Periodic Reporting for period 4 - TransfoPneumo (Structure and Function of the Bacterial Transformasome)
Período documentado: 2022-03-01 hasta 2023-08-31
Bacteria are extremely adaptable and able adjust their lifestyle very quickly when these changes occur. One dramatic illustration of this capacity is the spread of antibiotic resistance among bacterial pathogens. During the last decade, the emergence of multi-resistant bacteria, which are resistant to several treatments, led to increase mortality caused by common infections. The 2014 report on antimicrobial resistance from the World Health Organization warns against the beginning of a “post-antibiotic” era, when most of the bacterial pathogens will become resistant to all treatments available.
In this context, it is crucial to fully understand the molecular mechanism of bacterial adaptability to ultimately target and limit this ability. To survive in a changing environment, bacteria have to resist to stresses induced by these changes and ultimately to adapt their lifestyle if these changes persist. These two processes are almost contradictory since the first aims at maintaining cell integrity while the second allows long term variability through the acquisition of new traits.
In this project, we wanted to understand how DNA can be uptaken and recombined in the bacterial genome during bacterial transformation. Natural genetic transformation, first discovered in Streptococcus pneumoniae by F. Griffith in 1928, is observed in many Gram-negative and Gram-positive bacteria. This process promotes genome plasticity and adaptability. In particular, it enables many human pathogens such as Streptococcus pneumoniae, Neisseria gonorrhoeae or Vibrio Cholerae to acquire resistance to antibiotics and/or to escape vaccines through the binding and incorporation of new genetic material. While it is well established that this process requires the binding, internalization of external DNA and its recombination in the bacterial genome, the molecular details of these steps are unknown.
In this context, it is crucial to fully understand the molecular mechanism of bacterial adaptability to ultimately target and limit this ability. To survive in a changing environment, bacteria have to resist to stresses induced by these changes and ultimately to adapt their lifestyle if these changes persist. These two processes are almost contradictory since the first aims at maintaining cell integrity while the second allows long term variability through the acquisition of new traits.
In this project, we wanted to understand how DNA can be uptaken and recombined in the bacterial genome during bacterial transformation. Natural genetic transformation, first discovered in Streptococcus pneumoniae by F. Griffith in 1928, is observed in many Gram-negative and Gram-positive bacteria. This process promotes genome plasticity and adaptability. In particular, it enables many human pathogens such as Streptococcus pneumoniae, Neisseria gonorrhoeae or Vibrio Cholerae to acquire resistance to antibiotics and/or to escape vaccines through the binding and incorporation of new genetic material. While it is well established that this process requires the binding, internalization of external DNA and its recombination in the bacterial genome, the molecular details of these steps are unknown.
In this project, we aimed to gain a detailed understanding of each of these steps. We discovered a new appendage on the surface of S. pneumoniae cells and showed that this appendage is similar in morphology and composition to appendages called type IV pili, which are commonly found in Gram-negative bacteria. We showed that this new pneumococcal pilus is essential for transformation and that it directly binds DNA (PLOS Pathogens 2013 and 2015). We identified a new key ATPase involved in the recombination process, called RadA. In collaboration with Patrice Polard's team in Toulouse (France), we determined the crystal structure of this protein and identified its function in vitro and in vivo (Nature communications, 2017). We then trapped the enzyme bound to DNA and solved its structure using CryoEM. We also worked on its functional homologue found in the Gram-negative homologue called ComM. We also studied the initiation and molecular mechanism of the recombination event. We determined the structure of RecA filaments from S. pneumoniae and revealed the structural basis of its interaction with its loader on ssDNA during transformation (called DprA). Finally, we are also investigating the architecture of the transformation apparatus in its native cellular environment using cryo-tomography and correlative microscopy approaches.
In parallel, we serendipitously discovered filaments of the bifunctional aldehyde-alcohol dehydrogenase AdhE in Escherichia coli and Streptotoccus pneumoniae. We solved the structure of these filaments using cryo-EM and showed that filamentation is essential for the activity of this enzyme.
During the course of the project, we isolated type IV pili from Streptococcus sanguinis and solved their structure using CryoEM.
In parallel, we serendipitously discovered filaments of the bifunctional aldehyde-alcohol dehydrogenase AdhE in Escherichia coli and Streptotoccus pneumoniae. We solved the structure of these filaments using cryo-EM and showed that filamentation is essential for the activity of this enzyme.
During the course of the project, we isolated type IV pili from Streptococcus sanguinis and solved their structure using CryoEM.
Our aim was to understand the process of bacterial transformation at the molecular level. During this project, we were able to provide important insights into the structure and function of the molecular machines involved in this multi-step process. We have also implemented methods that will now help us to focus on the "in cellulo" structural biology of these systems and try to get an integrated view of the transformasome architecture within the cell. We will also seek to understand the molecular basis of DNA transfer across the bacterial membrane.