lasSoCocci

Infections caused by multi-drug resistant (MDR) bacteria are on the rise worldwide. Enterococcus faecalis, E. faecium and Staphylococcus aureus are major Gram-positive pathogens causing life-threatening nosocomial infections. These bacteria have developed resistance to the last resort antibiotic vancomycin and their infections are today difficult to treat. Next generation antibiotics are therefore urgently needed, especially for vancomycin-resistant E. faecium and S. aureus, which were recently ranked as high priority for the development of new antibiotics by the World Health Organization. Interesting molecules would be drugs targeting important virulence factors or resistance mechanisms without demonstrating toxicity to higher organisms. Recently we evidenced that two bacterial cyclic peptides belonging to the family of lasso peptides are promising candidates to fight MDR Gram-positive pathogens. These molecules, named sviceucin and siamycin, are able to completely abolish enterococcal virulence and vancomycin resistance, as well as S. aureus vancomycin resistance, without demonstrating toxic effects to higher animals. To our knowledge, this is the first example of molecules able to suppress virulence and antibiotic resistance, arguing that they are highly interesting molecules to study. Therefore understanding their mode of action, likely different from those of conventional antibiotics, is not only essential for their potential development as new drugs, but also will warrant the discovery of new pathways that can be targeted for antibacterial therapy. Thereby, our project aims at deciphering the underlying molecular mechanisms as well as the structure-activity relationship of sviceucin and siamycin. To achieve the goals, the highly interdisciplinary consortium will use numerous state-of-the art techniques. Based on literature data and on our thorough preliminary results, sviceucin and siamycin should have direct protein targets, i.e. histidine kinases of two-component systems. We suppose that interaction with some of these targets could trigger major changes in gene expression potentially at the basis of the observed decrease in virulence. Therefore, we will perform global transcriptomic analysis by RNA-sequencing in the presence and absence of the lasso peptides. Concerning the mechanism of the suppression of vancomycin resistance by sviceucin and siamycin, one of the strategies will be to isolate spontaneous mutants resistant to this effect of the peptides which will be analyzed by genome sequencing. This approach is a standard strategy for the identification of targets of antibacterial molecules. In addition, we will purify proteins suspected to be directly targeted by sviceucin and siamycin and analyze the interaction of these proteins with the peptide. This direct target identification will be completed by pull-down/immuno-precipitation approaches which are also complementary to the global approaches mentioned above to decipher the anti-virulence and anti-resistance mechanisms of sviceucin and siamycin. The potential targets identified will be verified using various in vivo and in vitro approaches. Based on the information obtained we will rationally design new peptides with modulated properties and examine their effects on the three pathogenic bacteria used in this study. The discovery of the potential of sviceucin or siamycin-derived peptides to fight MDR infections and their optimization in a context where the discovery of novel antibiotics is extremely rare will provide an alternative to the existing treatments against MDR Gram-positive pathogens.