Mechanisms of bacterial resistance

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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.