Periodic Reporting for period 1 - FISHnCRISPS (On the development of novel molecular and microscopy methodologies for the interrogation of host-pathogen interactions at the single-cell level)
Okres sprawozdawczy: 2020-06-01 do 2022-05-31
The first line of treatment against bacterial infection consists in the use of antibiotics, however antibiotic-resistant bacteria are emerging rapidly worldwide, endangering the efficacy of treatment and transforming what could be a simple infection in a serious threat. There is a pressing need for expanding our tools against pathogens, but how? Over the course of infectious disease evolution, both players in this tug-of-war (the pathogens and their hosts) have set out strategies to win over the other by adapting to challenging environments on the pathogen side, and by detecting and eradicating the intruder on the host side. Understanding these mechanisms on depth can identify how to aid our bodies to efficiently resolve an infection without the use of antibiotics. Here, I proposed the expansion of our toolbox to identify the crucial factors driving disease outcome, with the final purpose of discovering new potential drug targets.
This project aimed at the development and implementation of techniques that would allow the concurrent interrogation of host and pathogens at the single cell level,
The scientific objectives and goals have been addressed via three specific work packages: (1) develop a dual smFISH protocol to detect both host and pathogen transcripts; (2) single-cell CRISPRi genetic screens in Salmonella; (3) and their implementation in infection models.
For the first package, a successful protocol has been calibrated and applied to Salmonella infection in macrophages to follow the expression of virulence genes across time.
The second package has been accomplished by achieving down-regulation of Salmonella genes during infection in macrophages, and has been implemented as part of the third package. Moreover, to transfer the CRISPRi technology to the single-cell level, the scaffold region of the sgRNAs has been successfully engineered to include a capture sequence, not affecting efficiency of knock-down (KD) and allowing for direct recovery of the identity of the perturbation. A collection of perturbations targeting two-component systems was generated and used for subsequent experiments. The fellow has set up a protocol compatible with mainstream commercial single-cell technologies for recovering, at the single-cell level, both the gene expression profile of the host and the identity of Salmonella’s perturbations with a very high recovery rate. The protocols for these methods will be open access on online platforms, after manuscript submission. The CRISPRi plasmids have already been sent to addgene, and will be distributed following manuscript submission. These results were also disseminated in the several conferences the fellow attended.
The scientific objectives and goals have been addressed via three specific work packages: (1) develop a dual smFISH protocol to detect both host and pathogen transcripts; (2) single-cell CRISPRi genetic screens in Salmonella; (3) and their implementation in infection models.
For the first package, a successful protocol has been calibrated and applied to Salmonella infection in macrophages to follow the expression of virulence genes across time.
The second package has been accomplished by achieving down-regulation of Salmonella genes during infection in macrophages, and has been implemented as part of the third package. Moreover, to transfer the CRISPRi technology to the single-cell level, the scaffold region of the sgRNAs has been successfully engineered to include a capture sequence, not affecting efficiency of knock-down (KD) and allowing for direct recovery of the identity of the perturbation. A collection of perturbations targeting two-component systems was generated and used for subsequent experiments. The fellow has set up a protocol compatible with mainstream commercial single-cell technologies for recovering, at the single-cell level, both the gene expression profile of the host and the identity of Salmonella’s perturbations with a very high recovery rate. The protocols for these methods will be open access on online platforms, after manuscript submission. The CRISPRi plasmids have already been sent to addgene, and will be distributed following manuscript submission. These results were also disseminated in the several conferences the fellow attended.
With the research performed via this project, a novel, high-resolution method was established to study host-pathogen interactions. With it, it is possible to understand the dialogue between pathogenic intracellular bacteria and the host cells that they target. Previous techniques were limited by the resolution which did not achieve the individual cell level, while simultaneously perturbing the infectious mechanism of the bacteria. This work, in contrast, has established means to achieve both goals, and with this elucidate infection pathways, paving the way to the discovery of new potential drug targets, especially in light of the increasing emergence of antibiotic-resistant pathogens.