Programed genetic transformation: Staphylococcus aureus as a new model organism – GenTranSa

This project will be divided in two main parts based on the scientific questions asked and the methodologies proposed. These two halves of the project will generate complementary sets of data that will feed each other.
The aim of the first half of the project will be to investigate the role(s) of known and conserved actors. In every model organisms studied, competence development is highly regulated at the transcription level. This is why we will first characterize the transcription profile of early (sigH, comKs) and late competence genes (Task 1). To do this, we will use GFP and hopefully the luciferase as transcriptional reporters for competence and RNA-seq.
Then, an important part of this study will be dedicated to the characterization of natural transformation steps and actors, at the single cell level, in S. aureus (Task 2). To reach such goal, I propose to characterize the localization of conserved actors (ComGA, ComFA, ComEC, DprA, see Fig 1), by classic epifluorescence, TIRF or superresolution microscopy. Localization of these proteins will be also compared, at the single cell level in microfluidic chambers, to the transforming-DNA trajectory from the surface of the cell to its cytoplasm.
In the second half of the project, we will use two unbiased, high throughput techniques (RNA-sequencing, RNA-seq and yeast two-hybrid, Y2H) in order to identify new actors involved in competence development and genetic transformation in S. aureus (Task 3). The ‘Deep sequencing’ platform from the host institute will be essential to sequence the mRNA content in different experimental conditions. On the contrary, the choice of the bait used in Y2H screenings will surely orientate the investigation of identified partners function.
Importantly, this experimental pipeline, integrating gene expression, protein interaction, localization and function, should allow us to characterize this mode of HGT from the environmental signal to S. aureus genomic reorganization.

We obtained three major results over the first 18 months that open many lines of investigation for the second half of the project :

- We have been able to control the development of competence in a wild type S. aureus genetic background which means that we have the perfect experimental setup to investigate all the regulation pathways involved.

-We have clearly demonstrated that quorum sensing (QS) is an important part of the regulation of competence development in S. aureus. This characteristic would be common to all the main model organisms (i.e. S. pneumoniae, B. subtilis, V. cholera…) and confirm that cell-cell communication is key to somehow ‘synchronize’ part or the entire bacterial population into the environmental adaptation named genetic competence.

- We also have evidence for several potential stimuli (stress), that would be sensed by S. aureus, in response to which the bacteria would develop competence. We also have identified potential pathways that could detecte and transfer the signal to the main competence regulatory proteins.
When confirmed, these stimuli could help us to identify the conditions required in vivo for S. aureus to induce horizontal gene transfer through competence for natural transformation.

- Confirm our hypotheses concerning the stimuli (Task 1)
- Test our candidates for the signal transduction pathways (Task 1)
- Finish and analyze the constructs for microscopy (Task 2) involving fusions between natural transformation proteins and fluorescent proteins.
- Define with precision (RNA-seq) the main regulators' (SigH, ComK1 and ComK2) regulons (Task 3)

None for now.
At least 3 parts of the project could be quickly published.

Horizontal gene transfer (HGT) greatly contributes to the evolution of bacterial species. Acquisition of certain genes can completely change the fitness of a bacterium allowing the use of new substrates or the survival in environments otherwise considered as toxic. Analysis of sequence and functions in numerous genomes recently revealed that an important number of genes increasing virulence in major human pathogens have been acquired through HGT. In addition, several multi-resistant “super-bugs” have emerged over the last decades through the acquisition of antibiotic resistance genes. Staphylococcus aureus is the most emblematic example of the damages caused by these multi-resistant organisms and linked to outbreaks in hospitals but also in communities of healthy individuals.
Bacterial genetic transformation is one of the three main mechanisms allowing HGT in bacteria, with conjugation and transduction. Genetic transformation is defined as the capacity to capture exogenous DNA, transport it across the bacterial envelop and integrate it in the chromosome through homologous recombination. Unlike the two other mechanisms, genetic transformation is entirely controlled by the recipient cell and the proteins essential for these functions are encoded by the chromosome. Induction of expression of the genes controlling genetic transformation require that bacteria enter in a differentiated state called natural competence. The number of species that are known to be naturally transformable has almost doubled in twenty years and new bacteria displaying such aptitudes are often discovered and one of the latest is S. aureus.
The aim of this project will be to characterize competence and transformation in a new model organism, the human pathogen S. aureus. We will particularly attempt to achieve 5 main objectives: i) to identify the environmental signal(s) leading to competence development, ii) to decipher the transcriptional cascades transmitting this signal until the activation of natural transformation, iii) to characterize the steps and actors involved during natural transformation, iv) to define the singularities that make competence and transformation unique in S. aureus and v) to evaluate where, when and how does natural transformation participate to S. aureus genomic plasticity.
This pluridisciplinary project (transcriptional profiling, microscopy, genetic screens) relies on both hypothesis-driven and unbiased, high throughput approaches. Our hypotheses are based on known actors, present in other naturally transformable bacteria and conserved in S. aureus, but also on numerous preliminary results. Analysis of the transcriptional profile of conserved regulatory genes involved during competence development as well as the localization of conserved actors involved during natural transformation will allow us to establish temporal and spatial references necessary for the study of these processes in S. aureus. In parallel, we will use the “Transposon sequencing” and “yeast two hybrid” techniques to identify new actors involved during competence and genetic transformation that will complete our comprehension of this mode of HGT.
Finally, the knowledge accumulated during this project should allow us to understand how S. aureus became a major human pathogen. In addition, optimization of natural transformation in S. aureus will provide new ways to improve its genetic manipulation but also catalyze the rise of new genetic tools. On the contrary, inhibition of transformation should provide new strategies to fight against the transfer of antibiotic resistances.