Mechanisms of bacterial resistance

洋葱伯克霍尔德氏菌复合体（Bcc）是一种土壤细菌的集合，它们能够巧妙地降解各种污染物，但也会在植物和受损个体中引起疾病。BCC的一个特点是它的高抗生素耐药性。在这个提议中，我们研究了BCC对阳离子抗菌肽（CAMPs）如多粘菌素B（PMB）（一类抗生素和先天免疫系统的天然成分）的异常和极端抗性。早期的研究表明，Bcc LPS的独特结构负责排斥PMB活性，然而，一些证据表明，这不是Bcc抵抗CAMPs的唯一机制。为了进一步研究BCC的新的CAMP抗性特性，将进行几条研究线，这将导致确定CAMP抗性的机制。这些信息将有助于确定革兰氏阴性菌细胞超微结构的关键参与者，并了解它们在细菌抗生素耐药性中的作用。为了鉴定CAMP抗性中重要的细胞包膜成分，我们将采用遗传、基因组和生化分析。首先，我们将检查BCC细胞使用非必需的基因产物参与CAMP抗性的遗传技术，使用插入plasposon诱变。此外，我们将研究的功能基因组图谱的BCC菌株进行PMB应力使用基因表达启动子探针库。第二，最近测序的Bcc菌株249（对PMB有抗性）具有PMB敏感的衍生物Bcc菌株249-2，其估计具有200-500 kb的缺失。基因组探测将划定249-2缺失的终点，并帮助确定一个特定的机制参与Bcc PMB的阻力。第三，用CAMP爪蟾抗菌肽-2（唯一已知的对Bcc有效的CAMP）进行Bcc遗传选择将鉴定赋予CAMP抗性的必需和非必需上调基因。Bcc CAMP抗性的表征将导致发现新的细菌细胞包膜修饰或机制。了解Bcc CAMP耐药性将导致开发更有效的抗生素和治疗抗生素耐药革兰氏阴性菌。

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.