Investigating the regulation and function of translation factor MsrD

Overview & Synopsis

Antibiotics are low molecular weight organic molecules best known for their use in human health. Over the past decades, many anthropogenic factors have led to a massive dissemination of antibiotic-resistant bacterial strains thus constituting a major threat to our therapeutic arsenal. Among the wide diversity of antibiotic resistance mechanisms, factor-associated protection has recently attracted the attention of the scientific community. The ABC-F proteins are translation factors present in bacteria and eukaryotic organisms, with the exception of archaea. These factors have two ATP- binding cassettes, and they use hydrolysis of ATP to ensure their biological functions. Within the ABC-F family, some members termed antibiotic resistance ABC-Fs (ARE ABC-Fs), employ the mechanism of factor-associated protection to confer resistance to many ribosome-targeting antibiotics. Although some ARE ABC-Fs have been structurally characterized, the molecular mechanism by which they confer resistance once bound to the ribosome is not yet agreed upon. Furthermore, the involvement of ATP- binding cassettes is not fully understood, as well as the regulation of genes encoding these factors in response to exposure to antibiotics. The regulation and function of translation factor MsrD in response to certain antibiotics were investigated during my doctoral work, which combined the fields of microbiology, molecular biology, biochemistry and structural biology. First, I have demonstrated that gene msrD, which disseminates mainly in Streptococci, is inducible in presence of some macrolide antibiotics. This gene is regulated by an intrinsic transcriptional attenuator under the control of ORF msrDL. This ORF encodes a six amino acids-long leader peptide that is capable of inhibiting its own synthesis in the presence of certain macrolides. Thus, ligand-dependent ribosomal arrest on the ORF msrDL prevents folding of the intrinsic terminator, and consequently the msrD gene is then transcribed. Second, in order to understand the molecular arrest mechanism on the ORF msrDL, I obtained the structure of a ribosome, at near-atomic resolution, translating this ORF in presence of erythromycin by cryogenic electron microscopy. This structure reveals that in the presence of a ribosome-bound erythromycin molecule, the leader peptide MsrDL recognizes the latter by interacting via hydrophobic interactions, and it is forced to engage in a cavity of the tunnel not described in the literature. By doing so, MsrDL stabilizes several nucleotides composing the catalytic site of the ribosome in an inactive state leading to translational arrest. Finally, I have demonstrated that the association dynamics between translation factor MsrD and the ribosome is mediated by its two ATP-binding cassettes that are functionally asymmetric. This led me to propose a new resistance mechanism, where MsrD would dissociate antibiotic-stalled ribosomes. It has also been demonstrated that, in the presence of antibiotics, MsrD is able to regulate its own expression through a negative feedback loop. In addition, preliminary attempts to obtain the structure of ribosome-bound MsrD have been initiated.