Periodic Reporting for period 1 - ResistEpist (Dissection of the mechanisms causing the epistasis between antibiotic resistance mutations in Escherichia coli)
Période du rapport: 2018-12-01 au 2020-11-30
Antibiotic resistances allow bacteria to survive in the presence of antibiotics, but often cause a fitness disadvantage (called fitness cost) in the absence of the drug. Thus, the cost is the main biological parameter influencing the fate of resistances upon reducing antibiotic use. Antibiotic resistance mutations often interact with each other in a manner that the fitness cost resulting from combining different mutations in the same bacterium is different than the sum of the individual effects of these mutations. These interactions (called epistasis) often cause a reduction in the fitness cost, favoring their maintenance and dissemination in bacterial populations. Antibiotic resistance is the biggest health challenge in the 21st century, and epistatic interactions play a key role in the spread of resistances. Thus, a better undertanding of the epistasis between antibiotic resistance mutations is of paramount importance for global health.
This project aims to determine the molecular mechanisms involved in the interaction (epistasis) between antibiotic resistance mutations. We hypothesize that the fitness cost of resistance mutations is the result of the aggregated effects of diverse phenotypes caused by the interference of these mutations with different cellular functions.
Objective 1. To determine the phenotypic effects of resistance mutations.
Objective 2. Studying these phenotypic effects in evolved strains carrying compensatory mutations.
Objective 3. Integration of the results obtained into general hypotheses to explain the cause/s of the epistases. Test of the elaborated hypotheses.
Thus, in this project we are studying the effects of these mutations on phenotypes potentially affecting fitness, such as biosynthetic capacity, genome instability and proteostasis imbalance, in order to determine the causes of the fitness cost of single mutations and understanding the mechanisms causing the epistases between mutations. Importantly, some of the phenotypes that we are studying (e.g. genome instability) have never been associated with the cost of antibiotic resistance mutations; thus this research programme may unveil novel mechanisms involved in the fitness effects caused by resistance mutations. The mentioned phenotypes are being analyzed in single and double mutant ancestral strains and in derivatives that have been evolved in the lab, acquiring compensatory mutations that decrease the fitness cost of the resistance mutations. Determining the phenotypes affected in single or double ancestral strains and compensated in the corresponding evolved bacteria will help to identify the mechanisms underlying the epistasis between resistance mutations.
This project aims to determine the molecular mechanisms involved in the interaction (epistasis) between antibiotic resistance mutations. We hypothesize that the fitness cost of resistance mutations is the result of the aggregated effects of diverse phenotypes caused by the interference of these mutations with different cellular functions.
Objective 1. To determine the phenotypic effects of resistance mutations.
Objective 2. Studying these phenotypic effects in evolved strains carrying compensatory mutations.
Objective 3. Integration of the results obtained into general hypotheses to explain the cause/s of the epistases. Test of the elaborated hypotheses.
Thus, in this project we are studying the effects of these mutations on phenotypes potentially affecting fitness, such as biosynthetic capacity, genome instability and proteostasis imbalance, in order to determine the causes of the fitness cost of single mutations and understanding the mechanisms causing the epistases between mutations. Importantly, some of the phenotypes that we are studying (e.g. genome instability) have never been associated with the cost of antibiotic resistance mutations; thus this research programme may unveil novel mechanisms involved in the fitness effects caused by resistance mutations. The mentioned phenotypes are being analyzed in single and double mutant ancestral strains and in derivatives that have been evolved in the lab, acquiring compensatory mutations that decrease the fitness cost of the resistance mutations. Determining the phenotypes affected in single or double ancestral strains and compensated in the corresponding evolved bacteria will help to identify the mechanisms underlying the epistasis between resistance mutations.
Objective 1. Determination of the phenotypic effects of resistance mutations:
Task 1.1. Biosynthetic capacity and expression profile.
Biosynthesis tested via two parallel methods: fluorescence intensity of a constitutively expressed reporter, and using an inducible reporter. We observed that biosynthetic capacity does not correlate well with the fitness cost of resistance mutations.
Task 1.2. Genome instability.
We measured genome instability using a fluorescent reporter that activates upon the generation of DNA breaks. We observed a significant overlap between the epistatic effects of double resistance with respect to their fitness cost and the SOS induction, suggesting a causal relationship. Importantly, we found a strong correlation between the genome instability and the cost of resistance.
Task 1.3. Proteostasis imbalance.
Not completed. The limitations imposed by the Covid-19 crisis during half of the time of the fellowship, together with the extraordinary results obtained in Task 1.2 caused us to focus on the most relevant phenotype (genome instability), and therefore withdraw the experiments associated to this task from our plans.
Task 1.4. The effects of antibiotics on the above phenotypes in wild-type bacteria.
We analyzed the effects of rifampicin and streptomycin on two phenotypes (genome instability and fitness), obtaining two interesting conclusions: first, that antibiotics can indeed cause genome instability, and second, that the fitness cost of mutants affected in transcription can be increased by using sub-MIC concentrations of an antibiotic targeting translation, and vice-versa.
Objective 2. Study of the phenotypes in tasks 1.1-1.3 in evolved strains carrying compensatory mutations.
Task 2.1. Experimental evolution, sequencing and determination of compensatory mutations.
We evolved several resistant genotypes, analyzed the pace at which compensation occurs and identified compensatory mutations for all of them. As previously observed, in double mutants we found compensatory mutations compensating specific for the epistasis.
Task 2.2. Study of the phenotypes in tasks 1.1-1.3 in the evolved lines.
As explained above, task 1.3 was withdrawn from our plans. We did perform experimental evolution, and measured fitness cost and genome instability in the ancestral and evolved clones (see section 1.2.2.1). We concluded that the mechanisms causing genome instability are being repeatedly targeted by compensatory evolution.
Objective 3. Integration of the results obtained into general hypotheses to explain the cause/s of the epistases. Test of the elaborated hypotheses.
We concluded that coordination between transcription and translation, and its ultimate consequences (DNA breaks) are key factor both for the fitness cost of resistance mutations and for the epistasis between these mutations.
Task 1.1. Biosynthetic capacity and expression profile.
Biosynthesis tested via two parallel methods: fluorescence intensity of a constitutively expressed reporter, and using an inducible reporter. We observed that biosynthetic capacity does not correlate well with the fitness cost of resistance mutations.
Task 1.2. Genome instability.
We measured genome instability using a fluorescent reporter that activates upon the generation of DNA breaks. We observed a significant overlap between the epistatic effects of double resistance with respect to their fitness cost and the SOS induction, suggesting a causal relationship. Importantly, we found a strong correlation between the genome instability and the cost of resistance.
Task 1.3. Proteostasis imbalance.
Not completed. The limitations imposed by the Covid-19 crisis during half of the time of the fellowship, together with the extraordinary results obtained in Task 1.2 caused us to focus on the most relevant phenotype (genome instability), and therefore withdraw the experiments associated to this task from our plans.
Task 1.4. The effects of antibiotics on the above phenotypes in wild-type bacteria.
We analyzed the effects of rifampicin and streptomycin on two phenotypes (genome instability and fitness), obtaining two interesting conclusions: first, that antibiotics can indeed cause genome instability, and second, that the fitness cost of mutants affected in transcription can be increased by using sub-MIC concentrations of an antibiotic targeting translation, and vice-versa.
Objective 2. Study of the phenotypes in tasks 1.1-1.3 in evolved strains carrying compensatory mutations.
Task 2.1. Experimental evolution, sequencing and determination of compensatory mutations.
We evolved several resistant genotypes, analyzed the pace at which compensation occurs and identified compensatory mutations for all of them. As previously observed, in double mutants we found compensatory mutations compensating specific for the epistasis.
Task 2.2. Study of the phenotypes in tasks 1.1-1.3 in the evolved lines.
As explained above, task 1.3 was withdrawn from our plans. We did perform experimental evolution, and measured fitness cost and genome instability in the ancestral and evolved clones (see section 1.2.2.1). We concluded that the mechanisms causing genome instability are being repeatedly targeted by compensatory evolution.
Objective 3. Integration of the results obtained into general hypotheses to explain the cause/s of the epistases. Test of the elaborated hypotheses.
We concluded that coordination between transcription and translation, and its ultimate consequences (DNA breaks) are key factor both for the fitness cost of resistance mutations and for the epistasis between these mutations.
Before the completion of this project, the causes of the fitness cost of resistance mutations affecting transcription and translation were not completely known, and the factors contributing to the epistasis between resistances remained elusive. We unveiled DNA breaks as major contributors to the fitness cost of antibiotic resistance mutations affecting transcription and translation, an one of the factors contributing to the epistasis. Moreover, we revealed the involvement of R-loops (RNA:DNA hybrids typically formed during transcription) in this process, and demonstrated that RNase HI (the enzyme responsible for the specific degradation of R-loops) is a promising target for antimicrobial therapy specific against resistant bacteria.