Periodic Reporting for period 1 - Experimental Microbiome (“How mutualism evolves: Experimental microbiome evolution in gnotobiotic flies”)
Periodo di rendicontazione: 2015-05-01 al 2017-04-30
The research project “Experimental Microbiome” had the aim of uncovering the genetic basis of microbial benefit to animal growth. A large number of microbes live in association with animals. These microbes, called microbiota, profoundly affect the biology of their animal partners. In the gut, they enhance digestive efficiency, promoting host growth and development. Given their important role in shaping animal and human physiology, we are witnessing a blooming interest in the role of microbes, and specifically of “probiotics” (i.e. “Live microorganisms which confer a health benefit on the host"). Because of the current incidence of metabolic diseases, and the emerging link between these pathologies, nutrition and intestinal microbiota, the potential role of the microbiota in these pathologies is being revisited. In particular, until recently no study has informed us how the microbiome activities govern juvenile growth. The “Experimental Microbiome” research project addressed this issue. I focused on the identification of the bacterial genes involved into host growth promoting effect, by exploiting a simple one host/one bacterium model: Drosophila melanogaster associated with one of its natural commensals Lactobacillus plantarum. In order to disclose the genes involved in the growth promoting effect, I experimentally evolved a “poor” growth-promoting strain into a “potent” growth promoting strain, identifying the genetic variants responsible for the phenotype improvement. The present work allowed us to pinpoint the bacterial genes involved in Drosophila growth promotion, shedding light into the complex relationships between commensal bacteria and their animal partners.
The main aim of this research project was to dissect the genetic basis of the evolution of host-microbiota mutualism, with a particular focus on the microbial genetic signatures responsible of host growth promotion. To do this, we conducted two parallel lines of research:
1) to better apprehend L. plantarum genomics (“top-down” perspective),
2) to identify the genetic elements in L. plantarum genome shaping its impact on Drosophila growth (“bottom-up” approach).
First, we embarked on a large genome-sequencing project of 54 L. plantarum strains isolated from different habitats with the aim of probing the genomic diversity of the L. plantarum species. Although L. plantarum versatility was already recognized, until recently a clear link between its genomic evolution and ecological dispersal was unknown. We have now revealed that L. plantarum has evolved a “nomadic” lifestyle in which genomic adaptation was not driven by environmental specialization. This means that L. plantarum genomic and phenotypic characteristics appear unrelated to the actual source of isolation.
In parallel to this functional genomic approach, we developed a L. plantarum Experimental Evolution (EE) in order to genetically select bacterial strains improving host growth (main aim of this research project). The rationale of this approach was to test if we could "evolve" a poorly growth promoting L. plantarum strain into a "potent" growth promoting strain by fixating spontaneous genetic variants via artificial selection. Then identifying the nature of such fixed variants would inform us on the key bacterial genomic features sustaining Drosophila growth promotion.
This part of the project was divided in three main objectives:
1) Building the experimental microbiome
2) Identifying the genetic variants involved into microbial growth-promoting effect
3) Identifying the causative links between those genetic variants and their benefit
Objective 1. To this end, I mono-colonized a population of germ-free Drosophila embryos with a L. plantarum isolate which is poorly promoting Drosophila growth (LpNIZO2877). I let the mono-colonized individuals develop as larvae and grow in conditions of chronic undernutrition. Then, I selected the first adults (and their associated commensal bacteria with potentially spontaneous genetic variants) emerging from the mono-colonized larvae (only the 20% of first emerging individuals were selected), which were mated to propagate the next Drosophila generation (and their associated commensals with potentially spontaneous genetic variants). The L. plantarum EE lasted 320 days (20 fly generations and 330 bacterial generations in this setup). We started to obtain few bacterial isolates with an increased effect on Drosophila growth as compared to the ancestral LpNIZO2877 isolate, as early as after two fly generations. Those isolates had invaded the entire commensal bacterial community by Drosophila generation 9 since, at that stage, all the tested evolved isolates from the population of commensal L. plantarum were significantly more beneficial than the ancestral isolate.
Objective 2. Genome sequencing of individual and independently evolved isolates, showing improved Drosophila growth promotion capabilities, identified few bacterial genes affected by mutation. In particular, two genes (ackA and cheY) accumulated mutations in both independent replicates of evolution. Notably, the evolved strain (LpFlyG2) bearing only 1 mutation in the acetate kinase A (ackA) gene was more able to improve host growth compared to its ancestor. Remarkably, I discovered that L. plantarum improvement in growth promotion is uncoupled from the co-evolution with its host, as L. plantarum strains experimentally evolved in Drosophila nutritional medium without the fly also became more beneficial. Moreover, these strains accumulated mutations in the same ackA gene, showing the exceptional evolutionary convergence of L. plantarum in our nutritional environment and further proving the key role of ackA in host growth promotion (Martino et al., in preparation).
Objective 3. We also demonstrated the causative link between the ackA variant and L. plantarum increase in growth promotion. Specifically, the reversion of the ackA mutation from the evolved isolate (LpFlyG2) back to its ancestral state (thanks to CRISPR/Cas9 genome editing) led to the loss of growth promoting capability of the evolved isolate. This part of the project has been conducted in collaboration with the group of Prof. Chase Beisel (North Carolina State University – Raileigh, USA).
1) to better apprehend L. plantarum genomics (“top-down” perspective),
2) to identify the genetic elements in L. plantarum genome shaping its impact on Drosophila growth (“bottom-up” approach).
First, we embarked on a large genome-sequencing project of 54 L. plantarum strains isolated from different habitats with the aim of probing the genomic diversity of the L. plantarum species. Although L. plantarum versatility was already recognized, until recently a clear link between its genomic evolution and ecological dispersal was unknown. We have now revealed that L. plantarum has evolved a “nomadic” lifestyle in which genomic adaptation was not driven by environmental specialization. This means that L. plantarum genomic and phenotypic characteristics appear unrelated to the actual source of isolation.
In parallel to this functional genomic approach, we developed a L. plantarum Experimental Evolution (EE) in order to genetically select bacterial strains improving host growth (main aim of this research project). The rationale of this approach was to test if we could "evolve" a poorly growth promoting L. plantarum strain into a "potent" growth promoting strain by fixating spontaneous genetic variants via artificial selection. Then identifying the nature of such fixed variants would inform us on the key bacterial genomic features sustaining Drosophila growth promotion.
This part of the project was divided in three main objectives:
1) Building the experimental microbiome
2) Identifying the genetic variants involved into microbial growth-promoting effect
3) Identifying the causative links between those genetic variants and their benefit
Objective 1. To this end, I mono-colonized a population of germ-free Drosophila embryos with a L. plantarum isolate which is poorly promoting Drosophila growth (LpNIZO2877). I let the mono-colonized individuals develop as larvae and grow in conditions of chronic undernutrition. Then, I selected the first adults (and their associated commensal bacteria with potentially spontaneous genetic variants) emerging from the mono-colonized larvae (only the 20% of first emerging individuals were selected), which were mated to propagate the next Drosophila generation (and their associated commensals with potentially spontaneous genetic variants). The L. plantarum EE lasted 320 days (20 fly generations and 330 bacterial generations in this setup). We started to obtain few bacterial isolates with an increased effect on Drosophila growth as compared to the ancestral LpNIZO2877 isolate, as early as after two fly generations. Those isolates had invaded the entire commensal bacterial community by Drosophila generation 9 since, at that stage, all the tested evolved isolates from the population of commensal L. plantarum were significantly more beneficial than the ancestral isolate.
Objective 2. Genome sequencing of individual and independently evolved isolates, showing improved Drosophila growth promotion capabilities, identified few bacterial genes affected by mutation. In particular, two genes (ackA and cheY) accumulated mutations in both independent replicates of evolution. Notably, the evolved strain (LpFlyG2) bearing only 1 mutation in the acetate kinase A (ackA) gene was more able to improve host growth compared to its ancestor. Remarkably, I discovered that L. plantarum improvement in growth promotion is uncoupled from the co-evolution with its host, as L. plantarum strains experimentally evolved in Drosophila nutritional medium without the fly also became more beneficial. Moreover, these strains accumulated mutations in the same ackA gene, showing the exceptional evolutionary convergence of L. plantarum in our nutritional environment and further proving the key role of ackA in host growth promotion (Martino et al., in preparation).
Objective 3. We also demonstrated the causative link between the ackA variant and L. plantarum increase in growth promotion. Specifically, the reversion of the ackA mutation from the evolved isolate (LpFlyG2) back to its ancestral state (thanks to CRISPR/Cas9 genome editing) led to the loss of growth promoting capability of the evolved isolate. This part of the project has been conducted in collaboration with the group of Prof. Chase Beisel (North Carolina State University – Raileigh, USA).
The results of the research project “Experimental Microbiome” represent the first long-term in vivo model of host/microbiota evolution. We have been able to achieve all the objectives. In particular, we were able to:
1) Reveal the nomadic lifestyle of L. plantarum species, which was not known
2) Identify a single genetic variant of L. plantarum capable of improving its growth promotion
3) Revealing the key role of nutrition in shaping the microbial effect towards its animal partner.
The present project has greatly increased our knowledge of host/microbiota mutualism. It paves the way to studies focusing on how evolution shape animal-microbe interplay and raises new questions about the role of host, bacteria and the nutritional environment in determining their mutual association. The results achieved during this two-year project led to the publication of three articles, two reviews and two further manuscripts are in preparation. In addition, we deposited a patent to use Lactobacilli in medical and industrial applications (Publication n° WO2015173386 A1).
1) Reveal the nomadic lifestyle of L. plantarum species, which was not known
2) Identify a single genetic variant of L. plantarum capable of improving its growth promotion
3) Revealing the key role of nutrition in shaping the microbial effect towards its animal partner.
The present project has greatly increased our knowledge of host/microbiota mutualism. It paves the way to studies focusing on how evolution shape animal-microbe interplay and raises new questions about the role of host, bacteria and the nutritional environment in determining their mutual association. The results achieved during this two-year project led to the publication of three articles, two reviews and two further manuscripts are in preparation. In addition, we deposited a patent to use Lactobacilli in medical and industrial applications (Publication n° WO2015173386 A1).