Ribosomal protein S1 and novel antimicrobials

The extensive use of various antibiotics to treat bacterial infections throughout the last decades led to the appearance and rapid spread of drug-resistant pathogens, causing a major problem in public health care. A major target for clinically important antibiotics is the ribosome. Given its high complexity, it offers many sites for functional interference. Structural studies revealed that several antibiotics target predominantly the rRNA components at functional sites, which are conserved throughout the kingdoms. Consequently, this high cross species conservation of functional sites in rRNA limits the use of several antimicrobials due to their toxic side effects. In addition, despite the theoretically large number of binding opportunities, "ribosomal antibiotics" were shown to bind only to a few sites. Thus, a single resistance mechanism can confer resistance to several different antibiotics. Here, a new concept for the design of novel antimicrobials is presented. In contrast to antibiotics, which affect the function of the ribosome, the aim of this study is to identify molecular compounds, which interfere with assembly of ribosomal protein S1, which is known to be essential for growth of Gram-negative bacteria. In previous studies, it was shown that protein S1 requires ribosomal protein S2 for binding to the 30S subunit. Therefore, the first aim of the study entails the molecular characterization of the S1-S2 interaction surface by genetic and biophysical methods. Based on this information small peptides with the potential to interfere with the S1-S2 interaction will be designed. Upon validation of these peptides in preventing assembly of S1 to the ribosome, the second aim of the project is to design small molecule inhibitors, which interfere with S1-S2 interaction using these peptides as pharmacophores, and to test their potential antimicrobial activity. Since mitochondrial ribosomes lack functional homologues of S1 and eukaryotic ribosomes employ different mechanisms for translation initiation, it seems less likely that these inhibitors interfere with protein synthesis in eukaryotes. In addition, such a drug would be selective against a number of pathogenic Escherichia coli strains. Given that protein S1 is highly conserved in the gamma subdivision of proteobacteria it may have as well the potential to act as an antimicrobial agent against other Gram-negative pathogens, including Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella pneumoniae, without affecting the majority of the beneficial Gram-positive flora, as many of them do not possess a homologue of S1.

The extensive use of various antibiotics to treat bacterial infections throughout the last decades led to the appearance and rapid spread of drug-resistant pathogens, causing a major problem in public health care. One target for therapeutically important antibiotics is the ribosome that offers many sites for functional interference. However, the high cross species conservation of functional sites in the rRNA limits the use of several antimicrobials due to their toxic side effects. Thus, there appears to be a need for alternative antimicrobials, which exhibit a higher specificity. In the frame work of this project, we aimed to employ an alternative concept for the design of novel antimicrobial compounds. Our approach was based on the idea that compounds, which interfere with the assembly of essential proteins to the bacterial ribosome, could allow a more specific treatment of bacterial infections. In contrast to antibiotics targeting conserved functional sites in the ribosome, we anticipated that the inhibition of bacterial ribosome assembly would neither affect the eukaryotic nor the mitochondrial translation system of the human host. We focused specifically on the largest ribosomal protein in Bacteria, protein S1, which is essential for translation in Escherichia coli and most Gram-negative Bacteria. Due to the high conservation of S1 in the gamma subdivision of proteobacteria, a drug interfering with the S1-S2 interaction would act selectively against a number of Gram- negative pathogens. In addition, no functional S1 homologues are present in mitochondrial and eukaryotic ribosomes. During the course of the project, we were able to gain insights into the interaction site of S1 with the ribosome. Our results provide evidence that the protein primarily binds to the ribosome via a short, N-terminally located helix, which interacts directly with ribosomal protein S2. Since to date there is no structure of the native protein S1, and moreover the protein is missing in the high resolution structures available for the E. coli ribosome due to its intrinsic flexibility, the results of the project in addition significantly extended our basic scientific knowledge on the molecular mechanism of translation initiation. Moreover, they imply a pivotal role of S1 in the regulation of translation as it could contribute to the formation of specialized ribosomes under specific conditions. Thus, our results suggest new conceptions for possible novel functions of the important protein S1 in bacterial physiology.