够很好地降解各种污染物，但也会引起植物和受危害的个人的疾病。BCC的一个特点是它对抗生素有很高的耐药性。在这项建议中，我们研究了BCC对阳离子抗菌肽(CAMP)(如多粘菌素B(PMB))的不同寻常和极端的耐药性，多粘菌素B是一类抗生素和天然免疫系统的天然成分。早期的研究表明，BCC内毒素的独特结构是抵抗PMB活性的原因，但有几条证据表明，这并不是BCC抵抗CAMP的唯一机制。为了进一步研究BCC的新型cAMP抵抗特性，将进行几个方面的研究，以确定cAMP抵抗的机制。这些信息将有助于识别革兰氏阴性细菌细胞超微结构中的关键角色，并了解它们在细菌抗生素耐药性中的作用。为了确定在cAMP抗性中重要的细胞膜成分，我们将使用遗传、基因组和生化分析。首先，我们将使用插入质粒突变的方法，利用非必需基因产物的基因技术检测BCC细胞中与cAMP耐药相关的基因产物。此外，我们将使用基因表达启动子探针库来检测一株经受PMB应激的BCC菌株的功能基因组图谱。第二，最近测序的BCC株249(对PMB抗药性)具有PMB敏感的衍生BCC株249-2，估计有200-500kb的缺失。基因组探测将划定249-2缺失终点，并有助于确定参与BCC PMB耐药的特定机制。第三，用cAMP Mainin-2进行BCC基因选择，这是已知的唯一有效对抗BCC的cAMP，它将识别与cAMP抗性相关的必要和非必要上调基因。BCC cAMP耐药性的表征将导致发现新的细菌细胞被膜修饰或机制。了解BCC对cAMP的耐药性将有助于开发更有效的抗生素和治疗耐药的革兰氏阴性细菌。

The Burkholderia cepacia complex (Bcc) are a collection of soil bacteria that are exquisitely capable of degrading a wide range of pollutants but also causing disease in plants and compromised individuals. A hallmark of the Bcc is it's high antibiotic resistance. In this proposal, we examine the Bcc's unusual and extreme resistance to cationic antimicrobial peptides (CAMPs) like Polymyxin B (PMB), a class of antibiotics and natural components of the innate immune system. Earlier studies suggest that it is the unique structure of Bcc LPS that is responsible for repelling the PMB activity, however several lines of evidence suggest that this is not the only mechanism by which the Bcc resist CAMPs. To investigate the novel CAMP resistant properties of the Bcc further, several lines of investigation will be undertaken that will lead to determination of the mechanisms of CAMP resistance. This information will be useful in identifying key players in gram-negative bacteria cellular ultrastructure, and understanding their role in bacterial antibiotic resistance. To identify cell envelope components important in CAMP resistance, we will employ genetic, genomic, and biochemical analysis. First, we will examine Bcc cells using genetic techniques for non-essential gene products involved in CAMP resistance using insertion plasposon mutagenesis. In addition, we will examine the functional genomic profile of a Bcc strain undergoing PMB stress using a gene expression promoter probe library. Second, recently sequenced Bcc strain 249 (resistant to PMB) has a PMB sensitive derivative Bcc strain 249-2 estimated to have a 200-500 kb deletion. Genomic probing will demarcate the 249-2 deletion end-points and help identify a specific mechanism involved in Bcc PMB resistance. Third, Bcc genetic selection with CAMP magainin-2, the only known CAMP effective against the Bcc, will identify essential and non-essential upregulated genes conferring CAMP resistance. Characterization of Bcc CAMP resistance will lead to the discovery of novel bacterial cell envelope modifications or mechanisms. Understanding Bcc CAMP resistance will lead to the development of more effective antibiotics and treatments for antibiotic resistant gram-negative bacteria.