BIOGENESIS OF BACTERIAL MEMBRANES: A TARGET FOR NOVEL ANTIBIOTICS?

Antibiotic resistance is a major and growing problem in the world, making it more and more difficult to treat bacterial infections. If no action is taken the World Health Organization has predicted that by 2050 10 million people will die worldwide each year from the effects of antibiotic resistance. Currently, already 700,000 people worldwide die every year from an infection with resistant bacteria. Many factors contribute to the antibiotic resistance problem, including the widespread use of antibiotics in livestock and misuse of antibiotics in clinical settings. In addition, hardly any new class of antibiotics has been developed since 1980. To combat antibiotic resistance, it is essential to develop new antibiotics that target and kill bacteria using a novel mechanism of action. In this thesis we describe an innovative approach to identify new antibiotics, focusing mainly on the so-called BAM complex located in the outer membrane of Gram-negative bacteria, as explained in chapter 2 of this thesis. The BAM complex facilitates folding of outer membrane proteins and secretion of proteins across the outer membrane to the extracellular environment. To identifty novel antibiotics against the BAM complex we have developed a reporter assay that monitors induction of bacterial stress when the BAM complex is not functioning. Similar to a 'fight-or-flight' stress response in humans when approaching danger, bacteria also react with stress to impulses from the environment, such as the presence of toxic substances (e.g. antibiotics) or food shortages. In chapter 3 we describe the development of the reporter assay in which bacteria become green fluorescent as soon as they experience stress from a potential antibiotic. With this so-called 'stress assay' 1,600 compounds were tested and one compound (VUF15259) was found that probably targets the BAM complex. In chapter 4 we describe additional stress assays, which are used in chapter 5 to screen more than 316,953 compounds for antibacterial activity. This resulted in the identification of 2 compounds, CPD2 and CPD14, which weaken the outer membrane of bacteria and probably target the BAM complex. Finally, in chapter 6 we investigated the mechanism of action of another compound, called ES24, which in contrast to VUF15259, CPD2 and CPD4, acts on the inner membrane of bacteria. More specifically, we showed that ES24 targets the SecYEG translocon, a protein system in the inner membrane that facilitates protein transport. In summary, the results in this thesis show that our stress assay can identify novel antibacterial agents that act on the outer membrane of bacteria. One of the stress assays is currently being used by the European Lead Factory to screen their library of more than 500,000 compounds for antibacterial activity.