The role of cell-to-cell variability in pneumococcal virulence and antibiotic resistance

Project Information

PneumoCaTChER

Grant agreement ID: 771534

DOI

10.3030/771534

Project closed

EC signature date 13 April 2018

Start date 1 November 2018

End date 31 October 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 999 735,00

EU contribution

€ 1 999 735,00

Coordinated by

UNIVERSITE DE LAUSANNE
Switzerland

Periodic Reporting for period 4 - PneumoCaTChER (The role of cell-to-cell variability in pneumococcal virulence and antibiotic resistance)

Reporting period: 2023-05-01 to 2023-10-31

Summary of the context and overall objectives of the project

Phenotypic variation, the cell-to-cell variation in phenotype expression of genetically identical cells, can help pathogenic bacteria to elude the host immune response or resist antibiotic pressure. There is also large variation in the host immune response to pathogens. How the combined cellular heterogeneity of both host and microbe contribute to infection outcome is poorly understood and a better understanding of this might aid in better therapies.

In this ERC project we study the important opportunistic human pathogen Streptococcus pneumoniae (the pneumococcus). S. pneumoniae can cause severe life-threatening infections such as sepsis, pneumonia and meningitis and is annually responsible for over a million deaths. Here, we use innovative approaches for infection biology by combining synthetic biology and quantitative single cell biology including single cell RNA-seq, CRISPRi, engineered bistable switches and microfluidics. This will help us to reveal the molecular mechanisms underlying cell-to-cell variability and its importance in virulence and antibiotic resistance.

Insights obtained in this project will lead to a better understanding of phenotypic variation and might result in new treatment strategies for pneumococcal infections.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have tackled all major aims set out in this project. For aim 1, we have generated extensive RNA-seq and proteomics databases, which have now been analyzed and will be soon submitted for publication. In addition, we have setup CRISPRi-seq, which allows us to look for essential genes under various conditions (Liu et al. Cell Host & Microbe 2021). Within the framework of this project, we now have determined the essentialome of S. pneumoniae under the same conditions as we obtained transcriptome and proteome data. These datasets now have been integrated and will be soon submitted for publication.

For aim 2, we have setup the experimental methods to do host-pneumococcal interactions using A549 human cell lines. In addition, the first ever pneumococcal TIMER strains have been constructed. We have discovered that competence development is specifically upregulated during interaction with the host and we have obtained the first scRNA-seq data of A549 cells infected with wild type and competence mutants. This exciting new data confirms our hypothesis in that it shows that just a subpopulation of host cells mount an inflammatory response in reply to pneumococcal infection, demonstrating the presence of host phenotypic variation.

For aim 3, we now generated several synthetic gene-regulatory networks capable of demonstrating bimodal gene expression distributions. Specifically, we have generated an oscillator that can be coupled to virulence factor expression. We have setup and optimized microfluidics devices that now allow us to follow single pneumococci by live fluorescence microscopy for more than 24h (Rueff et al. Nature Comm. 2023. We have developed an advanced image analysis tools for microscopy data (BactMAP).

For aim 4, we have performed deep seq and SMRT-seq on heteroresisters and identified the CiaR/H system to be important in amoxicillin resistance development as well as determined the route to full amoxicillin resistance via horizontal gene transfer (Gibson et al. Plos Patho.2022). In addition, we have started several antibiotic challenges, in combination with CRISPRi-seq, to identify genes involved in antibiotic tolerance and resistance. Finally, by genetic interaction studies, we have identified several new cell division genes and pathways that might provide new therapeutic targets (de Wachter et al., eLife 2022).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Aim 1:
We have produced and will publish the first combined transcriptome/proteome/essentialome map of Streptococcus pneumoniae. This map provides invaluable new insights into which genes are important under which conditions. In addition, our single cell analysis on engineered reporters will tell us what the most important molecular factors are in generating cell-to-cell variability in pneumococcal gene expression.

Aim 2:
We will produce and publish scRNA-seq data of host cells infected with wild type and mutant pneumococci. These kind of experiments and data have so far not yet been publicly available. This will yield invaluable insights into the extend and importance of host cell variation to infection.

Aim 3:
Engineered synthetic gene-regulatory network have been used to test the hypothesis that heterogeneity in virulence factor gene expression is important for pathogenesis. Much heterogeneity in virulence factors production is lost upon cultivation of clinical isolates. By engineering such networks, we could generate oscillating virulence expression in genetically identical pneumococci for the first time. While pioneering these experiments, we have the developed microfluidics devices, that for the first time, provide robust growth and enables single cell analysis of live cells over time periods longer than 24h. In vitro experiments suggest that under certain conditions, heterogeneous capsule producers perform better than uniform producers. Finally, in collaboration, these synthetic strains have been tested for their colonization potential in a murine model of pneumococcal carriage. The tools and approaches pioneered here pave the way for analogous studies in other pathogens (Rueff et al. Nat. Comm. 2023).

Aim 4:
SMRT-seq in combination with experimental evolution has provided new insights into amoxicillin resistance in the pneumococcus. In addition, by genetic interaction screens, new cell division genes and new druggable targets have been identified. This might result in new therapies to treat antibiotic-resistant pneumococcal infections.

Synthetic gene regulatory network driving pneumococcal capsule production