Microbiota modulation through horizontal gene transfer.

Project Information

MiMoZa

Grant agreement ID: 895167

DOI

10.3030/895167

Project closed

EC signature date 9 March 2020

Start date 1 September 2020

End date 14 January 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 266 425,92

EU contribution

€ 266 425,92

Coordinated by

VIB VZW
Belgium

Periodic Reporting for period 2 - MiMoZa (Microbiota modulation through horizontal gene transfer.)

Reporting period: 2022-09-01 to 2023-08-31

Summary of the context and overall objectives of the project

My goal is to determine horizontal gene transfer (HGT) rates of specific functions within the gut microbiome. The microbes on and within our bodies, especially within our gastro-intestinal tract, are intimately connected with our immune system and promote health in myriad ways. Altering our gut microbiota (GM) composition and/or function might thus improve health. My research in the Jeroen Raes lab led to the realization that changing species abundances through dietary or lifestyle modulation may be difficult because of the small effect sizes of the factors influencing GM composition (e.g. diet, medication, or transit time). In addition, host-selection and competition of resident microbes, might limit the strain engraftment necessary for the effect of fecal transfers and probiotic cocktails. Despite initial successes (e.g. C. difficile infection treatment), current modulation strategies might therefore be less successful with other conditions. I contacted Ilana Brito, in order to find out whether HGT - a process in which prokaryotes exchange genetic material - may serve as an alternative way to modulate the residing GM. Little is known about the features that affect HGT rates within natural environments nor the role of selection in that process. If these were well-understood, it would allow to assess the possibilities of extending the gut microbiome with additional functions. During my research stay, I will experimentally determine HGT rates within the animal gut under natural and HGT-enhancing conditions. The knowledge generated through this MSCA-fellowship will stimulate the development of novel GM modulation strategies and will have important implications for human health and agricultural policies (e.g. antibiotic use, spread of transgenes).

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

I investigated horizontal gene transfer (HGT) rates of carbohydrate-degradation and antibiotic-resistance genes, both in vitro and in the mouse gut. To confirm activity of the carbohydrate-degradation genes, growth experiments were conducted with transformed E. coli strains. Subsequently, 36 mice were fed with E. coli strains carrying mobilizable or non-mobilizable plasmids, followed by exposure to ampicillin, human milk oligosaccharides (HMO), or saline solution for a week. Surprisingly, we observed fewer and narrower HGT events than anticipated, although substantial plasmid spread occurred initially. Our analysis linked higher species abundance with increased plasmid transfer rates, suggesting that cell density influences conjugation more significantly than phylogenetic distance. Additionally, we explored the effects of HMO on mouse health and gut microbiome composition by introducing HMO-degradation genes into E. coli. Despite the expected competitive advantage, HMO supplementation did not promote E. coli colonization in the mouse gut. We have presented our findings on plasmid conjugation at the Belgian Microbiology Symposium 2024 and are preparing manuscripts for publication on the plasmid conjugation results and the effects of plasmid and HMO supplementation on gut microbiota composition.

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)

Progress beyond the state of the art.
I here assessed HGT rates and associated changes in the gut microbiome under different sorts and levels of selection. Real-world investigations of this kind are scarce, and our work represents one of the few endeavors in this direction. To check HGT rates I used a newly developed method, OIL-PCR, and combined that with a phage treatment step to extend its applicability to settings where the HGT-agent is delivered through a donor-bacterium. In addition, I followed donor, plasmid and total bacterial numbers with qPCR. Where changes in the gut microbiome are currently only followed by marker gene sequencing, with relative abundance data as an outcome, I here used a combination of internal standard spike-ins and qPCR, obtaining absolute abundance data with its associated benefits for data analyses and interpretation. The availability of absolute data proved advantageous, enabling us to establish a correlation between plasmid transfer incidence and species abundance.
Next to antibiotic selection, I applied selective pressure through HMO supplementation, which provided the donor bacteria with an additional nutritional resource. To this end, I evaluated several carbohydrates and associated CAZymes for their specificity and created and tested an E. coli strain able to use this carbohydrate as the sole carbon source. I further tested the effects of this specific carbohydrate on the (mouse) gut microbiome, applying absolute abundance profiling, allowing an assessments of its safety for (mouse) health and gut microbiome composition, and the possibility to use this system for the creation of an exclusive metabolic niche.

Potential impact.
Little is known about the features that affect HGT rates within natural environments, such as the gut, nor the role of selection in that process. If these were well-understood, it would aid the development of predictive tools to guide health and environmental policies as well as current and novel GM modulation strategies.
For example, predicting the transferability of a gene/function and the role of selection in that process could help assessing the risk of spreading antibiotic or GMO genes within an animal microbiome. At this moment, we know herbicide-sprayed GMO consumption leads to the integration of herbicide resistance genes in animal GM and changes GM composition but it is currently not clear at which rates and conditions, nor whether certain taxa are preferentially affected. Such knowledge is however crucial to assess the safety of these compounds and organisms.
An increased insight into HGT rates of functions could also aid the development of next generation probiotics. Current probiotic strains often do not engraft and therefore fail to infer the health benefit. The creation of an exclusive metabolic niche would however stimulate strain engraftment and allow reversible engraftment by discontinuing the selective pressure. Furthermore, enhanced HGT mechanisms could adapt GM function in situ, circumventing the problems of current modulation strategies (e.g. prebiotics, probiotics or fecal material transplantation (FMT)). When these options fail, enhanced HGT mechanisms might be able to restore the functions lost through industrialization or disease.
Our results give crucial insights into the taxonomic breadth and dynamics of plasmid conjugation in a real-life system, elucidating the effect of species abundance and phylogenetic relationships, and therefore provide another step in the realization of these goals.

Graphic Summary of project work.