Streptococcus pneumoniae (pneumococcus) is a commensal gram-positive colonizer of the human nasopharynx capable of causing diseases including otitis media, pneumonia, bacteremia, and meningitis. Although it is often a harmless colonizer, there is a high rate of mortality and morbidity among the immunocompromised, elderly, and young children. While these infections can often be treated with antibiotics, resistance to numerous antibiotics is increasing. Antibiotic resistance is a well-studied dilemma; however, little information is known of how bacteria take up certain antibiotics. Because most antibiotics cannot diffuse freely across the bacterial cell wall, we hypothesize that metabolite transport proteins participate in the uptake of certain classes of antibiotics.

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics. In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin. In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro. To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>